{"gene":"PF3D7_1115600","run_date":"2026-09-08T12:51:28","timeline":{"discoveries":[{"year":1995,"finding":"PF3D7_1115600 (CYP19B) encodes an active peptidyl-prolyl cis-trans isomerase (PPIase); the recombinant mature protein expressed in E. coli displayed PPIase activity (kcat/Km = 2.3 × 10^6 s^-1 M^-1) that was inhibited by cyclosporin A (IC50 = 10 nM). The protein contains a putative N-terminal endoplasmic-reticulum-directed signal sequence; in vitro translation/translocation experiments showed the signal peptide is cleaved and the protein is glycosylated at two sites upon translocation across microsomes.","method":"Recombinant expression in E. coli, in vitro PPIase enzymatic assay, in vitro translation/translocation with microsome system","journal":"European journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro enzymatic assay with quantified kinetics, signal peptide cleavage and glycosylation demonstrated by reconstitution, single lab but multiple orthogonal methods","pmids":["7588714"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2003,"finding":"CYP19B (PfCyP-19B) was identified as one of the two major cyclosporin-binding proteins of P. falciparum by affinity chromatography on cyclosporin-Affigel, confirmed by SDS-PAGE, Western blotting, and peptide mass fingerprinting. It is an abundant parasite protein (up to 0.5% of total parasite protein), present at highest levels in schizont-stage parasites. Despite bearing an apparent signal sequence, CYP19B was located primarily in the cytosol of the parasite by subcellular fractionation. The PPIase activity of recombinant CYP19B showed the same profile of susceptibility to cyclosporin derivatives as bulk parasite PPIase activity.","method":"Affinity chromatography, SDS-PAGE, Western blotting, peptide mass fingerprinting, subcellular fractionation, recombinant PPIase assay","journal":"International journal for parasitology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (affinity chromatography, MS, fractionation, enzymatic assay) in a single focused study on this specific protein","pmids":["12906882"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2005,"finding":"In CsA-resistant P. falciparum clones, mutations were detected in CYP19B (Cyp19B) in functionally significant amino acid residues. One mutant CYP19A protein (a sibling cyclophilin) was unable to inhibit parasitic calcineurin in the presence of CsA; by analogy the CYP19B mutations are implicated in CsA resistance. This places CYP19B in the pathway by which the CsA–cyclophilin complex inhibits P. falciparum calcineurin.","method":"Sequencing of CsA-resistant clones, in vitro calcineurin phosphatase inhibition assay with purified mutant proteins","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — mutations in CYP19B identified in resistant clones and correlated with resistance, but in vitro functional rescue was demonstrated only for the sibling CYP19A; CYP19B-specific functional data are indirect, single lab","pmids":["15811524"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2012,"finding":"Recombinant CYP19B, assessed alongside 7 other P. falciparum cyclophilin family members using a common host/vector system, lacked PPIase isomerase activity but displayed molecular chaperone activity as judged by prevention of thermal aggregation of model substrates in vitro.","method":"Recombinant expression in E. coli (common system), PPIase enzymatic assay, thermal aggregation chaperone assay","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — direct in vitro assays for two activities, but single lab and the result for PPIase is negative (no activity detected for CYP19B specifically); chaperone activity is positive","pmids":["22546550"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2015,"finding":"PF3D7_1115600 (CYP19B) was identified as a putative interacting partner of the PP1c/Inhibitor-2 complex in P. berghei schizonts and gametocytes by mCherry-tagged affinity pulldown followed by mass spectrometry, suggesting CYP19B may be a target of the PP1c/I2 complex in both asexual and sexual stages.","method":"Affinity pulldown (mCherry-tagged PP1c/I2), mass spectrometry interactome mapping","journal":"International journal of molecular sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single pulldown/MS experiment with no reciprocal validation or functional follow-up for CYP19B specifically; membership in a large interactome list","pmids":["35162991"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2015,"finding":"Co-immunoprecipitation and yeast two-hybrid screening revealed that P. falciparum CYP19B (PfCYP19B) interacts with heat shock protein 70 (Hsp70). Treatment of parasites with CYP ligands (including CsA) disrupted transport of the rhoptry-associated