{"gene":"TG_246550","gene_symbol":"ASP3","run_date":"2026-09-08T12:58:25","timeline":{"discoveries":[{"year":2017,"finding":"TgASP3 resides in the endosomal-like compartment (post-Golgi) of T. gondii and acts as a maturase for microneme and rhoptry proteins; depletion of ASP3 blocks rhoptry discharge during invasion and host cell plasma membrane lysis during egress. Substrates were identified by N-terminome profiling (TAILS) comparing wild-type and ASP3-depleted parasites, and maturation was confirmed both in vivo and in vitro.","method":"Conditional depletion (knockdown), terminal amine isotopic labelling of substrates (TAILS/N-terminomics), in vitro protease assays, subcellular fractionation/localization imaging","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (N-terminomics, in vitro assay, genetic depletion with defined phenotypes, localization) in a single rigorous study","pmids":["28898199"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"TgASP3 phylogenetically clusters with Plasmodium falciparum Plasmepsins IX and X and shares a conserved phenylalanine residue (F344) in the flap region that is critical for binding the antimalarial inhibitor 49c; mutation of F344 increases IC50 of 49c 2–15-fold, and selection of resistant T. gondii strains consistently converts F344 to cysteine, establishing this flap residue as a key determinant of substrate/inhibitor recognition.","method":"Homology modeling, molecular docking, site-directed mutagenesis (F344 variants), drug-resistance selection in T. gondii, IC50 measurements","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis validated by docking model plus independent in-parasite resistance selection, single lab but multiple orthogonal approaches","pmids":["29519896"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"finding":"TgASP3 acts upstream of TgCPC1 in the secretory protein maturation pathway; TgASP3 (post-Golgi) and TgCPL (endolysosomal system) perform earlier proteolytic cleavages on micronemal effectors, while TgCPC1 mediates the final trimming step. Loss of TgCPC1 does not affect TgASP3 itself but blocks downstream maturation of effectors that TgASP3 had partially processed.","method":"Genetic deletion of TgCPC1, comparison of maturation states of secretory proteins relative to TgASP3/TgCPL-dependent cleavages, invasion/egress phenotypic assays","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis established by genetic deletion with defined substrate readouts, single lab, pathway position inferred from sequential cleavage evidence","pmids":["37326431"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgASP3 is an aspartyl protease that resides in the endosomal-like/post-Golgi compartment of Toxoplasma gondii, where it acts as an essential maturase that proteolytically processes microneme and rhoptry proteins prior to organellar storage and secretion; this activity is required for rhoptry discharge during invasion and host cell membrane lysis during egress, and the inhibitor-binding selectivity of TgASP3 is determined by a conserved flap-region phenylalanine (F344) that gates access to the catalytic cavity."},"narrative":{"mechanistic_narrative":"TgASP3 is an aspartyl protease of the endosomal-like/post-Golgi compartment of Toxoplasma gondii that functions as an essential maturase for microneme and rhoptry proteins, with its proteolytic activity required for rhoptry discharge during host cell invasion and for plasma membrane lysis during egress [PMID:28898199]. N-terminomic profiling of ASP3-depleted parasites established a defined set of secretory effector substrates whose maturation depends on this enzyme both in vivo and in vitro [PMID:28898199]. TgASP3 operates within a sequential maturation pathway, performing early cleavages of micronemal effectors upstream of the final trimming step carried out by TgCPC1, whose loss blocks downstream processing of ASP3-primed substrates without affecting ASP3 itself [PMID:37326431]. A conserved flap-region phenylalanine (F344) gates access to the catalytic cavity and determines inhibitor/substrate recognition: mutation of F344 raises the IC50 of the antimalarial inhibitor 49c, and drug-resistance selection reproducibly converts F344 to cysteine [PMID:29519896].","teleology":[{"year":2017,"claim":"Established that TgASP3 is the maturase responsible for processing microneme and rhoptry proteins, answering what