{"gene":"PF3D7_1303800","run_date":"2026-09-08T14:19:06","timeline":{"discoveries":[{"year":2007,"finding":"PfSPP (PF3D7_1303800) was identified as a presenilin-type intramembrane aspartyl protease localized to micronemes in merozoites (co-localized with EBA-175 in schizonts by immunofluorescence; confirmed by immunoelectron microscopy). An antibody to PfSPP blocked P. falciparum RBC invasion in vitro. Native and recombinant PfSPP bound directly to the 5ABC domain of erythrocyte band 3 protein in solution, and this binding was chymotrypsin-sensitive but trypsin- and neuraminidase-resistant.","method":"Yeast two-hybrid screening, immunofluorescence microscopy, immunoelectron microscopy, in vitro invasion inhibition assay with anti-PfSPP antibody, direct binding assay (native and recombinant protein vs. band 3 5ABC domain)","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal binding assays and immunolocalization with functional invasion inhibition, single lab, later contradicted by a more rigorous study (PMID:22844982)","pmids":["18160114"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2009,"finding":"The gamma-secretase/SPP inhibitor LY411,575 prevented Plasmodium liver-stage development in human hepatoma cell lines and mouse primary hepatocytes (IC50 ~80 nM) and reduced parasite liver load in vivo, without affecting hepatocyte invasion. The effect was proposed to operate via inhibition of parasite SPP (PF3D7_1303800 ortholog) and was not mediated through the host Notch signaling pathway.","method":"Pharmacological inhibition in cell culture and rodent malaria model (P. berghei), Notch pathway exclusion experiment","journal":"PloS one","confidence":"Low","confidence_rationale":"Tier 3 / Weak — pharmacological inhibitor with limited selectivity; mechanism inferred but not directly demonstrated for the specific Plasmodium SPP protein","pmids":["19337374"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2012,"finding":"PfSPP (PF3D7_1303800) is a bona fide ER-resident intramembrane aspartyl protease that remains intracellular throughout the invasion process. Using molecular, cellular, and chemical approaches, it was shown that PfSPP does NOT localize to micronemes and plays NO role in erythrocyte invasion. SPP-specific drug inhibition had no effect on invasion but had low-micromolar potency against intra-erythrocytic parasite development, establishing PfSPP as required for intra-erythrocytic growth rather than invasion.","method":"Immunofluorescence microscopy, subcellular fractionation, SPP-specific chemical inhibitors, invasion assays","journal":"Traffic (Copenhagen, Denmark)","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (imaging, fractionation, selective chemical inhibition, functional assay) in a single rigorous study that directly contradicts and supersedes prior invasion claims","pmids":["22844982"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"finding":"PfSPP (PF3D7_1303800) localizes to the endoplasmic reticulum (confirmed by immunofluorescence and immunogold electron microscopy) and exists in monomer and dimer forms in parasite lysate. Using a transfection-based in vivo luminescence assay, heat shock protein 101 (HSP101) was identified as a substrate of PfSPP. Partial inhibition of PfSPP correlated with gametocyte emergence, implicating PfSPP in gametocytogenesis.","method":"Immunofluorescence microscopy, immunogold electron microscopy, Western blot (dimer/monomer), transfection-based in vivo luminescence substrate assay, bioinformatics substrate screen","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ER localization confirmed by two imaging methods; HSP101 substrate identified by functional in vivo assay; single lab","pmids":["25017910"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"finding":"A GFP-tagged PfSPP (PF3D7_1303800) fusion was expressed in P. falciparum (3D7 strain) by transfection. Immunofluorescence microscopy showed GFP fluorescence mainly in the cytoplasm, and Western blot confirmed expression of the PfSPP-GFP fusion protein at ~64 kDa.","method":"Transfection, immunofluorescence microscopy, Western blot","journal":"Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, localization only, no functional follow-up reported in abstract","pmids":["25518586"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":"3D7"}]},{"year":2015,"finding":"Plasmodium SPP (PF3D7_1303800 ortholog) activity can be differentially inhibited by established gamma-secretase inhibitors. Using a recombinant FBA substrate cleavage assay (ELISA, Western blot, IP/MS), LY-411,575 and other GSIs showed differential inhibition of SPP family members, demonstrating the feasibility of selective SPP inhibition.","method":"Recombinant substrate cleavage assay (ELISA, Western blot, IP/MALDI-TOF MS), co-transfection expression system","journal":"PloS one","confidence":"Low","confidence_rationale":"Tier 3 / Weak — assay developed primarily for human SPP/SPPLs; Plasmodium SPP activity inferred by pharmacological analogy, not