protein 1 (RAP1), linking CYP19B-containing complexes to protein trafficking.","method":"Co-immunoprecipitation, yeast two-hybrid screening, drug treatment phenotypic assay for RAP1 trafficking","journal":"Parasitology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — two orthogonal interaction methods (Co-IP and Y2H) for Hsp70 binding; RAP1 trafficking disruption uses drug treatment affecting both CYP19A and CYP19B, so attribution to CYP19B specifically is partial","pmids":["26156578"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2023,"finding":"Overexpression of CYP19B in vitro conferred limited but significant resistance to both artemisinin and piperaquine in P. falciparum. CYP19B was shown to act as a negative regulator of the integrated stress response (ISR) pathway by modulating levels of phosphorylated eIF2α (eIF2α-P): artemisinin and piperaquine affect eIF2α-P in inverse directions, and both effects can be modulated by CYP19B overexpression towards resistance. A short tandem repeat (STR) polymorphism in the CYP19B promoter was linked to its transcriptional upregulation in drug-resistant field isolates.","method":"Stable overexpression of CYP19B in P. falciparum, in vitro drug susceptibility assay, immunoblot quantification of eIF2α-P, promoter polymorphism analysis","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Moderate — functional overexpression with quantified resistance phenotype plus mechanistic eIF2α-P readout, two orthogonal drug conditions tested, single lab but multiple methods","pmids":["36696458"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"PF3D7_1115600 (CYP19B) is a P. falciparum cyclophilin-family PPIase that, despite carrying an apparent signal sequence, localises primarily to the parasite cytosol; recombinant CYP19B binds cyclosporin A, exhibits molecular chaperone activity (thermal aggregation prevention), interacts with Hsp70, and acts as a negative regulator of the integrated stress response by dampening eIF2α phosphorylation, thereby contributing to artemisinin and piperaquine resistance when overexpressed; its PPIase activity and calcineurin-inhibitory function (as part of a CsA–cyclophilin complex) are implicated by mutations found in CsA-resistant parasites."},"narrative":{"mechanistic_narrative":"PF3D7_1115600 (CYP19B) is an abundant P. falciparum cyclophilin-family protein that participates in protein-folding quality control and stress signalling, and contributes to antimalarial drug resistance [PMID:7588714, PMID:36696458]. It was originally characterised as one of the two major cyclosporin A (CsA)-binding proteins of the parasite, comprising up to 0.5% of total parasite protein and peaking in schizont-stage parasites; despite carrying an apparent N-terminal ER-directed signal sequence, CYP19B localises primarily to the parasite cytosol [PMID:12906882]. Recombinant CYP19B was first shown to be an active peptidyl-prolyl cis-trans isomerase (PPIase) inhibited by CsA (IC50 ~10 nM) whose signal peptide can be cleaved and glycosylated in a microsomal translocation system [PMID:7588714]; a later comparative study of the parasite cyclophilin family instead detected molecular chaperone activity (prevention of thermal aggregation) rather than isomerase activity for recombinant CYP19B [PMID:22546550]. CYP19B interacts with Hsp70, and CsA-family ligands disrupt trafficking of the rhoptry protein RAP1, linking CYP19B-containing complexes to protein transport [PMID:26156578]. Functionally, CYP19B acts as a negative regulator of the integrated stress response: its overexpression modulates phosphorylated eIF2α levels and confers limited but significant resistance to artemisinin and piperaquine, with a promoter short-tandem-repeat polymorphism linked to its upregulation in drug-resistant field isolates [PMID:36696458]. Mutations in CYP19B recovered from CsA-resistant clones place it in the pathway by which a CsA–cyclophilin complex inhibits parasite calcineurin, although direct calcineurin-inhibition rescue in that study was demonstrated only for the sibling cyclophilin, not CYP19B itself [PMID:15811524].","teleology":[{"year":1995,"claim":"Establishing whether CYP19B is an enzyme addressed its basic biochemical identity: recombinant protein was shown to be a CsA-inhibitable PPIase, and its signal peptide was shown to be a functional ER-translocation/glycosylation signal.","evidence":"Recombinant expression in E. coli, quantified in vitro PPIase assay, in vitro translation/translocation across microsomes","pmids":["7588714"],"confidence":"High","gaps":["In vitro translocation/glycosylation does not establish the in vivo compartment of the native protein","No physiological PPIase substrate identified"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2003,"claim":"Whether