{"gene":"PF3D7_0932200","run_date":"2026-09-08T12:51:27","timeline":{"discoveries":[{"year":2008,"finding":"Crystal structure of P. falciparum PFN was solved alone and in complex with a polyproline ligand peptide, revealing a unique minidomain consisting of a large beta-hairpin extension common to all apicomplexan parasites and an acidic loop specific to Plasmodium species. Reverse genetics in P. berghei demonstrated PFN is essential for invasive blood stages.","method":"X-ray crystallography (crystal structure of PFN alone and with polyproline peptide); reverse genetics (P. berghei knockout)","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation by reverse genetics, two orthogonal methods in one study","pmids":["19000816"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2012,"finding":"P. falciparum PFN alone sequesters actin monomers, thereby inhibiting polymerization; however, its monomer-sequestering activity does not compete with the nucleating activity of formin 1 at an equimolar profilin-actin ratio.","method":"In vitro actin polymerization assay with recombinant P. falciparum PFN and formin 1 FH2 domain","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro biochemical assay, single lab, single method reported in abstract","pmids":["22428073"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2012,"finding":"Tetracycline-induced conditional knockdown of P. berghei PFN using an ApiAP2-based transactivator system resulted in a dramatic reduction in parasite viability, establishing PFN as essential.","method":"Conditional knockdown (TetR-aptamer regulatable system) in P. berghei blood stages; viability assay","journal":"Cell host & microbe","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean conditional KO with defined viability phenotype, single lab","pmids":["23245327"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2013,"finding":"Mature P. falciparum subtilisin-like protease 3 (PfSUB3) proteolysed recombinant P. falciparum PFN in a dose-dependent manner in vitro, identifying PFN as a substrate of PfSUB3. Additionally, recombinant PFN was found to induce TNF-α and IL-12 production from mouse bone marrow-derived dendritic cells, indicating a proinflammatory activity.","method":"In vitro protease activity assay (recombinant PfSUB3 + recombinant PFN); cytokine induction assay with mouse dendritic cells","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro enzymatic assay with recombinant proteins, single lab, no mutagenesis validation","pmids":["24080030"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2015,"finding":"P. falciparum PFN is a small globular protein that consists of two independently folding units (foldons), with binding sites for different protein ligands segregated in the two halves, suggesting evolution via an ancient gene fusion event. A domain-swapped mutant and half-profilins that can reassemble into full PFN were engineered to demonstrate this.","method":"Domain-swapped mutagenesis; expression of separate half-profilin fragments and reassembly assay; structural/biophysical characterization","journal":"Cellular and molecular life sciences","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — mutagenesis and reconstitution, single lab, mechanistic inference from domain-swapped construct","pmids":["26012696"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2016,"finding":"Overexpression of P. berghei PFN in sporozoites (but not in merozoites or ookinetes) impaired circular gliding motility and salivary gland invasion, while strong overexpression caused complete life cycle arrest. Overexpression correlated with altered actin accumulation at the parasite tip.","method":"Transgenic overexpression with stage-specific promoters in P. berghei; gliding motility assays; fluorescence imaging of actin at parasite tip","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transgenic overexpression with defined motility and invasion phenotypes, multiple stages examined, single lab","pmids":["27226484"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2017,"finding":"The apicomplexan-specific beta-hairpin motif of PFN is critically involved in actin binding. Mutations in this motif impaired gliding motility of P. berghei sporozoites. Force measurements using laser tweezers on migrating sporozoites, combined with molecular dynamics simulations, showed that the unique PFN-actin interface fine-tunes gliding motility speed.","method":"Site-directed mutagenesis of PFN beta-hairpin; P. berghei sporozoite gliding motility assay; laser tweezer force measurements; molecular dynamics simulations","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis validated in vivo motility phenotype, force measurements, and molecular dynamics; multiple orthogonal methods, single lab","pmids":["28552953"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2020,"finding":"Mutations in PFN that do not affect actin binding in vitro still reduce force generation during P. berghei sporozoite migration and increase retrograde flow, demonstrating