enzymatic step licenses these secretory effectors for organellar storage and discharge.","evidence":"Conditional depletion with TAILS N-terminomics, in vitro protease assays, and subcellular localization in T. gondii","pmids":["28898199"],"confidence":"High","gaps":["Structural basis of substrate cleavage not resolved","Mechanism coupling ASP3 maturation to rhoptry discharge and membrane lysis not defined","Full substrate repertoire beyond identified microneme/rhoptry proteins not delineated"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"claim":"Identified the flap-region residue F344 as the determinant of inhibitor and substrate recognition, explaining how the catalytic cavity is gated and how resistance arises.","evidence":"Homology modeling, docking, F344 site-directed mutagenesis, and drug-resistance selection with IC50 measurements in T. gondii","pmids":["29519896"],"confidence":"High","gaps":["No experimental crystal/cryo-EM structure to confirm the docking model","Effect of F344 variation on physiological substrate cleavage versus inhibitor binding not disentangled"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"claim":"Placed TgASP3 within an ordered proteolytic maturation cascade, showing it performs early cleavages upstream of the final TgCPC1 trimming step.","evidence":"Genetic deletion of TgCPC1 with substrate maturation-state readouts and invasion/egress assays in T. gondii","pmids":["37326431"],"confidence":"Medium","gaps":["Direct substrate handoff between ASP3, TgCPL, and TgCPC1 not biochemically reconstituted","Whether ASP3 and TgCPL act redundantly or on distinct sites not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The three-dimensional structure of TgASP3 and the molecular mechanism linking effector maturation to the physical events of rhoptry discharge and membrane lysis remain undefined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No experimentally determined enzyme structure","No reconstitution of the full ASP3-TgCPL-TgCPC1 cascade","Regulation of ASP3 activity and trafficking within the endosomal-like compartment unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["TGCPC1 (DOWNSTREAM MATURASE IN THE SAME SECRETORY PROCESSING CASCADE)","TGCPL (ENDOLYSOSOMAL PROTEASE ACTING IN THE SAME EFFECTOR MATURATION PATHWAY)"],"other_free_text":["Maturase for microneme and rhoptry proteins; activity required for rhoptry discharge during invasion and host cell plasma membrane lysis during egress [#0]","Flap-region phenylalanine F344 gates the catalytic cavity and determines inhibitor 49c binding; F344C substitution confers resistance [#1]","Phylogenetically clusters with Plasmodium falciparum Plasmepsins IX and X [#1]","Acts upstream of TgCPC1, performing early cleavages before final effector trimming [#2]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"28898199","id":"PMC_28898199","title":"A druggable secretory protein maturase of Toxoplasma essential for invasion and egress.","date":"2017","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/28898199","citation_count":80,"is_preprint":false},{"pmid":"15358257","id":"PMC_15358257","title":"Host cell invasion by the apicomplexans: the significance of microneme protein proteolysis.","date":"2004","source":"Current opinion in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/15358257","citation_count":72,"is_preprint":false},{"pmid":"28238685","id":"PMC_28238685","title":"Immunization with Toxoplasma gondii aspartic protease 3 increases survival time of infected mice.","date":"2017","source":"Acta tropica","url":"https://pubmed.ncbi.nlm.nih.gov/28238685","citation_count":17,"is_preprint":false},{"pmid":"29519896","id":"PMC_29519896","title":"Modeling and resistant alleles explain the selectivity of antimalarial compound 49c towards apicomplexan aspartyl proteases.","date":"2018","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/29519896","citation_count":14,"is_preprint":false},{"pmid":"37326431","id":"PMC_37326431","title":"A cathepsin C-like protease mediates the post-translation modification of Toxoplasma gondii secretory proteins for optimal invasion and egress.","date":"2023","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/37326431","citation_count":4,"is_preprint":false},{"pmid":"36712013","id":"PMC_36712013","title":"A cathepsin C-like protease post-translationally modifies Toxoplasma gondii secretory proteins for optimal invasion and egress.