CYP19B is a major CsA target in the parasite and where it resides was resolved by showing it is one of two dominant cyclosporin-binding proteins, highly abundant, schizont-enriched, and cytosolic despite its signal sequence.","evidence":"Cyclosporin-Affigel affinity chromatography, peptide mass fingerprinting, subcellular fractionation, recombinant PPIase assay","pmids":["12906882"],"confidence":"High","gaps":["Cytosolic localisation is inferred from fractionation, not imaging","Reason the ER signal sequence does not retain the protein in the secretory pathway is unexplained"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2005,"claim":"To connect CYP19B to CsA action, mutations in functionally significant residues were found in CsA-resistant clones, implicating CYP19B in the CsA–cyclophilin/calcineurin inhibition pathway.","evidence":"Sequencing of CsA-resistant clones plus in vitro calcineurin phosphatase inhibition assay","pmids":["15811524"],"confidence":"Medium","gaps":["Functional calcineurin-inhibition rescue was shown only for the sibling CYP19A, not CYP19B itself","Causal link between the CYP19B mutations and the resistance phenotype not demonstrated by allelic replacement"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2012,"claim":"A comparative family-wide biochemical survey re-examined CYP19B activity, detecting molecular chaperone (anti-aggregation) activity but no isomerase activity under the common assay conditions used.","evidence":"Recombinant expression in a common E. coli system, PPIase assay, thermal aggregation chaperone assay","pmids":["22546550"],"confidence":"Medium","gaps":["Negative PPIase result conflicts with the earlier positive assay and may reflect construct/assay differences","Chaperone substrates are model proteins, not physiological clients"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2015,"claim":"Interaction studies began to define CYP19B's protein-network context, showing direct binding to Hsp70 and linking CYP-ligand treatment to disruption of RAP1 trafficking.","evidence":"Co-immunoprecipitation and yeast two-hybrid for Hsp70; drug-treatment phenotypic assay for RAP1 transport","pmids":["26156578"],"confidence":"Medium","gaps":["RAP1 trafficking defect used ligands affecting both CYP19A and CYP19B, so the effect is not uniquely attributable to CYP19B","Functional consequence of the Hsp70 interaction not established"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2015,"claim":"An interactome screen placed CYP19B as a putative partner of the PP1c/Inhibitor-2 complex in both asexual and sexual stages, hinting at regulation by dephosphorylation.","evidence":"mCherry-tagged PP1c/I2 affinity pulldown with mass spectrometry in P. berghei schizonts and gametocytes","pmids":["35162991"],"confidence":"Low","gaps":["Single pulldown/MS without reciprocal validation for CYP19B","No functional consequence of the PP1c/I2 association tested","Shown in P. berghei, not P. falciparum"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2023,"claim":"The physiological role in drug resistance was defined by showing CYP19B is a negative regulator of the integrated stress response whose overexpression modulates eIF2α phosphorylation and confers resistance to artemisinin and piperaquine.","evidence":"Stable CYP19B overexpression in P. falciparum, in vitro drug susceptibility assays, eIF2α-P immunoblotting, promoter STR polymorphism analysis","pmids":["36696458"],"confidence":"High","gaps":["Direct enzymatic/biochemical mechanism by which CYP19B lowers eIF2α-P is not defined","Which kinase/phosphatase CYP19B acts upon is unidentified","Resistance conferred is limited in magnitude"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"How CYP19B's biochemical activity (isomerase versus chaperone), its Hsp70/PP1c-I2 associations, and its dampening of eIF2α phosphorylation are mechanistically integrated into a single molecular pathway remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No physiological PPIase or chaperone substrate identified","The molecular step linking CYP19B to eIF2α dephosphorylation is unknown","Reconciliation of positive and negative in vitro PPIase results is lacking"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["CsA–cyclophilin complex implicated in calcineurin inhibition [#2]","CYP19B–Hsp70 complex [#5]","Putative PP1c/Inhibitor-2 interactome (P. berghei) [#4]"],"partners":["HSP70 [#5]","PP1C/INHIBITOR-2 COMPLEX (PUTATIVE, P. BERGHEI) [#4]","CYCLOSPORIN A (LIGAND) [#0, #1]"],"other_free_text":["Highly abundant parasite protein (up to 0.5% of total), peaking in schizonts [#1]","Carries an N-terminal ER-directed signal sequence that is cleavable and glycosylatable in vitro, yet the native