{"gene":"TG_232960","gene_symbol":"PHYa","run_date":"2026-09-08T12:58:24","timeline":{"discoveries":[{"year":2012,"finding":"TgPhyA is required for prolyl hydroxylation of Toxoplasma Skp1; disruption of the TgphyA locus abolishes Skp1 glycosylation in parasites, and bacterially expressed TgPhyA prolyl-hydroxylates both Toxoplasma and Dictyostelium Skp1s in vitro using O2 and α-ketoglutarate as co-substrates.","method":"Genetic locus disruption (TgphyA knockout), in vitro prolyl hydroxylase assay with recombinant TgPhyA, kinetic analyses of O2 and α-ketoglutarate dependence, complementation in Dictyostelium phyA-null cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with recombinant enzyme, genetic knockout with defined biochemical phenotype, cross-species complementation, multiple orthogonal methods in one study","pmids":["22648409"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2012,"finding":"TgPhyA has a significantly higher apparent affinity for O2 compared to Dictyostelium DdPhyA, despite superimposable dependence on α-ketoglutarate concentration, suggesting that the O2-sensing threshold is tuned at the enzyme level.","method":"Kinetic analysis of recombinant TgPhyA vs DdPhyA with varying O2 and α-ketoglutarate concentrations","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — rigorous in vitro kinetics, but single lab, single paper","pmids":["22648409"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2012,"finding":"Parasites with disrupted TgphyA grow slowly at physiological O2 levels, establishing a functional role for PhyA-dependent Skp1 hydroxylation in parasite fitness.","method":"Genetic locus disruption (TgphyA knockout), growth assay under physiological O2 conditions","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic knockout with defined growth phenotype, corroborated by biochemical loss of Skp1 glycosylation","pmids":["22648409"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"PhyA-generated hydroxyproline on Toxoplasma Skp1 primes it for sequential glycosylation by a pathway involving at least four glycosyltransferases; glt1-knockout parasites exhibit a growth defect not rescued by catalytically inactive Glt1, demonstrating that the glycan downstream of PhyA activity is functionally required.","method":"Genetic knockout of glt1, catalytic-dead mutant rescue experiment, in vitro glycosylation assay with recombinant Glt1, NMR structure determination of sugar linkage","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution, NMR structural validation, genetic knockout with rescue test using catalytic-dead mutant, single lab but multiple orthogonal methods","pmids":["28928220"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"finding":"PHYa-dependent glycosylation of SKP1 controls the abundance of specific F-box proteins (FBXO13 and FBXO14) post-translationally; PHYaΔ parasites show reduced levels of these FBPs with no change in transcript levels, and proteasomal inhibitors rescue their abundance, indicating that the SKP1 glycosylation state regulates SCF-mediated FBP degradation.","method":"PHYa genetic knockout, SKP1 interactome analysis by mass spectrometry, epitope-tagged FBXO13/FBXO14 at genetic loci, proteasomal inhibitor rescue, transcript level measurement, parallel experiments in glycosylation-mutant cells and at 0.5% O2","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic knockout, interactome MS, proteasomal inhibitor rescue, transcript controls, replicated in glycosylation mutants and hypoxic conditions, multiple orthogonal methods","pmids":["39307307"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"finding":"The effects of PHYa on FBP abundance are phenocopied at 0.5% O2, linking O2 concentration to PHYa activity and downstream SCF-mediated protein degradation in living parasites.","method":"Growth of parasites at 0.5% O2 with measurement of FBP protein levels","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct physiological test correlating O2 concentration with PHYa-dependent FBP levels, single lab","pmids":["39307307"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2026,"finding":"PHYa-mediated hydroxylation of SKP1 followed by glycosylation inhibits SKP1 homodimerization and promotes interaction with select F-box proteins, thereby modulating SCF ubiquitin ligase assembly and the cellular proteome.","method":"Review synthesizing genetic and biochemical data from PhyA/SKP1 pathway studies in Toxoplasma and Dictyostelium, including