a function of PFN in force generation that is independent of its actin-binding activity.","method":"Site-directed mutagenesis of PFN (non-actin-binding mutants); laser tweezer force measurements on migrating P. berghei sporozoites; in vitro actin binding assays","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis combined with in vitro binding assays and biophysical force measurements, multiple orthogonal methods, single lab","pmids":["32034083"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2023,"finding":"Resistance mutations in P. falciparum PFN (N154Y, K124N) and actin-1 (M356L) were identified after selection with compound MMV020291. CRISPR-Cas9 engineering of these mutations into wild-type parasites conferred resistance. In vitro cosedimentation assays with recombinant P. falciparum proteins showed that potent MMV020291 analogues disrupt filamentous actin formation in the presence of PFN, establishing PFN as part of the drug target mechanism. The compound also inhibited actin-1-dependent apicoplast segregation.","method":"Whole genome sequencing of resistant lines; CRISPR-Cas9 engineering of resistance mutations; in vitro actin cosedimentation assay with recombinant PFN and actin-1; apicoplast segregation assay","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — CRISPR-validated resistance mutations plus in vitro biochemical reconstitution, multiple orthogonal methods, single lab","pmids":["37053271"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"P. falciparum/P. berghei PFN is an essential actin monomer-sequestering protein with a structurally unique apicomplexan-specific beta-hairpin motif that mediates actin binding and fine-tunes gliding motility; it functions not only by sequestering actin monomers (inhibiting spontaneous polymerization) but also contributes to force generation during sporozoite migration independently of its actin-binding activity, is proteolytically cleaved by PfSUB3, and its interaction with actin-1 is a target for antimalarial compounds that disrupt filamentous actin formation."},"narrative":{"mechanistic_narrative":"PF3D7_0932200 (PFN) is an essential actin-regulatory protein of malaria parasites that governs actin dynamics underlying invasive-stage motility [PMID:19000816, PMID:23245327]. It sequesters actin monomers to inhibit spontaneous polymerization, yet at equimolar ratios does not compete with the actin-nucleating activity of formin 1, allowing controlled filament assembly [PMID:22428073]. Structurally, PFN carries an apicomplexan-specific minidomain — a large beta-hairpin extension plus a Plasmodium-specific acidic loop — and folds as two independent units whose distinct halves segregate binding sites for different ligands [PMID:19000816, PMID:26012696]. The beta-hairpin motif forms a unique PFN–actin interface that is critical for actin binding and fine-tunes gliding-motility speed of P. berghei sporozoites [PMID:28552953]; beyond actin sequestration, PFN also contributes to force generation during sporozoite migration through a function that is independent of its actin-binding activity, since non-actin-binding mutants still reduce force and increase retrograde flow [PMID:32034083]. Overexpression in P. berghei sporozoites specifically disrupts circular gliding, salivary gland invasion, and tip actin accumulation, underscoring stage-specific dosage sensitivity [PMID:27226484]. PFN is proteolysed in vitro by the subtilisin-like protease PfSUB3 [PMID:24080030], and its interaction with actin-1 is the target of the antimalarial compound MMV020291, whose analogues disrupt filamentous actin formation and whose resistance mutations map to PFN and actin-1 [PMID:37053271].","teleology":[{"year":2008,"claim":"Establishing that PFN adopts a distinctive apicomplexan-specific fold and is genetically required answered whether it is a viable, parasite-selective target rather than a generic profilin.","evidence":"X-ray crystallography of PFN alone and with a polyproline peptide plus P. berghei reverse-genetics knockout","pmids":["19000816"],"confidence":"High","gaps":["Did not resolve the PFN-actin complex structure","Essentiality shown only for invasive blood stages"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2012,"claim":"Biochemical reconstitution defined PFN's core activity as actin-monomer sequestration and showed it does not antagonize formin 1 nucleation, framing how PFN can both restrain and permit polymerization.","evidence":"In vitro actin polymerization assays with recombinant PFN and the formin 1 FH2 domain","pmids":["22428073"],"confidence":"Medium","gaps":["Single in vitro method","Interplay quantified only at equimolar ratio","No cellular validation"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2012,"claim":"Conditional knockdown confirmed PFN essentiality in blood stages with a defined loss-of-viability phenotype, moving beyond gene-disruption inference.","evidence":"TetR-aptamer conditional knockdown in P. berghei blood stages with viability assay","pmids":["23245327"],"confidence":"Medium","gaps":["Molecular