{"gene":"TG_314500","gene_symbol":"SUB2","run_date":"2026-09-08T15:15:13","timeline":{"discoveries":[{"year":2003,"finding":"TgSUB2 is a subtilisin-like serine proteinase that localizes to rhoptry organelles in Toxoplasma gondii, as determined by direct localization experiments.","method":"Subcellular localization (direct experiment)","journal":"Molecular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiment with functional context, single lab, single paper","pmids":["12890015"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"finding":"TgSUB2 undergoes autocatalytic (self) processing as it traffics through the secretory pathway; site-directed mutagenesis of a sequence within TgSUB2 homologous to the ROP1 cleavage site (after Glu) abolished this autoprocessing, establishing TgSUB2 as a serine proteinase with cleavage specificity after Glu.","method":"Site-directed mutagenesis of the predicted active-site/cleavage motif; in vivo processing assay","journal":"Molecular microbiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — active-site mutagenesis abolishing autoprocessing, single lab but direct mechanistic experiment with mutagenesis validation","pmids":["12890015"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"finding":"TgSUB2 associates with the rhoptry protein ROP1, which is a potential substrate, suggesting TgSUB2 functions as a rhoptry protein maturase responsible for processing rhoptry organelle contents.","method":"Co-association/pulldown with ROP1; sequence homology to ROP1 cleavage site validated by mutagenesis","journal":"Molecular microbiology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single Co-IP/association experiment combined with mutagenesis supporting substrate identification, single lab","pmids":["12890015"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"finding":"Disruption of TgSUB2 was unsuccessful, implying that TgSUB2 is an essential gene in Toxoplasma gondii.","method":"Gene disruption attempt (negative result — failed knockout)","journal":"Molecular microbiology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — essentiality inferred from failed disruption, single lab, single method","pmids":["12890015"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgSUB2 is an essential, rhoptry-localized subtilisin-like serine proteinase in Toxoplasma gondii that undergoes autocatalytic processing during trafficking through the secretory pathway and functions as a rhoptry protein maturase — cleaving after Glu residues, as validated by mutagenesis — with ROP1 as a candidate substrate."},"narrative":{"mechanistic_narrative":"TgSUB2 is a subtilisin-like serine proteinase of Toxoplasma gondii that localizes to the rhoptry secretory organelles and functions as a candidate rhoptry protein maturase [PMID:12890015]. As it traffics through the secretory pathway, TgSUB2 undergoes autocatalytic processing; site-directed mutagenesis of a motif homologous to the ROP1 cleavage site abolished this autoprocessing, establishing that the enzyme cleaves after Glu residues [PMID:12890015]. Consistent with a maturase role, TgSUB2 associates with the rhoptry protein ROP1, a candidate substrate [PMID:12890015]. Beyond this single body of work, no further mechanistic detail — including a validated in vitro substrate repertoire or structural model — has been characterized in the available corpus.","teleology":[{"year":2003,"claim":"Establishing where an uncharacterized subtilisin-like protease acts localized TgSUB2 to the rhoptry organelles, placing it within the invasion-associated secretory machinery.","evidence":"Direct subcellular localization experiment in T. gondii","pmids":["12890015"],"confidence":"Medium","gaps":["Does not define the enzyme's substrates within the rhoptry","Trafficking route to the rhoptry not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"claim":"Whether TgSUB2 is a functional protease and what specificity it holds was answered by showing it autoprocesses during secretory trafficking and that mutating the ROP1-homologous cleavage motif abolishes this, defining cleavage after Glu.","evidence":"Site-directed mutagenesis of the predicted cleavage motif plus in vivo processing assay","pmids":["12890015"],"confidence":"High","gaps":["Cleavage specificity inferred from autoprocessing, not from a purified enzyme against defined peptide substrates","No structural basis for Glu specificity established"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"claim":"The biological role of TgSUB2 as a rhoptry protein maturase was supported by its association with ROP1 as a candidate