proteasomal inhibition experiments","journal":"Biochimica et biophysica acta. General subjects","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic model supported by multiple prior experiments from the same and related organisms, but this paper is a review synthesizing earlier data","pmids":["41796871"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2026,"finding":"The terminal SKP1 glycosyltransferase GAT1 (whose activity depends on PHYa-generated hydroxyproline substrate) physically complexes with SKP1 monomers, competing with F-box proteins and the SKP1 homodimer for the same core hydrophobic interface, with binding affinity and stoichiometry dictated by SKP1 glycostate; substoichiometric GAT1 promotes SKP1 monomerization in a CTR-dependent manner.","method":"Sedimentation velocity analytical ultracentrifugation, computational modeling validated by mutational probing, genetic knockout of gat1 with FBP interactome profiling","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — rigorous biophysical method (AUC) plus mutational validation, but preprint and single lab","pmids":["42327091"],"is_preprint":true,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"In Toxoplasma gondii, PHYa is an O2- and α-ketoglutarate-dependent prolyl 4-hydroxylase that hydroxylates a conserved proline on SKP1 (an E3-SCF ubiquitin ligase adaptor subunit); this hydroxyproline is then capped by a five-sugar glycan assembled by dedicated glycosyltransferases, and the glycosylated SKP1 is inhibited from homodimerization while preferentially engaging specific F-box proteins, thereby controlling SCF assembly and the proteasomal degradation of select substrates (including FBXO13 and FBXO14) in an O2-dependent manner that contributes to parasite fitness and virulence."},"narrative":{"mechanistic_narrative":"In Toxoplasma gondii, PHYa (TgPhyA) is an O2- and α-ketoglutarate-dependent prolyl 4-hydroxylase that initiates a post-translational modification cascade controlling SCF ubiquitin ligase assembly and thereby serving as a molecular oxygen sensor for the parasite [PMID:22648409, PMID:39307307]. PHYa hydroxylates a conserved proline on the E3-SCF adaptor SKP1; disruption of TgphyA abolishes Skp1 glycosylation, and recombinant TgPhyA hydroxylates both Toxoplasma and Dictyostelium Skp1 in vitro using O2 and α-ketoglutarate as co-substrates [PMID:22648409]. The PHYa-generated hydroxyproline primes SKP1 for sequential capping by a glycan built by dedicated glycosyltransferases, and this downstream glycan is itself functionally required for normal parasite growth [PMID:28928220]. Glycosylated SKP1 post-transcriptionally controls the abundance of specific F-box proteins (FBXO13 and FBXO14): PHYaΔ parasites lose these FBPs without transcript changes, and proteasome inhibitors restore them, showing that SKP1 glycostate governs SCF-mediated FBP degradation [PMID:39307307]. Because these effects are phenocopied at 0.5% O2, PHYa couples oxygen availability to SCF-dependent proteostasis in living parasites [PMID:39307307]. Consistent with a fitness role, TgphyA-disrupted parasites grow slowly at physiological O2 [PMID:22648409].","teleology":[{"year":2012,"claim":"Established that PHYa is the enzyme responsible for SKP1 prolyl hydroxylation, defining the entry point of the SKP1 modification pathway and its dependence on O2 and α-ketoglutarate.","evidence":"TgphyA genetic knockout abolishing Skp1 glycosylation, in vitro prolyl hydroxylase assay with recombinant enzyme, and cross-species complementation in Dictyostelium phyA-null cells","pmids":["22648409"],"confidence":"High","gaps":["The specific target proline and structural basis of substrate recognition were not resolved here","Downstream consequence of hydroxylation for SCF function not yet addressed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2012,"claim":"Showed the enzyme's O2 affinity is tuned at the protein level, supporting a role as a calibrated oxygen sensor rather than a constitutive modifier.","evidence":"Comparative kinetic analysis of recombinant TgPhyA versus DdPhyA across O2 and α-ketoglutarate concentrations","pmids":["22648409"],"confidence":"Medium","gaps":["Single lab, single study","Physiological O2 thresholds in vivo not directly mapped to enzyme kinetics"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2012,"claim":"Linked PHYa activity to parasite fitness by demonstrating a growth defect under physiological oxygen, connecting the biochemical modification