basis of lethality not dissected","Single lab/system"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2013,"claim":"Identifying PFN as a PfSUB3 substrate and detecting proinflammatory cytokine induction raised the possibility of regulatory processing and immune interactions beyond its cytoskeletal role.","evidence":"In vitro protease assay (recombinant PfSUB3 + PFN); cytokine induction in mouse bone marrow-derived dendritic cells","pmids":["24080030"],"confidence":"Medium","gaps":["No in vivo cleavage or mutagenesis of the cleavage site","Cytokine activity not linked to a parasite phenotype"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2015,"claim":"Demonstrating that PFN is built from two independently folding units with segregated ligand-binding sites explained its architecture as an apparent ancient gene fusion and mapped functional halves.","evidence":"Domain-swapped mutagenesis and reassembly of half-profilin fragments with biophysical characterization","pmids":["26012696"],"confidence":"Medium","gaps":["Physiological relevance of the fusion architecture untested in parasites","Specific ligands of each half not fully enumerated"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2016,"claim":"Stage-specific overexpression revealed PFN dosage is critical selectively in sporozoites, linking PFN levels to gliding, salivary gland invasion, and tip actin organization.","evidence":"Transgenic stage-specific overexpression in P. berghei with gliding and invasion assays and tip actin imaging","pmids":["27226484"],"confidence":"Medium","gaps":["Why sporozoites are uniquely sensitive unexplained","Overexpression is not a physiological perturbation"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2017,"claim":"Mutating the apicomplexan-specific beta-hairpin established this motif as the actin-binding determinant that fine-tunes gliding speed, connecting a unique structural feature to motor mechanics.","evidence":"Beta-hairpin mutagenesis, sporozoite gliding assays, laser-tweezer force measurements, and molecular dynamics in P. berghei","pmids":["28552953"],"confidence":"High","gaps":["Quantitative contribution of the acidic loop not isolated","Link to specific downstream motor components unresolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2020,"claim":"Showing that non-actin-binding PFN mutants still reduce force and increase retrograde flow uncoupled PFN's force-generation role from its canonical actin-binding activity.","evidence":"Non-actin-binding PFN mutants tested by laser-tweezer force measurements on migrating P. berghei sporozoites plus in vitro binding assays","pmids":["32034083"],"confidence":"High","gaps":["Molecular mechanism of the actin-binding-independent function unknown","Partner mediating force generation not identified"],"attribution":[{"species":"Plasmodium falciparum","strain":""},{"species":"Plasmodium berghei","strain":""}]},{"year":2023,"claim":"Mapping MMV020291 resistance to PFN and actin-1 and showing analogues disrupt F-actin formation in the presence of PFN validated the PFN–actin-1 interface as an antimalarial target.","evidence":"WGS of resistant lines, CRISPR-Cas9 mutation engineering, in vitro cosedimentation with recombinant PFN and actin-1, and apicoplast segregation assays in P. falciparum","pmids":["37053271"],"confidence":"High","gaps":["Direct compound-PFN binding not structurally resolved","Whether PFN or actin-1 is the primary drug-contact site unclear"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"The molecular basis of PFN's actin-binding-independent force-generation function and its structural interface with actin-1 during drug engagement remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of the PFN-actin-1 complex","Physiological role of PfSUB3 cleavage of PFN unknown","Mechanism coupling PFN to force generation not defined"],"attribution":[]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["ACTIN-1 (PFACT1)","FORMIN 1 (FH2 DOMAIN)","PFSUB3 (SUBTILISIN-LIKE PROTEASE 3)"],"other_free_text":["Contains an apicomplexan-specific beta-hairpin motif plus a Plasmodium-specific acidic loop that form a unique actin-binding interface [#0, #6]","Folds as two independent units with segregated ligand-binding sites, consistent with an ancient gene fusion [#4]","Has an actin-binding-independent role in force generation during sporozoite migration [#7]","Target of the antimalarial compound MMV020291 via the PFN-actin-1 interaction; resistance mutations N154Y and K124N in PFN [#8]","Recombinant PFN induces TNF-alpha and IL-12 from mouse dendritic cells [#3]"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"23245327","id":"PMC_23245327","title":"A tetracycline-repressible transactivator system to study essential genes in malaria parasites.","date":"2012","source":"Cell host & microbe","url":"https://pubmed.ncbi.nlm.nih.gov/23245327","citation_count":76,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["profilin"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"23184938","id":"PMC_23184938","title":"Mammalian