protein is cytosolic [#0, #1]","Mutations in functionally significant residues found in CsA-resistant clones [#2]","A promoter short-tandem-repeat polymorphism is linked to CYP19B upregulation in drug-resistant field isolates [#6]","Conflicting in vitro results: PPIase activity detected in 1995 [#0] but not in a 2012 family-wide survey [#3]"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"7520696","id":"PMC_7520696","title":"Roles of peptidyl-prolyl cis-trans isomerase and calcineurin in the mechanisms of antimalarial action of cyclosporin A, FK506, and rapamycin.","date":"1994","source":"Biochemical pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/7520696","citation_count":108,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidyl-prolyl cis-trans isomerase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"15811524","id":"PMC_15811524","title":"Plasmodium falciparum calcineurin and its association with heat shock protein 90: mechanisms for the antimalarial activity of cyclosporin A and synergism with geldanamycin.","date":"2005","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/15811524","citation_count":52,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["CYP19B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"15664653","id":"PMC_15664653","title":"A Plasmodium falciparum FK506-binding protein (FKBP) with peptidyl-prolyl cis-trans isomerase and chaperone activities.","date":"2005","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/15664653","citation_count":51,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidyl-prolyl cis-trans isomerase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"25986569","id":"PMC_25986569","title":"Immunophilins: Structures, Mechanisms and Ligands.","date":"2015","source":"Current molecular pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/25986569","citation_count":46,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidyl-prolyl cis-trans isomerase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"8577319","id":"PMC_8577319","title":"Cloning and characterization of a Plasmodium falciparum cyclophilin gene that is stage-specifically expressed.","date":"1995","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/8577319","citation_count":37,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidyl-prolyl cis-trans isomerase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"7588714","id":"PMC_7588714","title":"Molecular and biochemical characterization of a Plasmodium falciparum cyclophilin containing a cleavable signal sequence.","date":"1995","source":"European journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7588714","citation_count":37,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["curated_ref"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"12906882","id":"PMC_12906882","title":"Cyclosporin-binding proteins of Plasmodium falciparum.","date":"2003","source":"International journal for parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/12906882","citation_count":31,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidyl-prolyl cis-trans isomerase","PfCYP19B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"15776382","id":"PMC_15776382","title":"Antimalarial effects of macrolactones related to FK520 (ascomycin) are independent of the immunosuppressive properties of the compounds.","date":"2005","source":"The Journal of infectious diseases","url":"https://pubmed.ncbi.nlm.nih.gov/15776382","citation_count":31,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidyl-prolyl cis-trans isomerase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"22546550","id":"PMC_22546550","title":"A family of cyclophilin-like molecular chaperones in Plasmodium falciparum.","date":"2012","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/22546550","citation_count":23,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidyl-prolyl cis-trans isomerase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"21549842","id":"PMC_21549842","title":"Overexpression, purification and assessment of cyclosporin binding of a family of cyclophilins and cyclophilin-like proteins of the human malarial parasite Plasmodium falciparum.","date":"2011","source":"Protein expression and purification","url":"https://pubmed.ncbi.nlm.nih.gov/21549842","citation_count":14,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidyl-prolyl cis-trans isomerase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"26869851","id":"PMC_26869851","title":"Protein profiling of mefloquine resistant Plasmodium falciparum using mass spectrometry-based proteomics.","date":"2015","source":"International journal of mass spectrometry","url":"https://pubmed.ncbi.nlm.nih.gov/26869851","citation_count":10,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidyl-prolyl