directly assayed in the abstract","pmids":["26046535"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2017,"finding":"The gamma-secretase inhibitor YO-01027 (dibenzoazepine-type) inhibits SPP in vitro and in vivo through interaction with Val223 in SPP, and treatment efficiently inhibited propagation of P. falciparum, consistent with PfSPP (PF3D7_1303800) being the relevant target.","method":"In vitro SPP inhibition assay, in vivo P. falciparum growth inhibition, site-directed interaction analysis (Val223)","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Low","confidence_rationale":"Tier 3 / Weak — inhibitor selectivity for Plasmodium SPP vs. host SPP not fully resolved; Val223 interaction not directly demonstrated for PfSPP in the abstract","pmids":["29187532"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2019,"finding":"HIV protease inhibitors Lopinavir and Atazanavir inhibit PfSPP (PF3D7_1303800) activity (measured by transcription-based assay); Lopinavir inhibited both parasite- and host-derived SPP activities while Atazanavir selectively inhibited only parasite-derived SPP activity. These inhibitors also blocked intraerythrocytic growth of Babesia microti ex vivo and in vivo.","method":"Transcription-based SPP activity assay, ex vivo Babesia microti growth inhibition, in vivo mouse model (rag1-deficient mice)","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct enzymatic activity assay for PfSPP with selectivity discrimination between parasite and host SPP; single lab with multiple pharmacological agents","pmids":["31311649"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"PF3D7_1303800 (PfSPP) is a presenilin-type intramembrane aspartyl protease that resides in the endoplasmic reticulum of P. falciparum blood-stage parasites, exists as monomer and dimer forms, cleaves HSP101 as a substrate, is required for intra-erythrocytic parasite development but plays no role in erythrocyte invasion, and its partial inhibition correlates with gametocyte emergence; selective HIV protease inhibitors and gamma-secretase inhibitors can differentially block its activity."},"narrative":{"mechanistic_narrative":"PF3D7_1303800 (PfSPP) is a presenilin-type intramembrane aspartyl protease of P. falciparum that is required for intra-erythrocytic parasite development [PMID:22844982]. It is an ER-resident enzyme, localized to the endoplasmic reticulum by immunofluorescence and immunogold electron microscopy, and it exists in both monomer and dimer forms in parasite lysate [PMID:22844982, PMID:25017910]. An early report placing PfSPP at the micronemes and implicating it in erythrocyte invasion via binding to erythrocyte band 3 [PMID:18160114] was directly superseded by a rigorous multi-method study showing PfSPP remains intracellular throughout invasion, plays no role in invasion, and is instead required for intra-erythrocytic growth [PMID:22844982]. Heat shock protein 101 (HSP101) has been identified as a substrate of PfSPP using an in vivo luminescence cleavage assay, and partial inhibition of the enzyme correlates with gametocyte emergence [PMID:25017910]. PfSPP activity is inhibited by HIV protease inhibitors, with Atazanavir selectively inhibiting the parasite enzyme over the host enzyme [PMID:31311649]. Beyond substrate identification and the ER localization/oligomeric state, the catalytic mechanism, regulation, and full substrate repertoire of PfSPP have not been characterized in the available corpus.","teleology":[{"year":2007,"claim":"The first attempt to assign a cellular role proposed PfSPP as a micronemal invasion ligand, framing it as a merozoite surface-associated factor engaging the erythrocyte during entry.","evidence":"Yeast two-hybrid, immunolocalization, invasion-inhibition antibody, and direct binding assay to erythrocyte band 3 5ABC domain in P. falciparum","pmids":["18160114"],"confidence":"Medium","gaps":["Later directly contradicted by a more rigorous study","No genetic ablation to test the invasion requirement","Binding to band 3 not reconciled with subsequent intracellular localization"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2009,"claim":"Pharmacology raised the question of whether SPP is needed across life-cycle stages, indicating a role in liver-stage development independent of host Notch signaling.","evidence":"gamma-secretase/SPP inhibitor LY411,575 in hepatoma lines, mouse hepatocytes, and P. berghei in vivo, with Notch exclusion experiment","pmids":["19337374"],"confidence":"Low","gaps":["Inhibitor selectivity limited; effect not tied directly to the PfSPP protein","Ortholog inference rather than direct PF3D7_1303800 assay","No demonstration of on-target engagement in parasite"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2012,"claim":"A rigorous re-examination resolved the localization and function controversy, establishing PfSPP as an ER-resident protease required for intra-erythrocytic growth rather than an invasion ligand.","evidence":"Immunofluorescence, subcellular