{"gene":"PF3D7_0507200","run_date":"2026-09-07T10:55:52","timeline":{"discoveries":[{"year":2012,"finding":"The mature region of SUB3 (PfSUB3m, Thr516–Glu769) displays PMSF-sensitive serine protease activity in vitro; substitution of the active-site serine with alanine abolishes this activity, confirming the catalytic serine is essential. Both PfSUB3c and PfSUB3m undergo truncation to a ~25 kDa species that retains proteolytic activity. SUB3 is expressed at late asexual blood stages (confirmed by qRT-PCR, immunofluorescence assay, and Western blot).","method":"Recombinant expression in E. coli, in vitro proteolytic activity assay, PMSF inhibition, active-site serine-to-alanine mutagenesis, qRT-PCR, immunofluorescence assay, Western blot","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with active-site mutagenesis plus multiple orthogonal expression/localization methods in a single study","pmids":["22285468"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"finding":"Mature recombinant SUB3 cleaves recombinant P. falciparum profilin (PfPRF) in a dose-dependent manner in vitro, identifying PfPRF as a substrate of SUB3.","method":"In vitro proteolysis assay with recombinant mature SUB3 and recombinant PfPRF","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution with recombinant proteins, single lab, single method","pmids":["24080030"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2004,"finding":"SUB3 is one of three Plasmodium subtilisin-like serine proteases expressed in asexual blood stages; in at least two of the three family members the gene products localize to secretory organelles of the invasive merozoite (the review does not specify which two, so localization to secretory organelles is attributed to the family, not individually confirmed for SUB3 in this source).","method":"Review of experimental literature (no new primary experiment for SUB3 specifically reported)","journal":"Molecular microbiology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — review article summarising prior experimental data; no new primary experiment for SUB3 individually reported","pmids":["15225303"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"SUB3 (PF3D7_0507200) is a serine protease expressed at late asexual blood stages of P. falciparum whose catalytic activity depends on an essential active-site serine (Ser→Ala substitution abolishes activity); it cleaves P. falciparum profilin (PfPRF) in vitro, suggesting a possible role in processes requiring actin dynamics such as merozoite egress or invasion, but its precise in vivo substrates and mechanistic placement within egress/invasion pathways remain unresolved."},"narrative":{"mechanistic_narrative":"SUB3 (PF3D7_0507200) is a subtilisin-like serine protease expressed at late asexual blood stages of Plasmodium falciparum [PMID:22285468]. Its mature region (PfSUB3m, Thr516–Glu769) exhibits PMSF-sensitive proteolytic activity in vitro, and substitution of its active-site serine with alanine abolishes catalysis, establishing a canonical serine-protease mechanism dependent on this residue; both the full-construct and mature forms undergo truncation to an active ~25 kDa species [PMID:22285468]. In vitro, mature recombinant SUB3 cleaves recombinant P. falciparum profilin (PfPRF) in a dose-dependent manner, identifying PfPRF as a substrate and pointing toward a possible role in actin-dynamics-dependent processes [PMID:24080030]. Beyond its catalytic characterization and the in vitro PfPRF cleavage, the in vivo substrates, subcellular localization, and precise placement of SUB3 within merozoite egress or invasion pathways have not been characterized in the available corpus.","teleology":[{"year":2004,"claim":"Before any individual biochemical study, the question was whether SUB3 belonged to a functionally relevant protease family in blood-stage parasites; cataloguing it among three asexual-stage subtilisin-like proteases framed it as a candidate protease of the invasive parasite.","evidence":"Review of experimental literature on Plasmodium subtilisin-like proteases","pmids":["15225303"],"confidence":"Low","gaps":["Review attributes secretory-organelle localization to only two of three family members without specifying SUB3, so SUB3 localization is not individually established","No primary experiment on SUB3 reported in this source","No substrate or catalytic data for SUB3"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2012,"claim":"It was unknown whether SUB3 is a catalytically active enzyme and when it is expressed; recombinant expression with active-site mutagenesis showed the mature protein is a genuine serine protease whose activity depends on the catalytic serine, and it is expressed at late asexual blood stages.","evidence":"Recombinant expression in E. coli, in vitro proteolytic assay, PMSF inhibition, active-site serine-to-alanine mutagenesis, qRT-PCR, IFA, and Western blot","pmids":["22285468"],"confidence":"High","gaps":["No physiological substrate identified in this study","Subcellular localization of SUB3 not resolved","Functional role in the parasite life cycle not tested"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"claim":"With SUB3 established as an active protease, the open question was what it cleaves; an in vitro reconstitution identified P. falciparum profilin as a dose-dependent substrate, linking SUB3 to actin-regulatory machinery.","evidence":"In vitro proteolysis assay with