cis-trans isomerase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"26156578","id":"PMC_26156578","title":"Immunophilin-protein interactions in Plasmodium falciparum.","date":"2015","source":"Parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/26156578","citation_count":10,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfCYP19B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"33202000","id":"PMC_33202000","title":"A proteomic glimpse into the effect of antimalarial drugs on Plasmodium falciparum proteome towards highlighting possible therapeutic targets.","date":"2021","source":"Pathogens and disease","url":"https://pubmed.ncbi.nlm.nih.gov/33202000","citation_count":9,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidyl-prolyl cis-trans isomerase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"36696458","id":"PMC_36696458","title":"Short tandem repeat polymorphism in the promoter region of cyclophilin 19B drives its transcriptional upregulation and contributes to drug resistance in the malaria parasite Plasmodium falciparum.","date":"2023","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/36696458","citation_count":7,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PfCYP19B"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"26091727","id":"PMC_26091727","title":"Computational insights into the suicide inhibition of Plasmodium falciparum Fk506-binding protein 35.","date":"2015","source":"Bioorganic & medicinal chemistry letters","url":"https://pubmed.ncbi.nlm.nih.gov/26091727","citation_count":6,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidyl-prolyl cis-trans isomerase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"31120120","id":"PMC_31120120","title":"Expression and characterization of functional domains of FK506-binding protein 35 from Plasmodium knowlesi.","date":"2018","source":"Protein engineering, design & selection : PEDS","url":"https://pubmed.ncbi.nlm.nih.gov/31120120","citation_count":4,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidyl-prolyl cis-trans isomerase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"25978804","id":"PMC_25978804","title":"Molecular docking study of macrocycles as Fk506-binding protein inhibitors.","date":"2015","source":"Journal of molecular graphics & modelling","url":"https://pubmed.ncbi.nlm.nih.gov/25978804","citation_count":4,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidyl-prolyl cis-trans isomerase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"35162991","id":"PMC_35162991","title":"Mapping PP1c and Its Inhibitor 2 Interactomes Reveals Conserved and Specific Networks in Asexual and Sexual Stages of Plasmodium.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35162991","citation_count":3,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["peptidyl-prolyl cis-trans isomerase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11458,"output_tokens":2426,"usd":0.035382,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7791,"output_tokens":3704,"usd":0.065778,"stage2_stop_reason":"end_turn"},"total_usd":0.10116,"stage1_batch_id":"msgbatch_01DisAaTypwmC8QwdMBuePHo","stage2_batch_id":"msgbatch_0184zxZTqxiEfdYYfPMfkqjb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"PF3D7_1115600 (CYP19B) encodes an active peptidyl-prolyl cis-trans isomerase (PPIase); the recombinant mature protein expressed in E. coli displayed PPIase activity (kcat/Km = 2.3 × 10^6 s^-1 M^-1) that was inhibited by cyclosporin A (IC50 = 10 nM). The protein contains a putative N-terminal endoplasmic-reticulum-directed signal sequence; in vitro translation/translocation experiments showed the signal peptide is cleaved and the protein is glycosylated at two sites upon translocation across microsomes.\",\n      \"method\": \"Recombinant expression in E. coli, in vitro PPIase enzymatic assay, in vitro translation/translocation with microsome system\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro enzymatic assay with quantified kinetics, signal peptide cleavage and glycosylation demonstrated by reconstitution, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"7588714\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CYP19B (PfCyP-19B) was identified as one of the two major cyclosporin-binding proteins of P. falciparum by affinity chromatography on cyclosporin-Affigel, confirmed by SDS-PAGE, Western blotting, and peptide mass fingerprinting. It is an abundant parasite protein (up to 0.5% of total parasite protein), present at highest levels in schizont-stage parasites. Despite bearing an apparent signal sequence, CYP19B was located primarily in the cytosol of the parasite by subcellular fractionation. The PPIase activity of recombinant CYP19B showed the same profile of susceptibility to cyclosporin derivatives as bulk parasite PPIase activity.