{"gene":"TG_242720","gene_symbol":"ASP5","run_date":"2026-09-08T12:58:25","timeline":{"discoveries":[{"year":2015,"finding":"ASP5 is a Golgi-resident aspartyl protease that cleaves GRA16, GRA19, and GRA20 at their PEXEL-like (TEXEL) motifs; in the absence of ASP5, these GRAs fail to localize correctly to the parasitophorous vacuole membrane and GRA16/GRA24 are retained within the vacuolar space rather than reaching the host nucleus.","method":"Genetic deletion (Δasp5), epitope-tagging, immunofluorescence localization, and in vitro cleavage assays","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal epitope-tag experiments, genetic KO with multiple defined cellular phenotypes, replicated across two independent papers (PMID:26473595 and PMID:26270241)","pmids":["26473595"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"TgASP5 directly mediates proteolytic cleavage of GRA16 at its N-terminal PEXEL-like motif (RRLAE), and this cleavage is required for GRA16 export into the host cell; GRA24 export is also ASP5-dependent but its maturation/cleavage is ASP5-independent, indicating ASP5 has multiple export-related functions.","method":"N- and C-terminal epitope tagging to detect proteolysis, genetic deletion of TgASP5, western blot analysis of GRA16 processing","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — direct cleavage demonstrated with dual-tag proteolysis assay, confirmed in genetic KO; replicated in same timeframe by independent labs","pmids":["26270241"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"ASP5 cleaves a highly constrained PEXEL-like motif (TEXEL, RRL↓ consensus) at both N- and C-terminal ends of substrate proteins; ASP5 is Golgi-resident and also controls trafficking of effectors that lack this motif. ASP5 is required for establishment of the intravacuolar nanotubular network and for recruitment of host mitochondria to the vacuole.","method":"Genetic deletion (Δasp5), mass spectrometry of cleavage products, immunofluorescence, electron microscopy, host transcriptome analysis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (MS cleavage mapping, genetic KO, EM, host RNA-seq) in single rigorous study; consistent with two contemporaneous independent labs","pmids":["26576949"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"MYR1 requires TgASP5 for its proteolytic cleavage into two stable fragments, both of which localize to the parasitophorous vacuole and the PV membrane; MYR1 is essential for GRA16 and GRA24 translocation into the host cell.","method":"Whole-genome sequencing of mutants, genetic deletion, western blot for cleavage products, immunofluorescence localization","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with defined molecular phenotype (cleavage and localization), multiple effector translocation assays, independent replication in subsequent papers","pmids":["26838724"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2018,"finding":"ASP5 substrates identified by TAILS N-terminomics are exclusively dense granule proteins; validated substrates reside within the PV or PVM (not exported to the host cytosol), including two kinases (WNG1, WNG2) and one phosphatase, and are not processed in Δasp5 parasites or when the RRL motif is mutated to ARL.","method":"Terminal Amine Isotopic Labeling of Substrates (TAILS) N-terminomics comparing WT and Δasp5, site-directed mutagenesis (RRL→ARL), immunofluorescence, mouse virulence model","journal":"mBio","confidence":"High","confidence_rationale":"Tier 1 / Moderate — quantitative proteomics with mutagenesis validation, single lab but two enrichment techniques and rigorous controls","pmids":["30377279"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2018,"finding":"RNA-Seq of host cells infected with Δasp5 versus Δmyr1 tachyzoites shows that MYR1 function does not require ASP5 cleavage, indicating that a subset of effectors require only MYR1 (not ASP5) for export; ASP5 and MYR1 thus define partially overlapping but distinct export requirements.","method":"RNA-Seq transcriptome comparison of RH-WT, RHΔmyr1, and RHΔasp5 infections of human foreskin fibroblasts","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — defined pathway distinction (ASP5-independent MYR1 function) from transcriptomics, single lab, indirect readout of translocation","pmids":["29615509"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2018,"finding":"MYR1, which is cleaved by ASP5 into two disulfide-linked fragments, localizes to the PVM and is part of a novel protein complex with MYR2 and MYR3 required for GRA effector translocation; MYR3 but not MYR2 stably associates with MYR1, and all three are membrane-associated.","method":"Co-immunoprecipitation, immunofluorescence, western blot (disulfide linkage), genetic deletion, GRA16/GRA24 nuclear trafficking assays","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, localization, and functional translocation assays in single lab; ASP5 cleavage of MYR1 confirmed orthogonally","pmids":["29357375"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2020,"finding":"ASP5 is