to organismal phenotype.","evidence":"Growth assay of TgphyA knockout parasites under physiological O2","pmids":["22648409"],"confidence":"Medium","gaps":["Molecular basis linking growth defect to specific SCF substrates not yet defined","Virulence in vivo not tested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"Demonstrated that the hydroxyproline PHYa generates is the obligate substrate for a downstream glycan that is itself functionally essential, showing the modification is a multi-step, functionally coupled pathway.","evidence":"glt1 knockout with catalytic-dead mutant rescue test, in vitro glycosylation assay, and NMR determination of sugar linkage","pmids":["28928220"],"confidence":"High","gaps":["How the glycan mechanistically alters SKP1 function was not resolved here","Identity of regulated SCF substrates unknown at this stage"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"claim":"Identified the functional output of the pathway: SKP1 glycostate post-transcriptionally controls abundance of specific F-box proteins via proteasomal degradation, connecting PHYa to SCF-mediated proteostasis.","evidence":"PHYa knockout with SKP1 interactome MS, epitope-tagged FBXO13/FBXO14, proteasome inhibitor rescue, and transcript controls","pmids":["39307307"],"confidence":"High","gaps":["Full set of regulated F-box proteins and their substrates not enumerated","Mechanism by which glycosylation biases FBP engagement not structurally defined here"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"claim":"Confirmed that oxygen concentration directly gates the pathway in vivo by phenocopying PHYa loss at hypoxia, cementing PHYa as an O2-responsive regulator of SCF-mediated degradation.","evidence":"Parasite growth at 0.5% O2 with measurement of FBP protein levels","pmids":["39307307"],"confidence":"Medium","gaps":["Single lab","Quantitative relationship between O2 tension and FBP abundance not fully mapped"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2026,"claim":"Proposed a mechanistic model in which hydroxylation/glycosylation inhibits SKP1 homodimerization and promotes select F-box protein engagement to tune SCF assembly.","evidence":"Review synthesizing genetic and biochemical data from PhyA/SKP1 studies in Toxoplasma and Dictyostelium","pmids":["41796871"],"confidence":"Medium","gaps":["Review-level synthesis rather than new primary data","Direct structural demonstration of dimerization inhibition not provided in this entry"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2026,"claim":"Provided biophysical mechanism showing the terminal glycosyltransferase GAT1 competes with F-box proteins and SKP1 homodimers for a shared core interface in a glycostate-dependent manner, promoting SKP1 monomerization.","evidence":"Sedimentation velocity analytical ultracentrifugation, computational modeling with mutational probing, gat1 knockout with FBP interactome profiling (preprint)","pmids":["42327091"],"confidence":"Medium","gaps":["Preprint, single lab, not peer-reviewed","In vivo relevance of GAT1-driven monomerization to SCF output not established in cells"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"How the PHYa-dependent SKP1 modification cascade selects and regulates the broader repertoire of F-box proteins and their ubiquitination substrates, and whether it contributes to virulence in an animal model, remains open.","evidence":"No direct experimental evidence in the current corpus resolving substrate selection breadth or in vivo virulence","pmids":[],"confidence":"Low","gaps":["Full F-box protein/substrate repertoire controlled by SKP1 glycostate unknown","In vivo virulence contribution of PHYa not directly demonstrated in the corpus","Structural basis of glycan-directed FBP selectivity unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["SCF ubiquitin ligase complex (SKP1-F-box protein) [#4, #6]"],"partners":["SKP1 (SUBSTRATE) [#0]","FBXO13 (REGULATED F-BOX PROTEIN) [#4]","FBXO14 (REGULATED F-BOX PROTEIN) [#4]","GAT1 TERMINAL GLYCOSYLTRANSFERASE (DOWNSTREAM, GLYCOSTATE-DEPENDENT SKP1 PARTNER) [#7]","GLT1 GLYCOSYLTRANSFERASE (DOWNSTREAM PATHWAY) [#3]"],"other_free_text":["PHYa uses O2 and α-ketoglutarate as co-substrates and has higher apparent O2 affinity than Dictyostelium DdPhyA, tuning its role as an oxygen sensor [#0, #1]","PHYa-generated hydroxyproline is the obligate substrate for a five-sugar SKP1 glycan built by dedicated glycosyltransferases [#3]","SKP1 glycostate biases