and malaria parasite cyclase-associated proteins catalyze nucleotide exchange on G-actin through a conserved mechanism.","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23184938","citation_count":54,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["profilin"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"19000816","id":"PMC_19000816","title":"Structural basis for parasite-specific functions of the divergent profilin of Plasmodium falciparum.","date":"2008","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/19000816","citation_count":53,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["profilin"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"28552953","id":"PMC_28552953","title":"A unique profilin-actin interface is important for malaria parasite motility.","date":"2017","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/28552953","citation_count":40,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["profilin"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"31736970","id":"PMC_31736970","title":"The Liver-Stage Plasmodium Infection Is a Critical Checkpoint for Development of Experimental Cerebral Malaria.","date":"2019","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/31736970","citation_count":20,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["profilin"],"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":["profilin"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"22428073","id":"PMC_22428073","title":"The lasso segment is required for functional dimerization of the Plasmodium formin 1 FH2 domain.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/22428073","citation_count":18,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["profilin"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"37053271","id":"PMC_37053271","title":"Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics.","date":"2023","source":"PLoS biology","url":"https://pubmed.ncbi.nlm.nih.gov/37053271","citation_count":17,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["profilin"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"32034083","id":"PMC_32034083","title":"A function of profilin in force generation during malaria parasite motility that is independent of actin binding.","date":"2020","source":"Journal of cell 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CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/26012696","citation_count":7,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["profilin"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"31936739","id":"PMC_31936739","title":"Synthetic DNA Vaccines Adjuvanted with pIL-33 Drive Liver-Localized T Cells and Provide Protection from Plasmodium Challenge in a Mouse Model.","date":"2020","source":"Vaccines","url":"https://pubmed.ncbi.nlm.nih.gov/31936739","citation_count":4,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["PFN","profilin"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"29236299","id":"PMC_29236299","title":"Potential role of salt-bridges in the hinge-like movement of apicomplexa specific β-hairpin of Plasmodium and Toxoplasma profilins: A molecular dynamics simulation study.","date":"2018","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29236299","citation_count":3,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["profilin"],"competing_organism_flag":true,"competing_organism_match":"Toxoplasma","competing_organism_only":true},{"pmid":"41581407","id":"PMC_41581407","title":"Discovery of parasite proteins in the urine of vivax malaria patients through mass spectrometry.","date":"2026","source":"Diagnostic microbiology and infectious disease","url":"https://pubmed.ncbi.nlm.nih.gov/41581407","citation_count":1,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["profilin"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"42305886","id":"PMC_42305886","title":"Biological activity of Juglans regia leaf extract and in silico evaluation of its phytochemicals against Plasmodium berghei target proteins.","date":"2026","source":"Helminthologia","url":"https://pubmed.ncbi.nlm.nih.gov/42305886","citation_count":0,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["profilin"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9748,"output_tokens":2577,"usd":0.03395,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7947,"output_tokens":3564,"usd":0.064418,"stage2_stop_reason":"end_turn"},"total_usd":0.098368,"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\": 2008,\n      \"finding\": \"Crystal structure of P. falciparum PFN was solved alone and in complex with a polyproline ligand peptide, revealing a unique minidomain consisting of a large beta-hairpin extension common to all apicomplexan parasites and an acidic loop specific to Plasmodium species. Reverse genetics in P. berghei demonstrated PFN is essential for invasive blood stages.