required for translocation of GRA16 into host cells and for upregulation of host c-Myc and cyclin E1; GRA44 and GRA45, two MYR1-interacting proteins that contain ASP5 processing motifs, are essential for effector translocation, although ASP5 processing at these sites appears nonessential for their translocation function.","method":"Affinity purification/Co-IP of MYR1, genetic deletion, GRA16 nuclear translocation assay, c-Myc and cyclin E1 upregulation assays","journal":"mSphere","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP combined with genetic KO and functional translocation readout, single lab","pmids":["32075880"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2022,"finding":"In bradyzoites, ASP5 cleaves novel substrates identified by TAILS N-terminomics; Δasp5 bradyzoites are impaired in cyst wall formation in vitro and show significantly reduced cyst number and size in chronically infected mouse brains, establishing ASP5 as required for the establishment of the chronic infection niche.","method":"N-terminal amine isotopic labeling of substrates (TAILS) comparing WT and Δasp5 tachyzoites and bradyzoites, immunofluorescence, two-photon serial tomography of infected mouse brains","journal":"Molecular microbiology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — quantitative proteomics plus in vivo imaging with defined phenotype, single lab but multiple orthogonal methods","pmids":["36210525"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"finding":"Computational structural modeling of TgASP5 reveals a pepsin-like aspartyl protease active site; residues Ser505, Ala776, and Tyr689 in the S2 binding pocket confer specificity for Arg at the P2 position of the TEXEL substrate; the known inhibitor RRLStatine blocks the active site by forming a hydrogen bond with a catalytic aspartate.","method":"Molecular modeling, all-atom MD simulation, molecular docking, MM-PBSA binding energy calculations","journal":"Journal of biomolecular structure & dynamics","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational prediction only, no experimental structural or mutagenesis validation reported in the abstract","pmids":["38424737"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"finding":"GRA84 export to the host nucleus is dependent on TgASP5; GRA84 undergoes a separate N-terminal processing event (removing >90 amino acids) that is ASP5-independent, and mutagenesis shows this maturation is critical for transit across the vacuolar membrane via the MYR translocon.","method":"Genetic deletion of ASP5, site-directed mutagenesis of processing sites, epitope tagging, immunofluorescence localization","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — genetic KO plus mutagenesis with defined localization phenotype, single lab, single paper","pmids":["40464784"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"Deletion of ASP5 or MYR1 does not alter the inhibition of nitric oxide production in activated J774-A1 or RAW 264.7 macrophages infected with T. gondii, indicating this immune evasion phenotype is ASP5/MYR1-independent.","method":"Genetic deletion of ASP5 and MYR1, measurement of NO production and iNOS expression in infected macrophage lines","journal":"Frontiers in microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — negative result established by direct genetic KO with defined phenotypic readout, single lab","pmids":["30177926"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgASP5 is a Golgi-resident pepsin-like aspartyl protease that cleaves a constrained PEXEL-like motif (TEXEL, consensus RRL) in a defined set of dense granule proteins (including GRA16, GRA19, GRA20, MYR1, GRA44, GRA45, WNG1, WNG2, and others), thereby maturing these substrates for correct localization to or across the parasitophorous vacuole membrane; this processing is a prerequisite for assembly of the MYR1/2/3 translocon complex and for translocation of effectors such as GRA16 and GRA24 into the host cell nucleus, and ASP5 activity is additionally required for nanotubular network integrity, cyst wall formation in bradyzoites, and full virulence in vivo."},"narrative":{"mechanistic_narrative":"TgASP5 is a Golgi-resident pepsin-like aspartyl protease that matures a defined set of dense granule effector proteins by cleaving a constrained PEXEL-like motif (TEXEL, RRL consensus), thereby licensing effector trafficking to and across the parasitophorous vacuole membrane (PVM) and driving remodeling of the host cell [PMID:26473595, PMID:26576949]. ASP5 cleaves GRA16, GRA19, and GRA20 at their TEXEL motifs, and this processing is required for their correct PVM localization and for delivery of effectors such as GRA16 and GRA24 into the host nucleus [PMID:26473595, PMID:26270241]. N-terminomic profiling establishes that ASP5 substrates are exclusively dense granule proteins residing within the PV/PVM — including the kinases WNG1 and WNG2 — and that cleavage strictly depends on the RRL motif, since its mutation to ARL abolishes processing [PMID:30377279]. ASP5 additionally proteolytically activates the