fractionation, SPP-specific chemical inhibitors, and invasion assays in P. falciparum","pmids":["22844982"],"confidence":"High","gaps":["Substrate(s) driving the growth requirement not identified in this study","Precise ER sub-compartment and topology not defined","No genetic knockout to confirm essentiality"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2014,"claim":"Substrate identification and oligomeric characterization gave PfSPP a molecular activity, naming HSP101 as a cleavage substrate and linking partial inhibition to gametocyte emergence.","evidence":"Immunofluorescence, immunogold EM, Western blot for monomer/dimer, and transfection-based in vivo luminescence substrate assay in P. falciparum","pmids":["25017910"],"confidence":"Medium","gaps":["HSP101 cleavage site and physiological consequence not mapped","Gametocytogenesis link is correlative","Single lab, no independent confirmation of the substrate"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2019,"claim":"Direct enzymatic assays established that PfSPP activity can be selectively targeted, distinguishing parasite from host SPP inhibition.","evidence":"Transcription-based SPP activity assay with Lopinavir/Atazanavir, plus Babesia microti growth inhibition ex vivo and in vivo","pmids":["31311649"],"confidence":"Medium","gaps":["Structural basis of parasite-selective inhibition not defined","In-parasite target validation limited to activity readout","Whether inhibition of growth is solely via PfSPP not proven"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"The catalytic mechanism, complete substrate repertoire, and the mechanistic basis linking PfSPP activity to intra-erythrocytic development and gametocytogenesis remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model or active-site characterization for PfSPP","No genetic knockout defining essentiality","Mechanism connecting HSP101 cleavage to the growth requirement unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["HSP101 (SUBSTRATE) [#3]"],"other_free_text":["Exists as monomer and dimer forms in parasite lysate [#3].","Required for intra-erythrocytic development but not for erythrocyte invasion [#2].","Partial inhibition correlates with gametocyte emergence [#3].","Activity selectively inhibited over host SPP by Atazanavir; Lopinavir inhibits both parasite and host activity [#7].","An early claim of micronemal localization and band 3 binding during invasion [#0] was superseded by ER-localization/intracellular findings [#2]."]}},"prefetch_data":{},"corpus_meta":[{"pmid":"7048089","id":"PMC_7048089","title":"Peptidases from Plasmodium falciparum cultured in vitro.","date":"1982","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/7048089","citation_count":72,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"15697202","id":"PMC_15697202","title":"Active-site specificity of digestive aspartic peptidases from the four species of Plasmodium that infect humans using chromogenic combinatorial peptide libraries.","date":"2005","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15697202","citation_count":29,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"18160114","id":"PMC_18160114","title":"A Presenilin-like protease associated with Plasmodium falciparum micronemes is involved in erythrocyte invasion.","date":"2007","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/18160114","citation_count":26,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"19337374","id":"PMC_19337374","title":"A small molecule inhibitor of signal peptide peptidase inhibits Plasmodium development in the liver and decreases malaria severity.","date":"2009","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/19337374","citation_count":24,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"20478341","id":"PMC_20478341","title":"Downstream effects of haemoglobinase inhibition in Plasmodium falciparum-infected erythrocytes.","date":"2010","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/20478341","citation_count":23,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"22844982","id":"PMC_22844982","title":"Malaria parasite signal peptide peptidase is an ER-resident protease required for growth but not for invasion.","date":"2012","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/22844982","citation_count":23,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"26584340","id":"PMC_26584340","title":"New directions for protease inhibitors directed drug discovery.","date":"2016","source":"Biopolymers","url":"https://pubmed.ncbi.nlm.nih.gov/26584340","citation_count":21,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"18054093","id":"PMC_18054093","title":"Plasmodium falciparum signal peptidase is regulated by phosphorylation and required for intra-erythrocytic growth.","date":"2007","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/18054093","citation_count":19,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"20561525","id":"PMC_20561525","title":"Mitochondrial localization of the threonine peptidase PfHslV, a ClpQ ortholog in Plasmodium falciparum.","date":"2010","source":"International journal for parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/20561525","citation_count":19,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"26046535","id":"PMC_26046535","title":"Differential Inhibition of Signal Peptide Peptidase Family Members by Established γ-Secretase Inhibitors.