SKP1 away from homodimerization and toward select F-box proteins, controlling SCF assembly and proteasomal FBP turnover [#4, #6, #7]","Loss of PHYa causes slow growth at physiological O2 and is phenocopied at 0.5% O2 [#2, #5]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"22648409","id":"PMC_22648409","title":"The Skp1 protein from Toxoplasma is modified by a cytoplasmic prolyl 4-hydroxylase associated with oxygen sensing in the social amoeba Dictyostelium.","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22648409","citation_count":41,"is_preprint":false},{"pmid":"23098648","id":"PMC_23098648","title":"Role of the Skp1 prolyl-hydroxylation/glycosylation pathway in oxygen dependent submerged development of Dictyostelium.","date":"2012","source":"BMC developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/23098648","citation_count":21,"is_preprint":false},{"pmid":"28928220","id":"PMC_28928220","title":"Characterization of a cytoplasmic glucosyltransferase that extends the core trisaccharide of the Toxoplasma Skp1 E3 ubiquitin ligase subunit.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28928220","citation_count":18,"is_preprint":false},{"pmid":"39307307","id":"PMC_39307307","title":"Oxygen-dependent regulation of F-box proteins in Toxoplasma gondii is mediated by Skp1 glycosylation.","date":"2024","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/39307307","citation_count":12,"is_preprint":false},{"pmid":"31281925","id":"PMC_31281925","title":"Skp1 isoforms are differentially modified by a dual function prolyl 4-hydroxylase/N-acety lglucosaminyltransferase in a plant pathogen.","date":"2019","source":"Glycobiology","url":"https://pubmed.ncbi.nlm.nih.gov/31281925","citation_count":11,"is_preprint":false},{"pmid":"41796871","id":"PMC_41796871","title":"Protist oxygen-sensing and the role of prolyl hydroxylase-regulation of E3(SCF) ubiquitin ligases.","date":"2026","source":"Biochimica et biophysica acta. General subjects","url":"https://pubmed.ncbi.nlm.nih.gov/41796871","citation_count":0,"is_preprint":false},{"pmid":"42327091","id":"PMC_42327091","title":"Potential for the Terminal SKP1 Glycosyltransferase to Exert Non-Enzymatic Control of SKP1 in Toxoplasma gondii.","date":"2026","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/42327091","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6523,"output_tokens":2321,"usd":0.027192,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7794,"output_tokens":3781,"usd":0.066748,"stage2_stop_reason":"end_turn"},"total_usd":0.09394,"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\": 2012,\n      \"finding\": \"TgPhyA is required for prolyl hydroxylation of Toxoplasma Skp1; disruption of the TgphyA locus abolishes Skp1 glycosylation in parasites, and bacterially expressed TgPhyA prolyl-hydroxylates both Toxoplasma and Dictyostelium Skp1s in vitro using O2 and α-ketoglutarate as co-substrates.\",\n      \"method\": \"Genetic locus disruption (TgphyA knockout), in vitro prolyl hydroxylase assay with recombinant TgPhyA, kinetic analyses of O2 and α-ketoglutarate dependence, complementation in Dictyostelium phyA-null cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with recombinant enzyme, genetic knockout with defined biochemical phenotype, cross-species complementation, multiple orthogonal methods in one study\",\n      \"pmids\": [\"22648409\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TgPhyA has a significantly higher apparent affinity for O2 compared to Dictyostelium DdPhyA, despite superimposable dependence on α-ketoglutarate concentration, suggesting that the O2-sensing threshold is tuned at the enzyme level.\",\n      \"method\": \"Kinetic analysis of recombinant TgPhyA vs DdPhyA with varying O2 and α-ketoglutarate concentrations\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — rigorous in vitro kinetics, but single lab, single paper\",\n      \"pmids\": [\"22648409\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Parasites with disrupted TgphyA grow slowly at physiological O2 levels, establishing a functional role for PhyA-dependent Skp1 hydroxylation in parasite fitness.\",\n      \"method\": \"Genetic locus disruption (TgphyA knockout), growth assay under physiological O2 conditions\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic knockout with defined growth phenotype, corroborated by biochemical loss of Skp1 glycosylation\",\n      \"pmids\": [\"22648409\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PhyA-generated hydroxyproline on Toxoplasma Skp1 primes it for sequential glycosylation by a pathway involving at least four glycosyltransferases; glt1-knockout parasites exhibit a growth defect not rescued by catalytically inactive Glt1, demonstrating that the glycan downstream of PhyA activity is functionally required.