\",\n      \"method\": \"Affinity chromatography, SDS-PAGE, Western blotting, peptide mass fingerprinting, subcellular fractionation, recombinant PPIase assay\",\n      \"journal\": \"International journal for parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (affinity chromatography, MS, fractionation, enzymatic assay) in a single focused study on this specific protein\",\n      \"pmids\": [\"12906882\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"In CsA-resistant P. falciparum clones, mutations were detected in CYP19B (Cyp19B) in functionally significant amino acid residues. One mutant CYP19A protein (a sibling cyclophilin) was unable to inhibit parasitic calcineurin in the presence of CsA; by analogy the CYP19B mutations are implicated in CsA resistance. This places CYP19B in the pathway by which the CsA–cyclophilin complex inhibits P. falciparum calcineurin.\",\n      \"method\": \"Sequencing of CsA-resistant clones, in vitro calcineurin phosphatase inhibition assay with purified mutant proteins\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — mutations in CYP19B identified in resistant clones and correlated with resistance, but in vitro functional rescue was demonstrated only for the sibling CYP19A; CYP19B-specific functional data are indirect, single lab\",\n      \"pmids\": [\"15811524\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Recombinant CYP19B, assessed alongside 7 other P. falciparum cyclophilin family members using a common host/vector system, lacked PPIase isomerase activity but displayed molecular chaperone activity as judged by prevention of thermal aggregation of model substrates in vitro.\",\n      \"method\": \"Recombinant expression in E. coli (common system), PPIase enzymatic assay, thermal aggregation chaperone assay\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — direct in vitro assays for two activities, but single lab and the result for PPIase is negative (no activity detected for CYP19B specifically); chaperone activity is positive\",\n      \"pmids\": [\"22546550\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PF3D7_1115600 (CYP19B) was identified as a putative interacting partner of the PP1c/Inhibitor-2 complex in P. berghei schizonts and gametocytes by mCherry-tagged affinity pulldown followed by mass spectrometry, suggesting CYP19B may be a target of the PP1c/I2 complex in both asexual and sexual stages.\",\n      \"method\": \"Affinity pulldown (mCherry-tagged PP1c/I2), mass spectrometry interactome mapping\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single pulldown/MS experiment with no reciprocal validation or functional follow-up for CYP19B specifically; membership in a large interactome list\",\n      \"pmids\": [\"35162991\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Co-immunoprecipitation and yeast two-hybrid screening revealed that P. falciparum CYP19B (PfCYP19B) interacts with heat shock protein 70 (Hsp70). Treatment of parasites with CYP ligands (including CsA) disrupted transport of the rhoptry-associated protein 1 (RAP1), linking CYP19B-containing complexes to protein trafficking.\",\n      \"method\": \"Co-immunoprecipitation, yeast two-hybrid screening, drug treatment phenotypic assay for RAP1 trafficking\",\n      \"journal\": \"Parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — two orthogonal interaction methods (Co-IP and Y2H) for Hsp70 binding; RAP1 trafficking disruption uses drug treatment affecting both CYP19A and CYP19B, so attribution to CYP19B specifically is partial\",\n      \"pmids\": [\"26156578\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Overexpression of CYP19B in vitro conferred limited but significant resistance to both artemisinin and piperaquine in P. falciparum. CYP19B was shown to act as a negative regulator of the integrated stress response (ISR) pathway by modulating levels of phosphorylated eIF2α (eIF2α-P): artemisinin and piperaquine affect eIF2α-P in inverse directions, and both effects can be modulated by CYP19B overexpression towards resistance. A short tandem repeat (STR) polymorphism in the CYP19B promoter was linked to its transcriptional upregulation in drug-resistant field isolates.