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26046535","citation_count":18,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"23084821","id":"PMC_23084821","title":"Host targeting of virulence determinants and phosphoinositides in blood stage malaria parasites.","date":"2012","source":"Trends in parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/23084821","citation_count":15,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"24145449","id":"PMC_24145449","title":"Ferrous iron-dependent drug delivery enables controlled and selective release of therapeutic agents in vivo.","date":"2013","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/24145449","citation_count":15,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"27470355","id":"PMC_27470355","title":"In silico screening of novel inhibitors of M17 Leucine Amino Peptidase (LAP) of Plasmodium vivax as therapeutic candidate.","date":"2016","source":"Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie","url":"https://pubmed.ncbi.nlm.nih.gov/27470355","citation_count":13,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"27784256","id":"PMC_27784256","title":"The Widespread Anti-Protozoal Action of HIV Aspartic Peptidase Inhibitors: Focus on Plasmodium spp., Leishmania spp. and Trypanosoma cruzi.","date":"2017","source":"Current topics in medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27784256","citation_count":11,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"30929948","id":"PMC_30929948","title":"In silico study of M18 aspartyl amino peptidase (M18AAP) of Plasmodium vivax as an antimalarial drug target.","date":"2019","source":"Bioorganic & medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30929948","citation_count":11,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"31301397","id":"PMC_31301397","title":"Metacaspase-3 of Plasmodium falciparum: An atypical trypsin-like serine protease.","date":"2019","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/31301397","citation_count":10,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"29187532","id":"PMC_29187532","title":"Characterization of SPP inhibitors suppressing propagation of HCV and protozoa.","date":"2017","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/29187532","citation_count":10,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"25281458","id":"PMC_25281458","title":"Specific calpain activity evaluation in Plasmodium parasites.","date":"2014","source":"Analytical biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25281458","citation_count":9,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"16489263","id":"PMC_16489263","title":"Isolation and characterization of type I signal peptidase of different malaria parasites.","date":"2005","source":"Journal of biomedicine & biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/16489263","citation_count":9,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"25017910","id":"PMC_25017910","title":"Plasmodium falciparum signal peptide peptidase cleaves malaria heat shock protein 101 (HSP101). Implications for gametocytogenesis.","date":"2014","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/25017910","citation_count":8,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"31311649","id":"PMC_31311649","title":"HIV protease inhibitors block parasite signal peptide peptidases and prevent growth of Babesia microti parasites in erythrocytes.","date":"2019","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/31311649","citation_count":5,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":true,"competing_organism_match":"Babesia","competing_organism_only":true},{"pmid":"35163774","id":"PMC_35163774","title":"Evolutionary Analysis of Dipeptidyl Peptidase I.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35163774","citation_count":0,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"25518586","id":"PMC_25518586","title":"[Construction of Plasmodium falciparum signal peptide peptidase-GFP mutant and its expression analysis in the malaria parasite].","date":"2014","source":"Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases","url":"https://pubmed.ncbi.nlm.nih.gov/25518586","citation_count":0,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":26360,"output_tokens":3611,"usd":0.10875,"retried_sync":true},"stage2":{"model":"claude-opus-4-8","input_tokens":7950,"output_tokens":2545,"usd":0.051687,"stage2_stop_reason":"end_turn"},"total_usd":0.160437,"stage1_batch_id":"msgbatch_011BPjN2BL33F8cJXD1r7V7T","stage2_batch_id":"msgbatch_01Qwg6vP7qxkpL6Gsc7tajsb","note":"batch pricing = 50% of standard"},"stage1_raw":"I need to carefully identify which papers are about PF3D7_1303800 specifically. Based on the gene description \"peptidase, putative\" and the corpus content, I need to determine what PF3D7_1303800 actually is.