substrate.","evidence":"Co-association/pulldown with ROP1 combined with mutagenesis-validated cleavage-site homology","pmids":["12890015"],"confidence":"Medium","gaps":["Single Co-IP without reciprocal validation","Direct in vitro cleavage of ROP1 by TgSUB2 not demonstrated","Broader rhoptry substrate set unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"claim":"The requirement of TgSUB2 for parasite viability was inferred from an unsuccessful gene disruption, implying essentiality.","evidence":"Failed gene disruption attempt (negative result)","pmids":["12890015"],"confidence":"Low","gaps":["Essentiality inferred from failed knockout, not from a conditional/inducible knockout","Phenotype of depletion not characterized"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The full rhoptry substrate repertoire, the mechanism and timing of TgSUB2 activation, and a conditional-knockout demonstration of its essential function remain undefined.","evidence":"","pmids":[],"confidence":"Low","gaps":["No validated substrate beyond candidate ROP1","No structural model","Essentiality not confirmed by conditional depletion"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["ROP1 (CANDIDATE SUBSTRATE)"],"other_free_text":["Undergoes autocatalytic processing during secretory-pathway trafficking [#1]","Cleavage specificity after Glu residues, validated by mutagenesis of a ROP1-homologous cleavage motif [#1]","Candidate essential gene inferred from unsuccessful disruption [#3]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"12890015","id":"PMC_12890015","title":"TgSUB2 is a Toxoplasma gondii rhoptry organelle processing proteinase.","date":"2003","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/12890015","citation_count":69,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":2929,"output_tokens":940,"usd":0.011444,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":5808,"output_tokens":1846,"usd":0.037595,"stage2_stop_reason":"end_turn"},"total_usd":0.049039,"stage1_batch_id":"msgbatch_01S6GjoYQm8LTPUsdhASdqag","stage2_batch_id":"msgbatch_01VX63RTMdBJqsG6gQMqoCjz","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"TgSUB2 is a subtilisin-like serine proteinase that localizes to rhoptry organelles in Toxoplasma gondii, as determined by direct localization experiments.\",\n      \"method\": \"Subcellular localization (direct experiment)\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment with functional context, single lab, single paper\",\n      \"pmids\": [\"12890015\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"TgSUB2 undergoes autocatalytic (self) processing as it traffics through the secretory pathway; site-directed mutagenesis of a sequence within TgSUB2 homologous to the ROP1 cleavage site (after Glu) abolished this autoprocessing, establishing TgSUB2 as a serine proteinase with cleavage specificity after Glu.\",\n      \"method\": \"Site-directed mutagenesis of the predicted active-site/cleavage motif; in vivo processing assay\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — active-site mutagenesis abolishing autoprocessing, single lab but direct mechanistic experiment with mutagenesis validation\",\n      \"pmids\": [\"12890015\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"TgSUB2 associates with the rhoptry protein ROP1, which is a potential substrate, suggesting TgSUB2 functions as a rhoptry protein maturase responsible for processing rhoptry organelle contents.\",\n      \"method\": \"Co-association/pulldown with ROP1; sequence homology to ROP1 cleavage site validated by mutagenesis\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single Co-IP/association experiment combined with mutagenesis supporting substrate identification, single lab\",\n      \"pmids\": [\"12890015\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Disruption of TgSUB2 was unsuccessful, implying that TgSUB2 is an essential gene in Toxoplasma gondii.\",\n      \"method\": \"Gene disruption attempt (negative result — failed knockout)\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — essentiality inferred from failed disruption, single lab, single method\",\n      \"pmids\": [\"12890015\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgSUB2 is an essential, rhoptry-localized subtilisin-like serine proteinase in Toxoplasma gondii that undergoes autocatalytic processing during trafficking through the secretory pathway and functions as a rhoptry protein maturase — cleaving after Glu residues, as validated by mutagenesis — with ROP1 as a candidate substrate.