translocon component MYR1, cleaving it into two stable, disulfide-linked fragments that assemble with MYR2 and MYR3 into a PVM-associated complex required for GRA16/GRA24 nuclear translocation and for downstream host responses including c-Myc and cyclin E1 upregulation [PMID:26838724, PMID:29357375, PMID:32075880]. Beyond effector export, ASP5 is required for intravacuolar nanotubular network integrity and host mitochondrial recruitment [PMID:26576949], and for cyst wall formation and establishment of the chronic bradyzoite niche in vivo [PMID:36210525]. ASP5 acts through both cleavage-dependent and cleavage-independent routes: GRA24 export and GRA44/GRA45 translocation function require ASP5 without requiring processing at their own motifs, and GRA84 export depends on ASP5 despite an ASP5-independent maturation event [PMID:26270241, PMID:32075880, PMID:40464784].","teleology":[{"year":2015,"claim":"Established ASP5 as the Golgi protease that matures dense granule effectors, answering how PEXEL-like GRAs reach the PVM and the host nucleus.","evidence":"Genetic deletion, epitope-tagging, immunofluorescence, in vitro cleavage assays and MS cleavage mapping in T. gondii","pmids":["26473595","26270241","26576949"],"confidence":"High","gaps":["Direct enzymology of the recombinant protease not resolved","Mechanism of ASP5 control over motif-lacking effectors (e.g. GRA24) unexplained"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"Distinguished cleavage-dependent from cleavage-independent export, showing GRA16 requires ASP5 cleavage for export whereas GRA24 export is ASP5-dependent but processing-independent.","evidence":"Dual N-/C-terminal epitope tagging proteolysis assay with genetic ASP5 deletion in T. gondii","pmids":["26270241"],"confidence":"High","gaps":["Molecular basis of ASP5's cleavage-independent role in GRA24 export undefined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"Connected ASP5 to the export machinery by showing it cleaves MYR1 into two PVM-localized fragments essential for GRA16/GRA24 translocation.","evidence":"Whole-genome sequencing of mutants, genetic deletion, western blot and immunofluorescence in T. gondii RH","pmids":["26838724"],"confidence":"High","gaps":["Whether MYR1 cleavage per se (vs. simple presence) is required for translocation not fully resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2018,"claim":"Defined the ASP5 substrate repertoire as exclusively PV/PVM-resident dense granule proteins and confirmed strict RRL-motif dependence, refining the enzyme's specificity.","evidence":"TAILS N-terminomics comparing WT and Δasp5 with RRL→ARL mutagenesis and mouse virulence model in T. gondii RH","pmids":["30377279"],"confidence":"High","gaps":["Functional consequences of cleaving each individual substrate not dissected","Role of the WNG kinases downstream of cleavage undefined"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2018,"claim":"Resolved MYR1 as the core of a MYR1/MYR2/MYR3 PVM translocon and delineated overlapping-but-distinct ASP5 versus MYR1 export requirements.","evidence":"Co-IP, immunofluorescence, disulfide-linkage westerns, genetic deletion and translocation assays plus host RNA-Seq in T. gondii RH","pmids":["29357375","29615509"],"confidence":"High","gaps":["Stoichiometry and transport mechanism of the translocon unknown","How ASP5-independent MYR1 substrates traverse the PVM unclear"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2020,"claim":"Linked ASP5-dependent export to host cell cycle reprogramming and identified GRA44/GRA45 as essential translocon-associated factors whose ASP5 processing is dispensable for function.","evidence":"MYR1 affinity purification/Co-IP, genetic deletion, GRA16 nuclear translocation and c-Myc/cyclin E1 assays in T. gondii RH","pmids":["32075880"],"confidence":"Medium","gaps":["Direct role of GRA44/GRA45 within the translocon undefined","Why processing sites exist if processing is nonessential unexplained"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2022,"claim":"Extended ASP5 function to the chronic stage, showing it cleaves bradyzoite-specific substrates and is required for cyst wall formation and brain cyst establishment.","evidence":"TAILS N-terminomics of tachyzoites and bradyzoites, immunofluorescence and two-photon serial tomography of infected mouse brains in T. gondii","pmids":["36210525"],"confidence":"High","gaps":["Identity and mechanism of the specific bradyzoite substrates driving cyst wall assembly not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"claim":"Proposed a structural basis for TEXEL specificity, modeling a pepsin-like active site and S2 pocket residues (Ser505, Ala776, Tyr689) that select for P2 Arg.","evidence":"Molecular modeling, MD simulation, docking and MM-PBSA calculations (computational only)","pmids":["38424737"],"confidence":"Low","gaps":["No experimental structure or mutagenesis validation of the predicted specificity residues","Inhibitor binding mode inferred only from docking"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"claim":"Showed that