\",\n      \"method\": \"X-ray crystallography (crystal structure of PFN alone and with polyproline peptide); reverse genetics (P. berghei knockout)\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation by reverse genetics, two orthogonal methods in one study\",\n      \"pmids\": [\"19000816\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"P. falciparum PFN alone sequesters actin monomers, thereby inhibiting polymerization; however, its monomer-sequestering activity does not compete with the nucleating activity of formin 1 at an equimolar profilin-actin ratio.\",\n      \"method\": \"In vitro actin polymerization assay with recombinant P. falciparum PFN and formin 1 FH2 domain\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro biochemical assay, single lab, single method reported in abstract\",\n      \"pmids\": [\"22428073\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Tetracycline-induced conditional knockdown of P. berghei PFN using an ApiAP2-based transactivator system resulted in a dramatic reduction in parasite viability, establishing PFN as essential.\",\n      \"method\": \"Conditional knockdown (TetR-aptamer regulatable system) in P. berghei blood stages; viability assay\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean conditional KO with defined viability phenotype, single lab\",\n      \"pmids\": [\"23245327\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Mature P. falciparum subtilisin-like protease 3 (PfSUB3) proteolysed recombinant P. falciparum PFN in a dose-dependent manner in vitro, identifying PFN as a substrate of PfSUB3. Additionally, recombinant PFN was found to induce TNF-α and IL-12 production from mouse bone marrow-derived dendritic cells, indicating a proinflammatory activity.\",\n      \"method\": \"In vitro protease activity assay (recombinant PfSUB3 + recombinant PFN); cytokine induction assay with mouse dendritic cells\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro enzymatic assay with recombinant proteins, single lab, no mutagenesis validation\",\n      \"pmids\": [\"24080030\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"P. falciparum PFN is a small globular protein that consists of two independently folding units (foldons), with binding sites for different protein ligands segregated in the two halves, suggesting evolution via an ancient gene fusion event. A domain-swapped mutant and half-profilins that can reassemble into full PFN were engineered to demonstrate this.\",\n      \"method\": \"Domain-swapped mutagenesis; expression of separate half-profilin fragments and reassembly assay; structural/biophysical characterization\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — mutagenesis and reconstitution, single lab, mechanistic inference from domain-swapped construct\",\n      \"pmids\": [\"26012696\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Overexpression of P. berghei PFN in sporozoites (but not in merozoites or ookinetes) impaired circular gliding motility and salivary gland invasion, while strong overexpression caused complete life cycle arrest. Overexpression correlated with altered actin accumulation at the parasite tip.\",\n      \"method\": \"Transgenic overexpression with stage-specific promoters in P. berghei; gliding motility assays; fluorescence imaging of actin at parasite tip\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transgenic overexpression with defined motility and invasion phenotypes, multiple stages examined, single lab\",\n      \"pmids\": [\"27226484\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The apicomplexan-specific beta-hairpin motif of PFN is critically involved in actin binding. Mutations in this motif impaired gliding motility of P. berghei sporozoites. Force measurements using laser tweezers on migrating sporozoites, combined with molecular dynamics simulations, showed that the unique PFN-actin interface fine-tunes gliding motility speed.\",\n      \"method\": \"Site-directed mutagenesis of PFN beta-hairpin; P. berghei sporozoite gliding motility assay; laser tweezer force measurements; molecular dynamics simulations\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis validated in vivo motility phenotype, force measurements, and molecular dynamics; multiple orthogonal methods, single lab\",\n      \"pmids\": [\"28552953\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Mutations in PFN that do not affect actin binding in vitro still reduce force generation during P. berghei sporozoite migration and increase retrograde flow, demonstrating a function of PFN in force generation that is independent of its actin-binding activity.\",\n      \"method\": \"Site-directed mutagenesis of PFN (non-actin-binding mutants); laser tweezer force measurements on migrating P. berghei sporozoites; in vitro actin binding assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis combined with in vitro binding assays and biophysical force measurements, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"32034083\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"},\n        {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Resistance mutations in P. falciparum PFN (N154Y, K124N) and actin-1 (M356L) were identified after selection with compound MMV020291. CRISPR-Cas9 engineering of these mutations into wild-type parasites conferred resistance. In vitro cosedimentation assays with recombinant P. falciparum proteins showed that potent MMV020291 analogues disrupt filamentous actin formation in the presence of PFN, establishing PFN as part of the drug target mechanism. The compound also inhibited actin-1-dependent apicoplast segregation.