recombinant mature SUB3 and recombinant PfPRF","pmids":["24080030"],"confidence":"Medium","gaps":["Single-lab in vitro reconstitution with recombinant proteins; PfPRF cleavage not confirmed in vivo","Functional consequence of PfPRF cleavage for actin dynamics, egress, or invasion untested","No cleavage-site mapping or physiological timing established"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"The in vivo substrate repertoire, subcellular localization, and mechanistic role of SUB3 in merozoite egress or invasion remain unresolved.","evidence":"No timeline discovery addresses in vivo function, genetic requirement, or native localization of SUB3","pmids":[],"confidence":"Low","gaps":["No gene knockout/knockdown phenotype for SUB3","PfPRF cleavage not validated in the parasite","SUB3 native localization not individually confirmed"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["P. FALCIPARUM PROFILIN (PFPRF) — IN VITRO SUBSTRATE [#1]"],"other_free_text":["Catalytic activity depends on an essential active-site serine; Ser→Ala substitution abolishes activity [#0]","Mature region PfSUB3m (Thr516–Glu769) is active in vitro and both PfSUB3c and PfSUB3m truncate to an active ~25 kDa species [#0]","Expressed at late asexual blood stages [#0]","Member of a three-gene Plasmodium subtilisin-like protease family in asexual blood stages; secretory-organelle localization was attributed to the family, not individually confirmed for SUB3 [#2]"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"15225303","id":"PMC_15225303","title":"Subtilisin-like proteases of the malaria parasite.","date":"2004","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/15225303","citation_count":64,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"24080030","id":"PMC_24080030","title":"Proteolytic activity of Plasmodium falciparum subtilisin-like protease 3 on parasite profilin, a multifunctional protein.","date":"2013","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/24080030","citation_count":19,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfSUB3"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"22285468","id":"PMC_22285468","title":"Expression and characterization of catalytic domain of Plasmodium falciparum subtilisin-like protease 3.","date":"2012","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/22285468","citation_count":13,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfSUB3"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"41011189","id":"PMC_41011189","title":"Exploring Subtilisin Inhibition to Discover Antimalarial Drugs: Insights into Medicinal Chemistry and Drug Discovery.","date":"2025","source":"Pharmaceuticals (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/41011189","citation_count":0,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["SUB3"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":4622,"output_tokens":976,"usd":0.014253,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":5661,"output_tokens":1967,"usd":0.03874,"stage2_stop_reason":"end_turn"},"total_usd":0.052993,"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\": 2012,\n      \"finding\": \"The mature region of SUB3 (PfSUB3m, Thr516–Glu769) displays PMSF-sensitive serine protease activity in vitro; substitution of the active-site serine with alanine abolishes this activity, confirming the catalytic serine is essential. Both PfSUB3c and PfSUB3m undergo truncation to a ~25 kDa species that retains proteolytic activity. SUB3 is expressed at late asexual blood stages (confirmed by qRT-PCR, immunofluorescence assay, and Western blot).\",\n      \"method\": \"Recombinant expression in E. coli, in vitro proteolytic activity assay, PMSF inhibition, active-site serine-to-alanine mutagenesis, qRT-PCR, immunofluorescence assay, Western blot\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with active-site mutagenesis plus multiple orthogonal expression/localization methods in a single study\",\n      \"pmids\": [\"22285468\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Mature recombinant SUB3 cleaves recombinant P. falciparum profilin (PfPRF) in a dose-dependent manner in vitro, identifying PfPRF as a substrate of SUB3.\",\n      \"method\": \"In vitro proteolysis assay with recombinant mature SUB3 and recombinant PfPRF\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution with recombinant proteins, single lab, single method\",\n      \"pmids\": [\"24080030\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"SUB3 is one of three Plasmodium subtilisin-like serine proteases expressed in asexual blood stages; in at least two of the three family members the gene products localize to secretory organelles of the invasive merozoite (the review does not specify which two, so localization to secretory organelles is attributed to the family, not individually confirmed for SUB3 in this source).\",\n      \"method\": \"Review of experimental literature (no new primary experiment for SUB3 specifically reported)\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — review article summarising prior experimental data; no new primary experiment for SUB3 individually reported\",\n      \"pmids\": [\"15225303\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    }\n  ],\n  \"current_model\": \"SUB3 (PF3D7_0507200) is a serine protease expressed at late asexual blood stages of P. falciparum whose catalytic activity depends on an essential active-site serine (Ser→Ala substitution abolishes activity); it cleaves P. falciparum profilin (PfPRF) in vitro, suggesting a possible role in processes requiring actin dynamics such as merozoite egress or invasion, but its precise in vivo substrates and mechanistic placement within egress/invasion pathways remain unresolved.