ASP5-dependent export can be layered with a separate ASP5-independent maturation step, using GRA84 whose N-terminal processing is required for MYR-mediated PVM transit.","evidence":"Genetic ASP5 deletion, site-directed mutagenesis of processing sites and immunofluorescence localization in T. gondii","pmids":["40464784"],"confidence":"Medium","gaps":["Identity of the protease performing the ASP5-independent GRA84 cleavage unknown","Generality of dual-processing across other effectors untested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"How ASP5 governs export of substrates lacking a TEXEL motif, and the mechanistic link between substrate cleavage and translocon assembly, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No experimental structure of ASP5","Mechanism coupling cleavage to MYR translocon function undefined","Cleavage-independent roles of ASP5 in effector export mechanistically unexplained"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["MYR1/MYR2/MYR3 PVM translocon (ASP5 cleaves MYR1; MYR3 stably associates with MYR1)"],"partners":["MYR1 (SUBSTRATE; ASP5-CLEAVED)","MYR2","MYR3","GRA16 (SUBSTRATE)","GRA19 (SUBSTRATE)","GRA20 (SUBSTRATE)","GRA24 (ASP5-DEPENDENT EXPORT, CLEAVAGE-INDEPENDENT)","GRA44 (MYR1-INTERACTING, ASP5 PROCESSING NONESSENTIAL)","GRA45 (MYR1-INTERACTING, ASP5 PROCESSING NONESSENTIAL)","GRA84 (ASP5-DEPENDENT EXPORT)","WNG1 (SUBSTRATE KINASE)","WNG2 (SUBSTRATE KINASE)"],"other_free_text":["Cleaves a constrained TEXEL/PEXEL-like motif with RRL consensus; RRL→ARL mutation abolishes processing (#4).","Required for intravacuolar nanotubular network integrity and host mitochondrial recruitment to the vacuole (#2).","Required for bradyzoite cyst wall formation and establishment of chronic brain cysts in vivo (#8).","ASP5-dependent effector export drives host c-Myc and cyclin E1 upregulation (#7).","Inhibition of macrophage nitric oxide production is ASP5/MYR1-independent (#11).","Computationally predicted S2-pocket specificity residues Ser505, Ala776, Tyr689 and RRLStatine inhibitor binding (Low confidence, #9)."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"26838724","id":"PMC_26838724","title":"A Novel Secreted Protein, MYR1, Is Central to Toxoplasma's Manipulation of Host Cells.","date":"2016","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/26838724","citation_count":133,"is_preprint":false},{"pmid":"26473595","id":"PMC_26473595","title":"Fundamental Roles of the Golgi-Associated Toxoplasma Aspartyl Protease, ASP5, at the Host-Parasite Interface.","date":"2015","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/26473595","citation_count":95,"is_preprint":false},{"pmid":"26576949","id":"PMC_26576949","title":"An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell.","date":"2015","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/26576949","citation_count":91,"is_preprint":false},{"pmid":"26270241","id":"PMC_26270241","title":"The aspartyl protease TgASP5 mediates the export of the Toxoplasma GRA16 and GRA24 effectors into host cells.","date":"2015","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/26270241","citation_count":84,"is_preprint":false},{"pmid":"29357375","id":"PMC_29357375","title":"Identification of a novel protein complex essential for effector translocation across the parasitophorous vacuole membrane of Toxoplasma gondii.","date":"2018","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/29357375","citation_count":75,"is_preprint":false},{"pmid":"32075880","id":"PMC_32075880","title":"Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells.","date":"2020","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/32075880","citation_count":53,"is_preprint":false},{"pmid":"30377279","id":"PMC_30377279","title":"Aspartyl Protease 5 Matures Dense Granule Proteins That Reside at the Host-Parasite Interface in Toxoplasma gondii.","date":"2018","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/30377279","citation_count":45,"is_preprint":false},{"pmid":"29615509","id":"PMC_29615509","title":"MYR1-Dependent Effectors Are the Major Drivers of a Host Cell's Early Response to Toxoplasma, Including Counteracting MYR1-Independent Effects.","date":"2018","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/29615509","citation_count":44,"is_preprint":false},{"pmid":"30177926","id":"PMC_30177926","title":"Inhibition of Nitric Oxide Production in Activated Macrophages Caused by Toxoplasma gondii Infection Occurs by Distinct Mechanisms in Different Mouse Macrophage Cell Lines.","date":"2018","source":"Frontiers in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/30177926","citation_count":26,"is_preprint":false},{"pmid":"32853276","id":"PMC_32853276","title":"Naïve CD8 T cell IFNγ responses to a vacuolar antigen are regulated by an inflammasome-independent NLRP3 pathway and Toxoplasma gondii ROP5.","date":"2020","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/32853276","citation_count":16,"is_preprint":false},{"pmid":"36210525","id":"PMC_36210525","title":"Importance of aspartyl protease 5 in the establishment of the