\",\n      \"method\": \"Genetic knockout of glt1, catalytic-dead mutant rescue experiment, in vitro glycosylation assay with recombinant Glt1, NMR structure determination of sugar linkage\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution, NMR structural validation, genetic knockout with rescue test using catalytic-dead mutant, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"28928220\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PHYa-dependent glycosylation of SKP1 controls the abundance of specific F-box proteins (FBXO13 and FBXO14) post-translationally; PHYaΔ parasites show reduced levels of these FBPs with no change in transcript levels, and proteasomal inhibitors rescue their abundance, indicating that the SKP1 glycosylation state regulates SCF-mediated FBP degradation.\",\n      \"method\": \"PHYa genetic knockout, SKP1 interactome analysis by mass spectrometry, epitope-tagged FBXO13/FBXO14 at genetic loci, proteasomal inhibitor rescue, transcript level measurement, parallel experiments in glycosylation-mutant cells and at 0.5% O2\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic knockout, interactome MS, proteasomal inhibitor rescue, transcript controls, replicated in glycosylation mutants and hypoxic conditions, multiple orthogonal methods\",\n      \"pmids\": [\"39307307\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The effects of PHYa on FBP abundance are phenocopied at 0.5% O2, linking O2 concentration to PHYa activity and downstream SCF-mediated protein degradation in living parasites.\",\n      \"method\": \"Growth of parasites at 0.5% O2 with measurement of FBP protein levels\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct physiological test correlating O2 concentration with PHYa-dependent FBP levels, single lab\",\n      \"pmids\": [\"39307307\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"PHYa-mediated hydroxylation of SKP1 followed by glycosylation inhibits SKP1 homodimerization and promotes interaction with select F-box proteins, thereby modulating SCF ubiquitin ligase assembly and the cellular proteome.\",\n      \"method\": \"Review synthesizing genetic and biochemical data from PhyA/SKP1 pathway studies in Toxoplasma and Dictyostelium, including proteasomal inhibition experiments\",\n      \"journal\": \"Biochimica et biophysica acta. General subjects\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic model supported by multiple prior experiments from the same and related organisms, but this paper is a review synthesizing earlier data\",\n      \"pmids\": [\"41796871\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"The terminal SKP1 glycosyltransferase GAT1 (whose activity depends on PHYa-generated hydroxyproline substrate) physically complexes with SKP1 monomers, competing with F-box proteins and the SKP1 homodimer for the same core hydrophobic interface, with binding affinity and stoichiometry dictated by SKP1 glycostate; substoichiometric GAT1 promotes SKP1 monomerization in a CTR-dependent manner.\",\n      \"method\": \"Sedimentation velocity analytical ultracentrifugation, computational modeling validated by mutational probing, genetic knockout of gat1 with FBP interactome profiling\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — rigorous biophysical method (AUC) plus mutational validation, but preprint and single lab\",\n      \"pmids\": [\"42327091\"],\n      \"is_preprint\": true,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    }\n  ],\n  \"current_model\": \"In Toxoplasma gondii, PHYa is an O2- and α-ketoglutarate-dependent prolyl 4-hydroxylase that hydroxylates a conserved proline on SKP1 (an E3-SCF ubiquitin ligase adaptor subunit); this hydroxyproline is then capped by a five-sugar glycan assembled by dedicated glycosyltransferases, and the glycosylated SKP1 is inhibited from homodimerization while preferentially engaging specific F-box proteins, thereby controlling SCF assembly and the proteasomal degradation of select substrates (including FBXO13 and FBXO14) in an O2-dependent manner that contributes to parasite fitness and virulence.