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TgSUB2 is a subtilisin-like serine proteinase of Toxoplasma gondii that localizes to the rhoptry secretory organelles and functions as a candidate rhoptry protein maturase [#0, #2]. As it traffics through the secretory pathway, TgSUB2 undergoes autocatalytic processing; site-directed mutagenesis of a motif homologous to the ROP1 cleavage site abolished this autoprocessing, establishing that the enzyme cleaves after Glu residues [#1]. Consistent with a maturase role, TgSUB2 associates with the rhoptry protein ROP1, a candidate substrate [#2]. Beyond this single body of work, no further mechanistic detail — including a validated in vitro substrate repertoire or structural model — has been characterized in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Establishing where an uncharacterized subtilisin-like protease acts localized TgSUB2 to the rhoptry organelles, placing it within the invasion-associated secretory machinery.\",\n      \"evidence\": \"Direct subcellular localization experiment in T. gondii\",\n      \"pmids\": [\"12890015\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not define the enzyme's substrates within the rhoptry\", \"Trafficking route to the rhoptry not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Whether TgSUB2 is a functional protease and what specificity it holds was answered by showing it autoprocesses during secretory trafficking and that mutating the ROP1-homologous cleavage motif abolishes this, defining cleavage after Glu.\",\n      \"evidence\": \"Site-directed mutagenesis of the predicted cleavage motif plus in vivo processing assay\",\n      \"pmids\": [\"12890015\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cleavage specificity inferred from autoprocessing, not from a purified enzyme against defined peptide substrates\", \"No structural basis for Glu specificity established\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"The biological role of TgSUB2 as a rhoptry protein maturase was supported by its association with ROP1 as a candidate substrate.\",\n      \"evidence\": \"Co-association/pulldown with ROP1 combined with mutagenesis-validated cleavage-site homology\",\n      \"pmids\": [\"12890015\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP without reciprocal validation\", \"Direct in vitro cleavage of ROP1 by TgSUB2 not demonstrated\", \"Broader rhoptry substrate set unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"The requirement of TgSUB2 for parasite viability was inferred from an unsuccessful gene disruption, implying essentiality.\",\n      \"evidence\": \"Failed gene disruption attempt (negative result)\",\n      \"pmids\": [\"12890015\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Essentiality inferred from failed knockout, not from a conditional/inducible knockout\", \"Phenotype of depletion not characterized\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The full rhoptry substrate repertoire, the mechanism and timing of TgSUB2 activation, and a conditional-knockout demonstration of its essential function remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No validated substrate beyond candidate ROP1\", \"No structural model\", \"Essentiality not confirmed by conditional depletion\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004252\", \"supporting_discovery_ids\": [0, 1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0033644\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006508\", \"supporting_discovery_ids\": [1, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ROP1 (candidate substrate)\"],\n    \"other_free_text\": [\n      \"Undergoes autocatalytic processing during secretory-pathway trafficking [#1]\",\n      \"Cleavage specificity after Glu residues, validated by mutagenesis of a ROP1-homologous cleavage motif [#1]\",\n      \"Candidate essential gene inferred from unsuccessful disruption [#3]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}\n```","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_314500","name":"SUB2","product":"subtilisin SUB2","uniprot_acc":"A0A125YTZ4","chromosome":"TGME49_chrXI","start_pos":4146820},{"strain":"GT1","gene_id":"TGGT1_314500","name":null,"product":"subtilisin SUB2","uniprot_acc":null,"chromosome":"TGGT1_chrXI","start_pos":4111964},{"strain":"RH88","gene_id":"TGRH88_053560","name":null,"product":"subtilisin SUB2","uniprot_acc":null,"chromosome":"CM023092","start_pos":4198276},{"strain":"VEG","gene_id":"TGVEG_314500","name":null,"product":"subtilisin SUB2","uniprot_acc":"B6K9C6","chromosome":"TGVEG_chrXI","start_pos":4165356}]}