\",\n      \"method\": \"Whole genome sequencing of resistant lines; CRISPR-Cas9 engineering of resistance mutations; in vitro actin cosedimentation assay with recombinant PFN and actin-1; apicoplast segregation assay\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — CRISPR-validated resistance mutations plus in vitro biochemical reconstitution, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"37053271\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}\n      ]\n    }\n  ],\n  \"current_model\": \"P. falciparum/P. berghei PFN is an essential actin monomer-sequestering protein with a structurally unique apicomplexan-specific beta-hairpin motif that mediates actin binding and fine-tunes gliding motility; it functions not only by sequestering actin monomers (inhibiting spontaneous polymerization) but also contributes to force generation during sporozoite migration independently of its actin-binding activity, is proteolytically cleaved by PfSUB3, and its interaction with actin-1 is a target for antimalarial compounds that disrupt filamentous actin formation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PF3D7_0932200 (PFN) is an essential actin-regulatory protein of malaria parasites that governs actin dynamics underlying invasive-stage motility [#0, #2]. It sequesters actin monomers to inhibit spontaneous polymerization, yet at equimolar ratios does not compete with the actin-nucleating activity of formin 1, allowing controlled filament assembly [#1]. Structurally, PFN carries an apicomplexan-specific minidomain — a large beta-hairpin extension plus a Plasmodium-specific acidic loop — and folds as two independent units whose distinct halves segregate binding sites for different ligands [#0, #4]. The beta-hairpin motif forms a unique PFN–actin interface that is critical for actin binding and fine-tunes gliding-motility speed of P. berghei sporozoites [#6]; beyond actin sequestration, PFN also contributes to force generation during sporozoite migration through a function that is independent of its actin-binding activity, since non-actin-binding mutants still reduce force and increase retrograde flow [#7]. Overexpression in P. berghei sporozoites specifically disrupts circular gliding, salivary gland invasion, and tip actin accumulation, underscoring stage-specific dosage sensitivity [#5]. PFN is proteolysed in vitro by the subtilisin-like protease PfSUB3 [#3], and its interaction with actin-1 is the target of the antimalarial compound MMV020291, whose analogues disrupt filamentous actin formation and whose resistance mutations map to PFN and actin-1 [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Establishing that PFN adopts a distinctive apicomplexan-specific fold and is genetically required answered whether it is a viable, parasite-selective target rather than a generic profilin.\",\n      \"evidence\": \"X-ray crystallography of PFN alone and with a polyproline peptide plus P. berghei reverse-genetics knockout\",\n      \"pmids\": [\"19000816\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the PFN-actin complex structure\", \"Essentiality shown only for invasive blood stages\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Biochemical reconstitution defined PFN's core activity as actin-monomer sequestration and showed it does not antagonize formin 1 nucleation, framing how PFN can both restrain and permit polymerization.\",\n      \"evidence\": \"In vitro actin polymerization assays with recombinant PFN and the formin 1 FH2 domain\",\n      \"pmids\": [\"22428073\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single in vitro method\", \"Interplay quantified only at equimolar ratio\", \"No cellular validation\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Conditional knockdown confirmed PFN essentiality in blood stages with a defined loss-of-viability phenotype, moving beyond gene-disruption inference.\",\n      \"evidence\": \"TetR-aptamer conditional knockdown in P. berghei blood stages with viability assay\",\n      \"pmids\": [\"23245327\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of lethality not dissected\", \"Single lab/system\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identifying PFN as a PfSUB3 substrate and detecting proinflammatory cytokine induction raised the possibility of regulatory processing and immune interactions beyond its cytoskeletal role.