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"In Toxoplasma gondii, PHYa (TgPhyA) is an O2- and \\u03b1-ketoglutarate-dependent prolyl 4-hydroxylase that initiates a post-translational modification cascade controlling SCF ubiquitin ligase assembly and thereby serving as a molecular oxygen sensor for the parasite [#0, #4]. PHYa hydroxylates a conserved proline on the E3-SCF adaptor SKP1; disruption of TgphyA abolishes Skp1 glycosylation, and recombinant TgPhyA hydroxylates both Toxoplasma and Dictyostelium Skp1 in vitro using O2 and \\u03b1-ketoglutarate as co-substrates [#0]. The PHYa-generated hydroxyproline primes SKP1 for sequential capping by a glycan built by dedicated glycosyltransferases, and this downstream glycan is itself functionally required for normal parasite growth [#3]. Glycosylated SKP1 post-transcriptionally controls the abundance of specific F-box proteins (FBXO13 and FBXO14): PHYa\\u0394 parasites lose these FBPs without transcript changes, and proteasome inhibitors restore them, showing that SKP1 glycostate governs SCF-mediated FBP degradation [#4]. Because these effects are phenocopied at 0.5% O2, PHYa couples oxygen availability to SCF-dependent proteostasis in living parasites [#5]. Consistent with a fitness role, TgphyA-disrupted parasites grow slowly at physiological O2 [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Established that PHYa is the enzyme responsible for SKP1 prolyl hydroxylation, defining the entry point of the SKP1 modification pathway and its dependence on O2 and \\u03b1-ketoglutarate.\",\n      \"evidence\": \"TgphyA genetic knockout abolishing Skp1 glycosylation, in vitro prolyl hydroxylase assay with recombinant enzyme, and cross-species complementation in Dictyostelium phyA-null cells\",\n      \"pmids\": [\"22648409\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The specific target proline and structural basis of substrate recognition were not resolved here\", \"Downstream consequence of hydroxylation for SCF function not yet addressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed the enzyme's O2 affinity is tuned at the protein level, supporting a role as a calibrated oxygen sensor rather than a constitutive modifier.\",\n      \"evidence\": \"Comparative kinetic analysis of recombinant TgPhyA versus DdPhyA across O2 and \\u03b1-ketoglutarate concentrations\",\n      \"pmids\": [\"22648409\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, single study\", \"Physiological O2 thresholds in vivo not directly mapped to enzyme kinetics\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Linked PHYa activity to parasite fitness by demonstrating a growth defect under physiological oxygen, connecting the biochemical modification to organismal phenotype.\",\n      \"evidence\": \"Growth assay of TgphyA knockout parasites under physiological O2\",\n      \"pmids\": [\"22648409\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis linking growth defect to specific SCF substrates not yet defined\", \"Virulence in vivo not tested\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrated that the hydroxyproline PHYa generates is the obligate substrate for a downstream glycan that is itself functionally essential, showing the modification is a multi-step, functionally coupled pathway.\",\n      \"evidence\": \"glt1 knockout with catalytic-dead mutant rescue test, in vitro glycosylation assay, and NMR determination of sugar linkage\",\n      \"pmids\": [\"28928220\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the glycan mechanistically alters SKP1 function was not resolved here\", \"Identity of regulated SCF substrates unknown at this stage\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified the functional output of the pathway: SKP1 glycostate post-transcriptionally controls abundance of specific F-box proteins via proteasomal degradation, connecting PHYa to SCF-mediated proteostasis.\",\n      \"evidence\": \"PHYa knockout with SKP1 interactome MS, epitope-tagged FBXO13/FBXO14, proteasome inhibitor rescue, and transcript controls\",\n      \"pmids\": [\"39307307\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full set of regulated F-box proteins and their substrates not enumerated\", \"Mechanism by which glycosylation biases FBP engagement not structurally defined here\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Confirmed that oxygen concentration directly gates the pathway in vivo by phenocopying PHYa loss at hypoxia, cementing PHYa as an O2-responsive regulator of SCF-mediated degradation.