\",\n      \"method\": \"Stable overexpression of CYP19B in P. falciparum, in vitro drug susceptibility assay, immunoblot quantification of eIF2α-P, promoter polymorphism analysis\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional overexpression with quantified resistance phenotype plus mechanistic eIF2α-P readout, two orthogonal drug conditions tested, single lab but multiple methods\",\n      \"pmids\": [\"36696458\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"PF3D7_1115600 (CYP19B) is a P. falciparum cyclophilin-family PPIase that, despite carrying an apparent signal sequence, localises primarily to the parasite cytosol; recombinant CYP19B binds cyclosporin A, exhibits molecular chaperone activity (thermal aggregation prevention), interacts with Hsp70, and acts as a negative regulator of the integrated stress response by dampening eIF2α phosphorylation, thereby contributing to artemisinin and piperaquine resistance when overexpressed; its PPIase activity and calcineurin-inhibitory function (as part of a CsA–cyclophilin complex) are implicated by mutations found in CsA-resistant parasites.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PF3D7_1115600 (CYP19B) is an abundant P. falciparum cyclophilin-family protein that participates in protein-folding quality control and stress signalling, and contributes to antimalarial drug resistance [#0, #6]. It was originally characterised as one of the two major cyclosporin A (CsA)-binding proteins of the parasite, comprising up to 0.5% of total parasite protein and peaking in schizont-stage parasites; despite carrying an apparent N-terminal ER-directed signal sequence, CYP19B localises primarily to the parasite cytosol [#1]. Recombinant CYP19B was first shown to be an active peptidyl-prolyl cis-trans isomerase (PPIase) inhibited by CsA (IC50 ~10 nM) whose signal peptide can be cleaved and glycosylated in a microsomal translocation system [#0]; a later comparative study of the parasite cyclophilin family instead detected molecular chaperone activity (prevention of thermal aggregation) rather than isomerase activity for recombinant CYP19B [#3]. CYP19B interacts with Hsp70, and CsA-family ligands disrupt trafficking of the rhoptry protein RAP1, linking CYP19B-containing complexes to protein transport [#5]. Functionally, CYP19B acts as a negative regulator of the integrated stress response: its overexpression modulates phosphorylated eIF2\\u03b1 levels and confers limited but significant resistance to artemisinin and piperaquine, with a promoter short-tandem-repeat polymorphism linked to its upregulation in drug-resistant field isolates [#6]. Mutations in CYP19B recovered from CsA-resistant clones place it in the pathway by which a CsA\\u2013cyclophilin complex inhibits parasite calcineurin, although direct calcineurin-inhibition rescue in that study was demonstrated only for the sibling cyclophilin, not CYP19B itself [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Establishing whether CYP19B is an enzyme addressed its basic biochemical identity: recombinant protein was shown to be a CsA-inhibitable PPIase, and its signal peptide was shown to be a functional ER-translocation/glycosylation signal.\",\n      \"evidence\": \"Recombinant expression in E. coli, quantified in vitro PPIase assay, in vitro translation/translocation across microsomes\",\n      \"pmids\": [\"7588714\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro translocation/glycosylation does not establish the in vivo compartment of the native protein\", \"No physiological PPIase substrate identified\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Whether CYP19B is a major CsA target in the parasite and where it resides was resolved by showing it is one of two dominant cyclosporin-binding proteins, highly abundant, schizont-enriched, and cytosolic despite its signal sequence.\",\n      \"evidence\": \"Cyclosporin-Affigel affinity chromatography, peptide mass fingerprinting, subcellular fractionation, recombinant PPIase assay\",\n      \"pmids\": [\"12906882\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cytosolic localisation is inferred from fractionation, not imaging\", \"Reason the ER signal sequence does not retain the protein in the secretory pathway is unexplained\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"To connect CYP19B to CsA action, mutations in functionally significant residues were found in CsA-resistant clones, implicating CYP19B in the CsA\\u2013cyclophilin/calcineurin inhibition pathway.\",\n      \"evidence\": \"Sequencing of CsA-resistant clones plus in vitro calcineurin phosphatase inhibition assay\",\n      \"pmids\": [\"15811524\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional calcineurin-inhibition rescue was shown only for the sibling CYP19A, not CYP19B itself\", \"Causal link between the CYP19B mutations and the resistance phenotype not demonstrated by allelic replacement\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"A comparative family-wide biochemical survey re-examined CYP19B activity, detecting molecular chaperone (anti-aggregation) activity but no isomerase activity under the common assay conditions used.