intracellular niche during acute and chronic infection of Toxoplasma gondii.","date":"2022","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/36210525","citation_count":6,"is_preprint":false},{"pmid":"40464784","id":"PMC_40464784","title":"Proteolytic processing and host nuclear targeting of the novel effector GRA84 in Toxoplasma gondii.","date":"2025","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/40464784","citation_count":1,"is_preprint":false},{"pmid":"42288483","id":"PMC_42288483","title":"β-catenin-driven innate and metabolic reprograming in macrophages fuel T-cell-dependent inflammation in Toxoplasma gondii infection: implications for therapeutic intervention.","date":"2026","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/42288483","citation_count":0,"is_preprint":false},{"pmid":"38424737","id":"PMC_38424737","title":"Toxoplasma gondii aspartic protease 5: structural basis of substrate binding and inhibition mechanism.","date":"2024","source":"Journal of biomolecular structure & dynamics","url":"https://pubmed.ncbi.nlm.nih.gov/38424737","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10525,"output_tokens":3402,"usd":0.041302,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9284,"output_tokens":4202,"usd":0.075735,"stage2_stop_reason":"end_turn"},"total_usd":0.117037,"stage1_batch_id":"msgbatch_01TEPNZ4MCzJ7xtsNwosiDRk","stage2_batch_id":"msgbatch_01FQShM7n1sfCr4pWTDShmki","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2015,\n      \"finding\": \"ASP5 is a Golgi-resident aspartyl protease that cleaves GRA16, GRA19, and GRA20 at their PEXEL-like (TEXEL) motifs; in the absence of ASP5, these GRAs fail to localize correctly to the parasitophorous vacuole membrane and GRA16/GRA24 are retained within the vacuolar space rather than reaching the host nucleus.\",\n      \"method\": \"Genetic deletion (Δasp5), epitope-tagging, immunofluorescence localization, and in vitro cleavage assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal epitope-tag experiments, genetic KO with multiple defined cellular phenotypes, replicated across two independent papers (PMID:26473595 and PMID:26270241)\",\n      \"pmids\": [\"26473595\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TgASP5 directly mediates proteolytic cleavage of GRA16 at its N-terminal PEXEL-like motif (RRLAE), and this cleavage is required for GRA16 export into the host cell; GRA24 export is also ASP5-dependent but its maturation/cleavage is ASP5-independent, indicating ASP5 has multiple export-related functions.\",\n      \"method\": \"N- and C-terminal epitope tagging to detect proteolysis, genetic deletion of TgASP5, western blot analysis of GRA16 processing\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — direct cleavage demonstrated with dual-tag proteolysis assay, confirmed in genetic KO; replicated in same timeframe by independent labs\",\n      \"pmids\": [\"26270241\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ASP5 cleaves a highly constrained PEXEL-like motif (TEXEL, RRL↓ consensus) at both N- and C-terminal ends of substrate proteins; ASP5 is Golgi-resident and also controls trafficking of effectors that lack this motif. ASP5 is required for establishment of the intravacuolar nanotubular network and for recruitment of host mitochondria to the vacuole.\",\n      \"method\": \"Genetic deletion (Δasp5), mass spectrometry of cleavage products, immunofluorescence, electron microscopy, host transcriptome analysis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (MS cleavage mapping, genetic KO, EM, host RNA-seq) in single rigorous study; consistent with two contemporaneous independent labs\",\n      \"pmids\": [\"26576949\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MYR1 requires TgASP5 for its proteolytic cleavage into two stable fragments, both of which localize to the parasitophorous vacuole and the PV membrane; MYR1 is essential for GRA16 and GRA24 translocation into the host cell.\",\n      \"method\": \"Whole-genome sequencing of mutants, genetic deletion, western blot for cleavage products, immunofluorescence localization\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with defined molecular phenotype (cleavage and localization), multiple effector translocation assays, independent replication in subsequent papers\",\n      \"pmids\": [\"26838724\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ASP5 substrates identified by TAILS N-terminomics are exclusively dense granule proteins; validated substrates reside within the PV or PVM (not exported to the host cytosol), including two kinases (WNG1, WNG2) and one phosphatase, and are not processed in Δasp5 parasites or when the RRL motif is mutated to ARL.\",\n      \"method\": \"Terminal Amine Isotopic Labeling of Substrates (TAILS) N-terminomics comparing WT and Δasp5, site-directed mutagenesis (RRL→ARL), immunofluorescence, mouse virulence model\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative proteomics with mutagenesis validation, single lab but two enrichment techniques and rigorous controls\",\n      \"pmids\": [\"30377279\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RNA-Seq of host cells infected with Δasp5 versus Δmyr1 tachyzoites shows that MYR1 function does not require ASP5 cleavage, indicating that a subset of effectors require only MYR1 (not ASP5) for export; ASP5 and MYR1 thus define partially overlapping but distinct export requirements.