\",\n      \"evidence\": \"In vitro protease assay (recombinant PfSUB3 + PFN); cytokine induction in mouse bone marrow-derived dendritic cells\",\n      \"pmids\": [\"24080030\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vivo cleavage or mutagenesis of the cleavage site\", \"Cytokine activity not linked to a parasite phenotype\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrating that PFN is built from two independently folding units with segregated ligand-binding sites explained its architecture as an apparent ancient gene fusion and mapped functional halves.\",\n      \"evidence\": \"Domain-swapped mutagenesis and reassembly of half-profilin fragments with biophysical characterization\",\n      \"pmids\": [\"26012696\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological relevance of the fusion architecture untested in parasites\", \"Specific ligands of each half not fully enumerated\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Stage-specific overexpression revealed PFN dosage is critical selectively in sporozoites, linking PFN levels to gliding, salivary gland invasion, and tip actin organization.\",\n      \"evidence\": \"Transgenic stage-specific overexpression in P. berghei with gliding and invasion assays and tip actin imaging\",\n      \"pmids\": [\"27226484\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Why sporozoites are uniquely sensitive unexplained\", \"Overexpression is not a physiological perturbation\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Mutating the apicomplexan-specific beta-hairpin established this motif as the actin-binding determinant that fine-tunes gliding speed, connecting a unique structural feature to motor mechanics.\",\n      \"evidence\": \"Beta-hairpin mutagenesis, sporozoite gliding assays, laser-tweezer force measurements, and molecular dynamics in P. berghei\",\n      \"pmids\": [\"28552953\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of the acidic loop not isolated\", \"Link to specific downstream motor components unresolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showing that non-actin-binding PFN mutants still reduce force and increase retrograde flow uncoupled PFN's force-generation role from its canonical actin-binding activity.\",\n      \"evidence\": \"Non-actin-binding PFN mutants tested by laser-tweezer force measurements on migrating P. berghei sporozoites plus in vitro binding assays\",\n      \"pmids\": [\"32034083\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of the actin-binding-independent function unknown\", \"Partner mediating force generation not identified\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}, {\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Mapping MMV020291 resistance to PFN and actin-1 and showing analogues disrupt F-actin formation in the presence of PFN validated the PFN–actin-1 interface as an antimalarial target.\",\n      \"evidence\": \"WGS of resistant lines, CRISPR-Cas9 mutation engineering, in vitro cosedimentation with recombinant PFN and actin-1, and apicoplast segregation assays in P. falciparum\",\n      \"pmids\": [\"37053271\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct compound-PFN binding not structurally resolved\", \"Whether PFN or actin-1 is the primary drug-contact site unclear\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular basis of PFN's actin-binding-independent force-generation function and its structural interface with actin-1 during drug engagement remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of the PFN-actin-1 complex\", \"Physiological role of PfSUB3 cleavage of PFN unknown\", \"Mechanism coupling PFN to force generation not defined\"],\n      \"attribution\": []\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003785\", \"supporting_discovery_ids\": [1, 6, 8]},\n      {\"term_id\": \"GO:0042989\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0071973\", \"supporting_discovery_ids\": [5, 6, 7]},\n      {\"term_id\": \"GO:0030041\", \"supporting_discovery_ids\": [1, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"actin-1 (PfACT1)\",\n      \"formin 1 (FH2 domain)\",\n      \"PfSUB3 (subtilisin-like protease 3)\"\n    ],\n    \"other_free_text\": [\n      \"Contains an apicomplexan-specific beta-hairpin motif plus a Plasmodium-specific acidic loop that form a unique actin-binding interface [#0, #6]\",\n      \"Folds as two independent units with segregated ligand-binding sites, consistent with an ancient gene fusion [#4]\",\n      \"Has an actin-binding-independent role in force generation during sporozoite migration [#7]\",\n      \"Target of the antimalarial compound MMV020291 via the PFN-actin-1 interaction; resistance mutations N154Y and K124N in PFN [#8]\",\n      \"Recombinant PFN induces TNF-alpha and IL-12 from mouse dendritic cells [#3]\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0932200","name":null,"product":"profilin","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_0833000","name":null,"product":"profilin","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_0730900","name":null,"product":"profilin, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"PFN","evaluation":null}