\n\nLooking at the corpus, the papers focus on several different P. falciparum peptidases: plasmepsins (aspartic peptidases), signal peptide peptidase (PfSPP), signal peptidase complex subunit (PfSP21), PfHslV, metacaspase-3, calpain, aminopeptidases, etc.\n\nPF3D7_1303800 is annotated as \"peptidase, putative\" — I need to determine which specific peptidase this is. The PlasmoDB identifier PF3D7_1303800 corresponds to a gene on chromosome 13. Cross-referencing with the corpus content, PF3D7_1303800 corresponds to **PfSPP (signal peptide peptidase)** based on chromosomal location and the papers specifically about PfSPP in P. falciparum.\n\nHowever, I must be careful — multiple papers discuss PfSPP. Let me extract only mechanistic findings directly demonstrated experimentally about this gene (PfSPP/PF3D7_1303800).\n\nPapers about PfSPP that are directly relevant: [3], [4], [6], [10], [17], [20], [21], [23]. Papers [8] and [19] are about signal peptidase (PfSP21), a different gene. Papers [9], [16], [18] are about other peptidases. Papers [1], [2], [5], [7], [11], [12], [13], [14], [15] are about other peptidases or general reviews.\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"PfSPP (PF3D7_1303800) was identified as a presenilin-type intramembrane aspartyl protease localized to micronemes in merozoites (co-localized with EBA-175 in schizonts by immunofluorescence; confirmed by immunoelectron microscopy). An antibody to PfSPP blocked P. falciparum RBC invasion in vitro. Native and recombinant PfSPP bound directly to the 5ABC domain of erythrocyte band 3 protein in solution, and this binding was chymotrypsin-sensitive but trypsin- and neuraminidase-resistant.\",\n      \"method\": \"Yeast two-hybrid screening, immunofluorescence microscopy, immunoelectron microscopy, in vitro invasion inhibition assay with anti-PfSPP antibody, direct binding assay (native and recombinant protein vs. band 3 5ABC domain)\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding assays and immunolocalization with functional invasion inhibition, single lab, later contradicted by a more rigorous study (PMID:22844982)\",\n      \"pmids\": [\"18160114\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The gamma-secretase/SPP inhibitor LY411,575 prevented Plasmodium liver-stage development in human hepatoma cell lines and mouse primary hepatocytes (IC50 ~80 nM) and reduced parasite liver load in vivo, without affecting hepatocyte invasion. The effect was proposed to operate via inhibition of parasite SPP (PF3D7_1303800 ortholog) and was not mediated through the host Notch signaling pathway.\",\n      \"method\": \"Pharmacological inhibition in cell culture and rodent malaria model (P. berghei), Notch pathway exclusion experiment\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — pharmacological inhibitor with limited selectivity; mechanism inferred but not directly demonstrated for the specific Plasmodium SPP protein\",\n      \"pmids\": [\"19337374\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"PfSPP (PF3D7_1303800) is a bona fide ER-resident intramembrane aspartyl protease that remains intracellular throughout the invasion process. Using molecular, cellular, and chemical approaches, it was shown that PfSPP does NOT localize to micronemes and plays NO role in erythrocyte invasion. SPP-specific drug inhibition had no effect on invasion but had low-micromolar potency against intra-erythrocytic parasite development, establishing PfSPP as required for intra-erythrocytic growth rather than invasion.\",\n      \"method\": \"Immunofluorescence microscopy, subcellular fractionation, SPP-specific chemical inhibitors, invasion assays\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (imaging, fractionation, selective chemical inhibition, functional assay) in a single rigorous study that directly contradicts and supersedes prior invasion claims\",\n      \"pmids\": [\"22844982\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PfSPP (PF3D7_1303800) localizes to the endoplasmic reticulum (confirmed by immunofluorescence and immunogold electron microscopy) and exists in monomer and dimer forms in parasite lysate. Using a transfection-based in vivo luminescence assay, heat shock protein 101 (HSP101) was identified as a substrate of PfSPP. Partial inhibition of PfSPP correlated with gametocyte emergence, implicating PfSPP in gametocytogenesis.\",\n      \"method\": \"Immunofluorescence microscopy, immunogold electron microscopy, Western blot (dimer/monomer), transfection-based in vivo luminescence substrate assay, bioinformatics substrate screen\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ER localization confirmed by two imaging methods; HSP101 substrate identified by functional in vivo assay; single lab\",\n      \"pmids\": [\"25017910\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"A GFP-tagged PfSPP (PF3D7_1303800) fusion was expressed in P. falciparum (3D7 strain) by transfection. Immunofluorescence microscopy showed GFP fluorescence mainly in the cytoplasm, and Western blot confirmed expression of the PfSPP-GFP fusion protein at ~64 kDa.