\",\n      \"evidence\": \"Parasite growth at 0.5% O2 with measurement of FBP protein levels\",\n      \"pmids\": [\"39307307\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Quantitative relationship between O2 tension and FBP abundance not fully mapped\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Proposed a mechanistic model in which hydroxylation/glycosylation inhibits SKP1 homodimerization and promotes select F-box protein engagement to tune SCF assembly.\",\n      \"evidence\": \"Review synthesizing genetic and biochemical data from PhyA/SKP1 studies in Toxoplasma and Dictyostelium\",\n      \"pmids\": [\"41796871\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Review-level synthesis rather than new primary data\", \"Direct structural demonstration of dimerization inhibition not provided in this entry\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Provided biophysical mechanism showing the terminal glycosyltransferase GAT1 competes with F-box proteins and SKP1 homodimers for a shared core interface in a glycostate-dependent manner, promoting SKP1 monomerization.\",\n      \"evidence\": \"Sedimentation velocity analytical ultracentrifugation, computational modeling with mutational probing, gat1 knockout with FBP interactome profiling (preprint)\",\n      \"pmids\": [\"42327091\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, single lab, not peer-reviewed\", \"In vivo relevance of GAT1-driven monomerization to SCF output not established in cells\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the PHYa-dependent SKP1 modification cascade selects and regulates the broader repertoire of F-box proteins and their ubiquitination substrates, and whether it contributes to virulence in an animal model, remains open.\",\n      \"evidence\": \"No direct experimental evidence in the current corpus resolving substrate selection breadth or in vivo virulence\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Full F-box protein/substrate repertoire controlled by SKP1 glycostate unknown\", \"In vivo virulence contribution of PHYa not directly demonstrated in the corpus\", \"Structural basis of glycan-directed FBP selectivity unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0031545\", \"supporting_discovery_ids\": [0, 1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0051213\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006511\", \"supporting_discovery_ids\": [4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0070979\", \"supporting_discovery_ids\": [4, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0071456\", \"supporting_discovery_ids\": [1, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\"SCF ubiquitin ligase complex (SKP1-F-box protein) [#4, #6]\"],\n    \"partners\": [\"SKP1 (substrate) [#0]\", \"FBXO13 (regulated F-box protein) [#4]\", \"FBXO14 (regulated F-box protein) [#4]\", \"GAT1 terminal glycosyltransferase (downstream, glycostate-dependent SKP1 partner) [#7]\", \"Glt1 glycosyltransferase (downstream pathway) [#3]\"],\n    \"other_free_text\": [\n      \"PHYa uses O2 and \\u03b1-ketoglutarate as co-substrates and has higher apparent O2 affinity than Dictyostelium DdPhyA, tuning its role as an oxygen sensor [#0, #1]\",\n      \"PHYa-generated hydroxyproline is the obligate substrate for a five-sugar SKP1 glycan built by dedicated glycosyltransferases [#3]\",\n      \"SKP1 glycostate biases SKP1 away from homodimerization and toward select F-box proteins, controlling SCF assembly and proteasomal FBP turnover [#4, #6, #7]\",\n      \"Loss of PHYa causes slow growth at physiological O2 and is phenocopied at 0.5% O2 [#2, #5]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}\n```","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_232960","name":"PHYa","product":"prolyl hydroxylase PHYa","uniprot_acc":"A0A125YFP7","chromosome":"TGME49_chrVIII","start_pos":2294236},{"strain":"GT1","gene_id":"TGGT1_232960","name":null,"product":"oxidoreductase, 2OG-Fe(II) oxygenase family protein","uniprot_acc":null,"chromosome":"TGGT1_chrVIII","start_pos":2290628},{"strain":"RH88","gene_id":"TGRH88_076950","name":null,"product":"oxidoreductase, 2OG-Fe(II) oxygenase family protein","uniprot_acc":null,"chromosome":"CM023089","start_pos":7400891},{"strain":"VEG","gene_id":"TGVEG_232960","name":null,"product":"oxidoreductase, 2OG-Fe(II) oxygenase family protein","uniprot_acc":"B6KJY2","chromosome":"TGVEG_chrVIII","start_pos":2303856}]}