\",\n      \"evidence\": \"Recombinant expression in a common E. coli system, PPIase assay, thermal aggregation chaperone assay\",\n      \"pmids\": [\"22546550\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Negative PPIase result conflicts with the earlier positive assay and may reflect construct/assay differences\", \"Chaperone substrates are model proteins, not physiological clients\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Interaction studies began to define CYP19B's protein-network context, showing direct binding to Hsp70 and linking CYP-ligand treatment to disruption of RAP1 trafficking.\",\n      \"evidence\": \"Co-immunoprecipitation and yeast two-hybrid for Hsp70; drug-treatment phenotypic assay for RAP1 transport\",\n      \"pmids\": [\"26156578\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"RAP1 trafficking defect used ligands affecting both CYP19A and CYP19B, so the effect is not uniquely attributable to CYP19B\", \"Functional consequence of the Hsp70 interaction not established\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"An interactome screen placed CYP19B as a putative partner of the PP1c/Inhibitor-2 complex in both asexual and sexual stages, hinting at regulation by dephosphorylation.\",\n      \"evidence\": \"mCherry-tagged PP1c/I2 affinity pulldown with mass spectrometry in P. berghei schizonts and gametocytes\",\n      \"pmids\": [\"35162991\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single pulldown/MS without reciprocal validation for CYP19B\", \"No functional consequence of the PP1c/I2 association tested\", \"Shown in P. berghei, not P. falciparum\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The physiological role in drug resistance was defined by showing CYP19B is a negative regulator of the integrated stress response whose overexpression modulates eIF2\\u03b1 phosphorylation and confers resistance to artemisinin and piperaquine.\",\n      \"evidence\": \"Stable CYP19B overexpression in P. falciparum, in vitro drug susceptibility assays, eIF2\\u03b1-P immunoblotting, promoter STR polymorphism analysis\",\n      \"pmids\": [\"36696458\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct enzymatic/biochemical mechanism by which CYP19B lowers eIF2\\u03b1-P is not defined\", \"Which kinase/phosphatase CYP19B acts upon is unidentified\", \"Resistance conferred is limited in magnitude\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CYP19B's biochemical activity (isomerase versus chaperone), its Hsp70/PP1c-I2 associations, and its dampening of eIF2\\u03b1 phosphorylation are mechanistically integrated into a single molecular pathway remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No physiological PPIase or chaperone substrate identified\", \"The molecular step linking CYP19B to eIF2\\u03b1 dephosphorylation is unknown\", \"Reconciliation of positive and negative in vitro PPIase results is lacking\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003755\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0016018\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0051082\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0030291\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0140469\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0006417\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0015031\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [\n      \"CsA\\u2013cyclophilin complex implicated in calcineurin inhibition [#2]\",\n      \"CYP19B\\u2013Hsp70 complex [#5]\",\n      \"Putative PP1c/Inhibitor-2 interactome (P. berghei) [#4]\"\n    ],\n    \"partners\": [\n      \"Hsp70 [#5]\",\n      \"PP1c/Inhibitor-2 complex (putative, P. berghei) [#4]\",\n      \"Cyclosporin A (ligand) [#0, #1]\"\n    ],\n    \"other_free_text\": [\n      \"Highly abundant parasite protein (up to 0.5% of total), peaking in schizonts [#1]\",\n      \"Carries an N-terminal ER-directed signal sequence that is cleavable and glycosylatable in vitro, yet the native protein is cytosolic [#0, #1]\",\n      \"Mutations in functionally significant residues found in CsA-resistant clones [#2]\",\n      \"A promoter short-tandem-repeat polymorphism is linked to CYP19B upregulation in drug-resistant field isolates [#6]\",\n      \"Conflicting in vitro results: PPIase activity detected in 1995 [#0] but not in a 2012 family-wide survey [#3]\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_1115600","name":null,"product":"peptidyl-prolyl cis-trans isomerase","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_0932200","name":null,"product":"peptidyl-prolyl cis-trans isomerase, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_0913200","name":null,"product":"peptidylprolyl isomerase precursor, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"CYP19B","evaluation":null}