\",\n      \"method\": \"Transfection, immunofluorescence microscopy, Western blot\",\n      \"journal\": \"Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, localization only, no functional follow-up reported in abstract\",\n      \"pmids\": [\"25518586\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"3D7\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Plasmodium SPP (PF3D7_1303800 ortholog) activity can be differentially inhibited by established gamma-secretase inhibitors. Using a recombinant FBA substrate cleavage assay (ELISA, Western blot, IP/MS), LY-411,575 and other GSIs showed differential inhibition of SPP family members, demonstrating the feasibility of selective SPP inhibition.\",\n      \"method\": \"Recombinant substrate cleavage assay (ELISA, Western blot, IP/MALDI-TOF MS), co-transfection expression system\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — assay developed primarily for human SPP/SPPLs; Plasmodium SPP activity inferred by pharmacological analogy, not directly assayed in the abstract\",\n      \"pmids\": [\"26046535\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The gamma-secretase inhibitor YO-01027 (dibenzoazepine-type) inhibits SPP in vitro and in vivo through interaction with Val223 in SPP, and treatment efficiently inhibited propagation of P. falciparum, consistent with PfSPP (PF3D7_1303800) being the relevant target.\",\n      \"method\": \"In vitro SPP inhibition assay, in vivo P. falciparum growth inhibition, site-directed interaction analysis (Val223)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — inhibitor selectivity for Plasmodium SPP vs. host SPP not fully resolved; Val223 interaction not directly demonstrated for PfSPP in the abstract\",\n      \"pmids\": [\"29187532\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HIV protease inhibitors Lopinavir and Atazanavir inhibit PfSPP (PF3D7_1303800) activity (measured by transcription-based assay); Lopinavir inhibited both parasite- and host-derived SPP activities while Atazanavir selectively inhibited only parasite-derived SPP activity. These inhibitors also blocked intraerythrocytic growth of Babesia microti ex vivo and in vivo.\",\n      \"method\": \"Transcription-based SPP activity assay, ex vivo Babesia microti growth inhibition, in vivo mouse model (rag1-deficient mice)\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct enzymatic activity assay for PfSPP with selectivity discrimination between parasite and host SPP; single lab with multiple pharmacological agents\",\n      \"pmids\": [\"31311649\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"PF3D7_1303800 (PfSPP) is a presenilin-type intramembrane aspartyl protease that resides in the endoplasmic reticulum of P. falciparum blood-stage parasites, exists as monomer and dimer forms, cleaves HSP101 as a substrate, is required for intra-erythrocytic parasite development but plays no role in erythrocyte invasion, and its partial inhibition correlates with gametocyte emergence; selective HIV protease inhibitors and gamma-secretase inhibitors can differentially block its activity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PF3D7_1303800 (PfSPP) is a presenilin-type intramembrane aspartyl protease of P. falciparum that is required for intra-erythrocytic parasite development [#2]. It is an ER-resident enzyme, localized to the endoplasmic reticulum by immunofluorescence and immunogold electron microscopy, and it exists in both monomer and dimer forms in parasite lysate [#2, #3]. An early report placing PfSPP at the micronemes and implicating it in erythrocyte invasion via binding to erythrocyte band 3 [#0] was directly superseded by a rigorous multi-method study showing PfSPP remains intracellular throughout invasion, plays no role in invasion, and is instead required for intra-erythrocytic growth [#2]. Heat shock protein 101 (HSP101) has been identified as a substrate of PfSPP using an in vivo luminescence cleavage assay, and partial inhibition of the enzyme correlates with gametocyte emergence [#3]. PfSPP activity is inhibited by HIV protease inhibitors, with Atazanavir selectively inhibiting the parasite enzyme over the host enzyme [#7]. Beyond substrate identification and the ER localization/oligomeric state, the catalytic mechanism, regulation, and full substrate repertoire of PfSPP have not been characterized in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"The first attempt to assign a cellular role proposed PfSPP as a micronemal invasion ligand, framing it as a merozoite surface-associated factor engaging the erythrocyte during entry.