\",\n      \"method\": \"RNA-Seq transcriptome comparison of RH-WT, RHΔmyr1, and RHΔasp5 infections of human foreskin fibroblasts\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — defined pathway distinction (ASP5-independent MYR1 function) from transcriptomics, single lab, indirect readout of translocation\",\n      \"pmids\": [\"29615509\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MYR1, which is cleaved by ASP5 into two disulfide-linked fragments, localizes to the PVM and is part of a novel protein complex with MYR2 and MYR3 required for GRA effector translocation; MYR3 but not MYR2 stably associates with MYR1, and all three are membrane-associated.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, western blot (disulfide linkage), genetic deletion, GRA16/GRA24 nuclear trafficking assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, localization, and functional translocation assays in single lab; ASP5 cleavage of MYR1 confirmed orthogonally\",\n      \"pmids\": [\"29357375\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ASP5 is required for translocation of GRA16 into host cells and for upregulation of host c-Myc and cyclin E1; GRA44 and GRA45, two MYR1-interacting proteins that contain ASP5 processing motifs, are essential for effector translocation, although ASP5 processing at these sites appears nonessential for their translocation function.\",\n      \"method\": \"Affinity purification/Co-IP of MYR1, genetic deletion, GRA16 nuclear translocation assay, c-Myc and cyclin E1 upregulation assays\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP combined with genetic KO and functional translocation readout, single lab\",\n      \"pmids\": [\"32075880\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In bradyzoites, ASP5 cleaves novel substrates identified by TAILS N-terminomics; Δasp5 bradyzoites are impaired in cyst wall formation in vitro and show significantly reduced cyst number and size in chronically infected mouse brains, establishing ASP5 as required for the establishment of the chronic infection niche.\",\n      \"method\": \"N-terminal amine isotopic labeling of substrates (TAILS) comparing WT and Δasp5 tachyzoites and bradyzoites, immunofluorescence, two-photon serial tomography of infected mouse brains\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — quantitative proteomics plus in vivo imaging with defined phenotype, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"36210525\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Computational structural modeling of TgASP5 reveals a pepsin-like aspartyl protease active site; residues Ser505, Ala776, and Tyr689 in the S2 binding pocket confer specificity for Arg at the P2 position of the TEXEL substrate; the known inhibitor RRLStatine blocks the active site by forming a hydrogen bond with a catalytic aspartate.\",\n      \"method\": \"Molecular modeling, all-atom MD simulation, molecular docking, MM-PBSA binding energy calculations\",\n      \"journal\": \"Journal of biomolecular structure & dynamics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational prediction only, no experimental structural or mutagenesis validation reported in the abstract\",\n      \"pmids\": [\"38424737\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"GRA84 export to the host nucleus is dependent on TgASP5; GRA84 undergoes a separate N-terminal processing event (removing >90 amino acids) that is ASP5-independent, and mutagenesis shows this maturation is critical for transit across the vacuolar membrane via the MYR translocon.\",\n      \"method\": \"Genetic deletion of ASP5, site-directed mutagenesis of processing sites, epitope tagging, immunofluorescence localization\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — genetic KO plus mutagenesis with defined localization phenotype, single lab, single paper\",\n      \"pmids\": [\"40464784\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Deletion of ASP5 or MYR1 does not alter the inhibition of nitric oxide production in activated J774-A1 or RAW 264.7 macrophages infected with T. gondii, indicating this immune evasion phenotype is ASP5/MYR1-independent.\",\n      \"method\": \"Genetic deletion of ASP5 and MYR1, measurement of NO production and iNOS expression in infected macrophage lines\",\n      \"journal\": \"Frontiers in microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — negative result established by direct genetic KO with defined phenotypic readout, single lab\",\n      \"pmids\": [\"30177926\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgASP5 is a Golgi-resident pepsin-like aspartyl protease that cleaves a constrained PEXEL-like motif (TEXEL, consensus RRL) in a defined set of dense granule proteins (including GRA16, GRA19, GRA20, MYR1, GRA44, GRA45, WNG1, WNG2, and others), thereby maturing these substrates for correct localization to or across the parasitophorous vacuole membrane; this processing is a prerequisite for assembly of the MYR1/2/3 translocon complex and for translocation of effectors such as GRA16 and GRA24 into the host cell nucleus, and ASP5 activity is additionally required for nanotubular network integrity, cyst wall formation in bradyzoites, and full virulence in vivo.