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SUB3 (PF3D7_0507200) is a subtilisin-like serine protease expressed at late asexual blood stages of Plasmodium falciparum [#0]. Its mature region (PfSUB3m, Thr516–Glu769) exhibits PMSF-sensitive proteolytic activity in vitro, and substitution of its active-site serine with alanine abolishes catalysis, establishing a canonical serine-protease mechanism dependent on this residue; both the full-construct and mature forms undergo truncation to an active ~25 kDa species [#0]. In vitro, mature recombinant SUB3 cleaves recombinant P. falciparum profilin (PfPRF) in a dose-dependent manner, identifying PfPRF as a substrate and pointing toward a possible role in actin-dynamics-dependent processes [#1]. Beyond its catalytic characterization and the in vitro PfPRF cleavage, the in vivo substrates, subcellular localization, and precise placement of SUB3 within merozoite egress or invasion pathways have not been characterized in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Before any individual biochemical study, the question was whether SUB3 belonged to a functionally relevant protease family in blood-stage parasites; cataloguing it among three asexual-stage subtilisin-like proteases framed it as a candidate protease of the invasive parasite.\",\n      \"evidence\": \"Review of experimental literature on Plasmodium subtilisin-like proteases\",\n      \"pmids\": [\"15225303\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Review attributes secretory-organelle localization to only two of three family members without specifying SUB3, so SUB3 localization is not individually established\",\n        \"No primary experiment on SUB3 reported in this source\",\n        \"No substrate or catalytic data for SUB3\"\n      ],\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"It was unknown whether SUB3 is a catalytically active enzyme and when it is expressed; recombinant expression with active-site mutagenesis showed the mature protein is a genuine serine protease whose activity depends on the catalytic serine, and it is expressed at late asexual blood stages.\",\n      \"evidence\": \"Recombinant expression in E. coli, in vitro proteolytic assay, PMSF inhibition, active-site serine-to-alanine mutagenesis, qRT-PCR, IFA, and Western blot\",\n      \"pmids\": [\"22285468\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No physiological substrate identified in this study\",\n        \"Subcellular localization of SUB3 not resolved\",\n        \"Functional role in the parasite life cycle not tested\"\n      ],\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"With SUB3 established as an active protease, the open question was what it cleaves; an in vitro reconstitution identified P. falciparum profilin as a dose-dependent substrate, linking SUB3 to actin-regulatory machinery.\",\n      \"evidence\": \"In vitro proteolysis assay with recombinant mature SUB3 and recombinant PfPRF\",\n      \"pmids\": [\"24080030\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab in vitro reconstitution with recombinant proteins; PfPRF cleavage not confirmed in vivo\",\n        \"Functional consequence of PfPRF cleavage for actin dynamics, egress, or invasion untested\",\n        \"No cleavage-site mapping or physiological timing established\"\n      ],\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The in vivo substrate repertoire, subcellular localization, and mechanistic role of SUB3 in merozoite egress or invasion remain unresolved.\",\n      \"evidence\": \"No timeline discovery addresses in vivo function, genetic requirement, or native localization of SUB3\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No gene knockout/knockdown phenotype for SUB3\",\n        \"PfPRF cleavage not validated in the parasite\",\n        \"SUB3 native localization not individually confirmed\"\n      ],\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004252\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0008236\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [],\n    \"pathway\": [],\n    \"complexes\": [],\n    \"partners\": [\"P. falciparum profilin (PfPRF) — in vitro substrate [#1]\"],\n    \"other_free_text\": [\n      \"Catalytic activity depends on an essential active-site serine; Ser→Ala substitution abolishes activity [#0]\",\n      \"Mature region PfSUB3m (Thr516–Glu769) is active in vitro and both PfSUB3c and PfSUB3m truncate to an active ~25 kDa species [#0]\",\n      \"Expressed at late asexual blood stages [#0]\",\n      \"Member of a three-gene Plasmodium subtilisin-like protease family in asexual blood stages; secretory-organelle localization was attributed to the family, not individually confirmed for SUB3 [#2]\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0507200","name":null,"product":"subtilisin-like protease 3","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1106800","name":null,"product":"subtilisin-like protease 3, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1026400","name":null,"product":"subtilisin-like protease 3, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"SUB3","evaluation":null}