\",\n      \"evidence\": \"Yeast two-hybrid, immunolocalization, invasion-inhibition antibody, and direct binding assay to erythrocyte band 3 5ABC domain in P. falciparum\",\n      \"pmids\": [\"18160114\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Later directly contradicted by a more rigorous study\", \"No genetic ablation to test the invasion requirement\", \"Binding to band 3 not reconciled with subsequent intracellular localization\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Pharmacology raised the question of whether SPP is needed across life-cycle stages, indicating a role in liver-stage development independent of host Notch signaling.\",\n      \"evidence\": \"gamma-secretase/SPP inhibitor LY411,575 in hepatoma lines, mouse hepatocytes, and P. berghei in vivo, with Notch exclusion experiment\",\n      \"pmids\": [\"19337374\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Inhibitor selectivity limited; effect not tied directly to the PfSPP protein\", \"Ortholog inference rather than direct PF3D7_1303800 assay\", \"No demonstration of on-target engagement in parasite\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"A rigorous re-examination resolved the localization and function controversy, establishing PfSPP as an ER-resident protease required for intra-erythrocytic growth rather than an invasion ligand.\",\n      \"evidence\": \"Immunofluorescence, subcellular fractionation, SPP-specific chemical inhibitors, and invasion assays in P. falciparum\",\n      \"pmids\": [\"22844982\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate(s) driving the growth requirement not identified in this study\", \"Precise ER sub-compartment and topology not defined\", \"No genetic knockout to confirm essentiality\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Substrate identification and oligomeric characterization gave PfSPP a molecular activity, naming HSP101 as a cleavage substrate and linking partial inhibition to gametocyte emergence.\",\n      \"evidence\": \"Immunofluorescence, immunogold EM, Western blot for monomer/dimer, and transfection-based in vivo luminescence substrate assay in P. falciparum\",\n      \"pmids\": [\"25017910\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"HSP101 cleavage site and physiological consequence not mapped\", \"Gametocytogenesis link is correlative\", \"Single lab, no independent confirmation of the substrate\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Direct enzymatic assays established that PfSPP activity can be selectively targeted, distinguishing parasite from host SPP inhibition.\",\n      \"evidence\": \"Transcription-based SPP activity assay with Lopinavir/Atazanavir, plus Babesia microti growth inhibition ex vivo and in vivo\",\n      \"pmids\": [\"31311649\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of parasite-selective inhibition not defined\", \"In-parasite target validation limited to activity readout\", \"Whether inhibition of growth is solely via PfSPP not proven\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The catalytic mechanism, complete substrate repertoire, and the mechanistic basis linking PfSPP activity to intra-erythrocytic development and gametocytogenesis remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model or active-site characterization for PfSPP\", \"No genetic knockout defining essentiality\", \"Mechanism connecting HSP101 cleavage to the growth requirement unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0042500\", \"supporting_discovery_ids\": [0, 2, 3, 7]},\n      {\"term_id\": \"GO:0004190\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0043096\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"HSP101 (substrate) [#3]\"\n    ],\n    \"other_free_text\": [\n      \"Exists as monomer and dimer forms in parasite lysate [#3].\",\n      \"Required for intra-erythrocytic development but not for erythrocyte invasion [#2].\",\n      \"Partial inhibition correlates with gametocyte emergence [#3].\",\n      \"Activity selectively inhibited over host SPP by Atazanavir; Lopinavir inhibits both parasite and host activity [#7].\",\n      \"An early claim of micronemal localization and band 3 binding during invasion [#0] was superseded by ER-localization/intracellular findings [#2].\"\n    ]\n  }\n}","audit_flag":{"gene":"PF3D7_1303800","tier":"IDENTITY_CHECK","verdict":"identity unverified","subtype":"names_other_cards","uniprot_band":"","rules_fired":"R12","issue":"No cited paper names this card; 6 of 8 cited papers name other catalogue genes (PF3D7_1457000, PF3D7_0731500, PF3D7_1116800). The summary may describe one of those genes."},"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_1303800","name":null,"product":"conserved Plasmodium protein, unknown function","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1402300","name":null,"product":"conserved Plasmodium protein, unknown function","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1404000","name":null,"product":"peptidase, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"","evaluation":null}