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TgASP5 is a Golgi-resident pepsin-like aspartyl protease that matures a defined set of dense granule effector proteins by cleaving a constrained PEXEL-like motif (TEXEL, RRL consensus), thereby licensing effector trafficking to and across the parasitophorous vacuole membrane (PVM) and driving remodeling of the host cell [#0, #2]. ASP5 cleaves GRA16, GRA19, and GRA20 at their TEXEL motifs, and this processing is required for their correct PVM localization and for delivery of effectors such as GRA16 and GRA24 into the host nucleus [#0, #1]. N-terminomic profiling establishes that ASP5 substrates are exclusively dense granule proteins residing within the PV/PVM — including the kinases WNG1 and WNG2 — and that cleavage strictly depends on the RRL motif, since its mutation to ARL abolishes processing [#4]. ASP5 additionally proteolytically activates the translocon component MYR1, cleaving it into two stable, disulfide-linked fragments that assemble with MYR2 and MYR3 into a PVM-associated complex required for GRA16/GRA24 nuclear translocation and for downstream host responses including c-Myc and cyclin E1 upregulation [#3, #6, #7]. Beyond effector export, ASP5 is required for intravacuolar nanotubular network integrity and host mitochondrial recruitment [#2], and for cyst wall formation and establishment of the chronic bradyzoite niche in vivo [#8]. ASP5 acts through both cleavage-dependent and cleavage-independent routes: GRA24 export and GRA44/GRA45 translocation function require ASP5 without requiring processing at their own motifs, and GRA84 export depends on ASP5 despite an ASP5-independent maturation event [#1, #7, #10].\",\n  \"teleology\": [\n    {\n      \"year\": 2015,\n      \"claim\": \"Established ASP5 as the Golgi protease that matures dense granule effectors, answering how PEXEL-like GRAs reach the PVM and the host nucleus.\",\n      \"evidence\": \"Genetic deletion, epitope-tagging, immunofluorescence, in vitro cleavage assays and MS cleavage mapping in T. gondii\",\n      \"pmids\": [\"26473595\", \"26270241\", \"26576949\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct enzymology of the recombinant protease not resolved\", \"Mechanism of ASP5 control over motif-lacking effectors (e.g. GRA24) unexplained\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Distinguished cleavage-dependent from cleavage-independent export, showing GRA16 requires ASP5 cleavage for export whereas GRA24 export is ASP5-dependent but processing-independent.\",\n      \"evidence\": \"Dual N-/C-terminal epitope tagging proteolysis assay with genetic ASP5 deletion in T. gondii\",\n      \"pmids\": [\"26270241\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of ASP5's cleavage-independent role in GRA24 export undefined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Connected ASP5 to the export machinery by showing it cleaves MYR1 into two PVM-localized fragments essential for GRA16/GRA24 translocation.\",\n      \"evidence\": \"Whole-genome sequencing of mutants, genetic deletion, western blot and immunofluorescence in T. gondii RH\",\n      \"pmids\": [\"26838724\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MYR1 cleavage per se (vs. simple presence) is required for translocation not fully resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined the ASP5 substrate repertoire as exclusively PV/PVM-resident dense granule proteins and confirmed strict RRL-motif dependence, refining the enzyme's specificity.\",\n      \"evidence\": \"TAILS N-terminomics comparing WT and Δasp5 with RRL→ARL mutagenesis and mouse virulence model in T. gondii RH\",\n      \"pmids\": [\"30377279\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequences of cleaving each individual substrate not dissected\", \"Role of the WNG kinases downstream of cleavage undefined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Resolved MYR1 as the core of a MYR1/MYR2/MYR3 PVM translocon and delineated overlapping-but-distinct ASP5 versus MYR1 export requirements.\",\n      \"evidence\": \"Co-IP, immunofluorescence, disulfide-linkage westerns, genetic deletion and translocation assays plus host RNA-Seq in T. gondii RH\",\n      \"pmids\": [\"29357375\", \"29615509\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and transport mechanism of the translocon unknown\", \"How ASP5-independent MYR1 substrates traverse the PVM unclear\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linked ASP5-dependent export to host cell cycle reprogramming and identified GRA44/GRA45 as essential translocon-associated factors whose ASP5 processing is dispensable for function.