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/36712013","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":5705,"output_tokens":1074,"usd":0.016612,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":5948,"output_tokens":2094,"usd":0.041045,"stage2_stop_reason":"end_turn"},"total_usd":0.057657,"stage1_batch_id":"msgbatch_01TEPNZ4MCzJ7xtsNwosiDRk","stage2_batch_id":"msgbatch_01FQShM7n1sfCr4pWTDShmki","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2017,\n      \"finding\": \"TgASP3 resides in the endosomal-like compartment (post-Golgi) of T. gondii and acts as a maturase for microneme and rhoptry proteins; depletion of ASP3 blocks rhoptry discharge during invasion and host cell plasma membrane lysis during egress. Substrates were identified by N-terminome profiling (TAILS) comparing wild-type and ASP3-depleted parasites, and maturation was confirmed both in vivo and in vitro.\",\n      \"method\": \"Conditional depletion (knockdown), terminal amine isotopic labelling of substrates (TAILS/N-terminomics), in vitro protease assays, subcellular fractionation/localization imaging\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (N-terminomics, in vitro assay, genetic depletion with defined phenotypes, localization) in a single rigorous study\",\n      \"pmids\": [\"28898199\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TgASP3 phylogenetically clusters with Plasmodium falciparum Plasmepsins IX and X and shares a conserved phenylalanine residue (F344) in the flap region that is critical for binding the antimalarial inhibitor 49c; mutation of F344 increases IC50 of 49c 2–15-fold, and selection of resistant T. gondii strains consistently converts F344 to cysteine, establishing this flap residue as a key determinant of substrate/inhibitor recognition.\",\n      \"method\": \"Homology modeling, molecular docking, site-directed mutagenesis (F344 variants), drug-resistance selection in T. gondii, IC50 measurements\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis validated by docking model plus independent in-parasite resistance selection, single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"29519896\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TgASP3 acts upstream of TgCPC1 in the secretory protein maturation pathway; TgASP3 (post-Golgi) and TgCPL (endolysosomal system) perform earlier proteolytic cleavages on micronemal effectors, while TgCPC1 mediates the final trimming step. Loss of TgCPC1 does not affect TgASP3 itself but blocks downstream maturation of effectors that TgASP3 had partially processed.\",\n      \"method\": \"Genetic deletion of TgCPC1, comparison of maturation states of secretory proteins relative to TgASP3/TgCPL-dependent cleavages, invasion/egress phenotypic assays\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis established by genetic deletion with defined substrate readouts, single lab, pathway position inferred from sequential cleavage evidence\",\n      \"pmids\": [\"37326431\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgASP3 is an aspartyl protease that resides in the endosomal-like/post-Golgi compartment of Toxoplasma gondii, where it acts as an essential maturase that proteolytically processes microneme and rhoptry proteins prior to organellar storage and secretion; this activity is required for rhoptry discharge during invasion and host cell membrane lysis during egress, and the inhibitor-binding selectivity of TgASP3 is determined by a conserved flap-region phenylalanine (F344) that gates access to the catalytic cavity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TgASP3 is an aspartyl protease of the endosomal-like/post-Golgi compartment of Toxoplasma gondii that functions as an essential maturase for microneme and rhoptry proteins, with its proteolytic activity required for rhoptry discharge during host cell invasion and for plasma membrane lysis during egress [#0]. N-terminomic profiling of ASP3-depleted parasites established a defined set of secretory effector substrates whose maturation depends on this enzyme both in vivo and in vitro [#0]. TgASP3 operates within a sequential maturation pathway, performing early cleavages of micronemal effectors upstream of the final trimming step carried out by TgCPC1, whose loss blocks downstream processing of ASP3-primed substrates without affecting ASP3 itself [#2]. A conserved flap-region phenylalanine (F344) gates access to the catalytic cavity and determines inhibitor/substrate recognition: mutation of F344 raises the IC50 of the antimalarial inhibitor 49c, and drug-resistance selection reproducibly converts F344 to cysteine [#1].