\",\n      \"evidence\": \"MYR1 affinity purification/Co-IP, genetic deletion, GRA16 nuclear translocation and c-Myc/cyclin E1 assays in T. gondii RH\",\n      \"pmids\": [\"32075880\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct role of GRA44/GRA45 within the translocon undefined\", \"Why processing sites exist if processing is nonessential unexplained\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended ASP5 function to the chronic stage, showing it cleaves bradyzoite-specific substrates and is required for cyst wall formation and brain cyst establishment.\",\n      \"evidence\": \"TAILS N-terminomics of tachyzoites and bradyzoites, immunofluorescence and two-photon serial tomography of infected mouse brains in T. gondii\",\n      \"pmids\": [\"36210525\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity and mechanism of the specific bradyzoite substrates driving cyst wall assembly not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Proposed a structural basis for TEXEL specificity, modeling a pepsin-like active site and S2 pocket residues (Ser505, Ala776, Tyr689) that select for P2 Arg.\",\n      \"evidence\": \"Molecular modeling, MD simulation, docking and MM-PBSA calculations (computational only)\",\n      \"pmids\": [\"38424737\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No experimental structure or mutagenesis validation of the predicted specificity residues\", \"Inhibitor binding mode inferred only from docking\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showed that ASP5-dependent export can be layered with a separate ASP5-independent maturation step, using GRA84 whose N-terminal processing is required for MYR-mediated PVM transit.\",\n      \"evidence\": \"Genetic ASP5 deletion, site-directed mutagenesis of processing sites and immunofluorescence localization in T. gondii\",\n      \"pmids\": [\"40464784\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the protease performing the ASP5-independent GRA84 cleavage unknown\", \"Generality of dual-processing across other effectors untested\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ASP5 governs export of substrates lacking a TEXEL motif, and the mechanistic link between substrate cleavage and translocon assembly, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experimental structure of ASP5\", \"Mechanism coupling cleavage to MYR translocon function undefined\", \"Cleavage-independent roles of ASP5 in effector export mechanistically unexplained\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004190\", \"supporting_discovery_ids\": [0, 1, 2, 4, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006508\", \"supporting_discovery_ids\": [0, 1, 3, 4, 8], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0051701\", \"supporting_discovery_ids\": [0, 1, 2, 7], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0009306\", \"supporting_discovery_ids\": [3, 6, 10], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]}\n    ],\n    \"complexes\": [\n      \"MYR1/MYR2/MYR3 PVM translocon (ASP5 cleaves MYR1; MYR3 stably associates with MYR1)\"\n    ],\n    \"partners\": [\n      \"MYR1 (substrate; ASP5-cleaved)\",\n      \"MYR2\",\n      \"MYR3\",\n      \"GRA16 (substrate)\",\n      \"GRA19 (substrate)\",\n      \"GRA20 (substrate)\",\n      \"GRA24 (ASP5-dependent export, cleavage-independent)\",\n      \"GRA44 (MYR1-interacting, ASP5 processing nonessential)\",\n      \"GRA45 (MYR1-interacting, ASP5 processing nonessential)\",\n      \"GRA84 (ASP5-dependent export)\",\n      \"WNG1 (substrate kinase)\",\n      \"WNG2 (substrate kinase)\"\n    ],\n    \"other_free_text\": [\n      \"Cleaves a constrained TEXEL/PEXEL-like motif with RRL consensus; RRL→ARL mutation abolishes processing (#4).\",\n      \"Required for intravacuolar nanotubular network integrity and host mitochondrial recruitment to the vacuole (#2).\",\n      \"Required for bradyzoite cyst wall formation and establishment of chronic brain cysts in vivo (#8).\",\n      \"ASP5-dependent effector export drives host c-Myc and cyclin E1 upregulation (#7).\",\n      \"Inhibition of macrophage nitric oxide production is ASP5/MYR1-independent (#11).\",\n      \"Computationally predicted S2-pocket specificity residues Ser505, Ala776, Tyr689 and RRLStatine inhibitor binding (Low confidence, #9).\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_242720","name":"ASP5","product":"aspartyl protease ASP5","uniprot_acc":"S8F6T7","chromosome":"TGME49_chrVI","start_pos":2205049},{"strain":"GT1","gene_id":"TGGT1_242720","name":null,"product":"aspartyl protease ASP5","uniprot_acc":null,"chromosome":"TGGT1_chrVI","start_pos":2116490},{"strain":"RH88","gene_id":"TGRH88_029500","name":null,"product":"aspartyl protease ASP5","uniprot_acc":null,"chromosome":"CM023087","start_pos":1511180},{"strain":"VEG","gene_id":"TGVEG_242720","name":null,"product":"aspartyl protease ASP5","uniprot_acc":"A0A0F7UST4","chromosome":"TGVEG_chrVI","start_pos":2208912}]}