\",\n  \"teleology\": [\n    {\n      \"year\": 2017,\n      \"claim\": \"Established that TgASP3 is the maturase responsible for processing microneme and rhoptry proteins, answering what enzymatic step licenses these secretory effectors for organellar storage and discharge.\",\n      \"evidence\": \"Conditional depletion with TAILS N-terminomics, in vitro protease assays, and subcellular localization in T. gondii\",\n      \"pmids\": [\"28898199\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of substrate cleavage not resolved\",\n        \"Mechanism coupling ASP3 maturation to rhoptry discharge and membrane lysis not defined\",\n        \"Full substrate repertoire beyond identified microneme/rhoptry proteins not delineated\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified the flap-region residue F344 as the determinant of inhibitor and substrate recognition, explaining how the catalytic cavity is gated and how resistance arises.\",\n      \"evidence\": \"Homology modeling, docking, F344 site-directed mutagenesis, and drug-resistance selection with IC50 measurements in T. gondii\",\n      \"pmids\": [\"29519896\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No experimental crystal/cryo-EM structure to confirm the docking model\",\n        \"Effect of F344 variation on physiological substrate cleavage versus inhibitor binding not disentangled\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Placed TgASP3 within an ordered proteolytic maturation cascade, showing it performs early cleavages upstream of the final TgCPC1 trimming step.\",\n      \"evidence\": \"Genetic deletion of TgCPC1 with substrate maturation-state readouts and invasion/egress assays in T. gondii\",\n      \"pmids\": [\"37326431\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct substrate handoff between ASP3, TgCPL, and TgCPC1 not biochemically reconstituted\",\n        \"Whether ASP3 and TgCPL act redundantly or on distinct sites not resolved\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The three-dimensional structure of TgASP3 and the molecular mechanism linking effector maturation to the physical events of rhoptry discharge and membrane lysis remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No experimentally determined enzyme structure\",\n        \"No reconstitution of the full ASP3-TgCPL-TgCPC1 cascade\",\n        \"Regulation of ASP3 activity and trafficking within the endosomal-like compartment unknown\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004190\", \"supporting_discovery_ids\": [0, 1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0016485\", \"supporting_discovery_ids\": [0, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006909\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"TgCPC1 (downstream maturase in the same secretory processing cascade)\",\n      \"TgCPL (endolysosomal protease acting in the same effector maturation pathway)\"\n    ],\n    \"other_free_text\": [\n      \"Maturase for microneme and rhoptry proteins; activity required for rhoptry discharge during invasion and host cell plasma membrane lysis during egress [#0]\",\n      \"Flap-region phenylalanine F344 gates the catalytic cavity and determines inhibitor 49c binding; F344C substitution confers resistance [#1]\",\n      \"Phylogenetically clusters with Plasmodium falciparum Plasmepsins IX and X [#1]\",\n      \"Acts upstream of TgCPC1, performing early cleavages before final effector trimming [#2]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_246550","name":"ASP3","product":"aspartyl protease ASP3","uniprot_acc":"A0A125YQD9","chromosome":"TGME49_chrXII","start_pos":2845768},{"strain":"GT1","gene_id":"TGGT1_246550","name":null,"product":"aspartyl protease ASP3","uniprot_acc":null,"chromosome":"TGGT1_chrXII","start_pos":2777006},{"strain":"RH88","gene_id":"TGRH88_060810","name":null,"product":"aspartyl protease ASP3","uniprot_acc":null,"chromosome":"CM023093","start_pos":2875441},{"strain":"VEG","gene_id":"TGVEG_246550","name":null,"product":"aspartyl protease ASP3","uniprot_acc":"B6KGS0","chromosome":"TGVEG_chrXII","start_pos":2814548}]}