{"gene":"TG_214620","gene_symbol":"PHYb","run_date":"2026-09-08T11:06:27","timeline":{"discoveries":[{"year":2019,"finding":"TgPHYb is a 2-oxoglutarate-dependent prolyl hydroxylase required for optimal parasite growth and protein synthesis at high, but not low, O2 tensions, functioning as a sensor of elevated oxygen levels.","method":"Conditional expression strain (knockdown) with growth and invasion assays under varying oxygen conditions","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean conditional knockdown with defined cellular phenotypes (growth, invasion, protein synthesis) under multiple oxygen conditions in a single lab study","pmids":["30914506"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"TgPHYb promotes the activity of translation elongation factor eEF2; depletion of TgPHYb leads to hyper-phosphorylation and inactivation of eEF2, decreased rates of translational elongation, and reduced abundance of parasite adhesins.","method":"Conditional knockdown of TgPHYb with measurement of eEF2 phosphorylation status, translational elongation rates, and adhesin protein levels","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KD with multiple orthogonal readouts (phosphorylation, translation rate, protein abundance), single lab","pmids":["30914506"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"finding":"PHYb acts upstream of CDPK3 in a stress-response pathway: PHYb promotes eEF2 activity by preventing CDPK3-mediated hyper-phosphorylation of eEF2; in the absence of PHYb, eEF2 is hyper-phosphorylated by CDPK3, inhibiting eEF2 interaction with the ribosome and blocking translational elongation.","method":"Genetic epistasis (CDPK3 loss-of-function in extracellularly stressed parasites), eEF2 phosphorylation assays, and co-immunoprecipitation of CDPK3 with eEF2","journal":"mSphere","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis plus co-IP and phosphorylation assays, single lab, two orthogonal methods placing PHYb in the pathway","pmids":["37272703"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgPHYb is a 2-oxoglutarate-dependent prolyl hydroxylase that senses high oxygen levels in Toxoplasma gondii and supports translational elongation by suppressing CDPK3-mediated hyper-phosphorylation of eEF2, thereby maintaining eEF2 ribosome engagement and parasite protein synthesis during extracellular stress."},"narrative":{"mechanistic_narrative":"TgPHYb is a 2-oxoglutarate-dependent prolyl hydroxylase in Toxoplasma gondii that acts as a sensor of elevated oxygen levels, supporting parasite growth, invasion, and protein synthesis specifically at high but not low O2 tensions [PMID:30914506]. Its mechanistic role is to sustain translational elongation: PHYb maintains translation elongation factor eEF2 in an active, ribosome-engaged state, and its depletion causes eEF2 hyper-phosphorylation, reduced elongation rates, and lower abundance of parasite adhesins [PMID:30914506]. This regulation operates through a stress-response pathway in which PHYb acts upstream of CDPK3, preventing CDPK3-mediated hyper-phosphorylation of eEF2 that would otherwise block eEF2-ribosome interaction and halt elongation [PMID:37272703]. Beyond this oxygen-sensing/eEF2-CDPK3 axis, the direct hydroxylation substrate of PHYb has not been characterized in the available corpus.","teleology":[{"year":2019,"claim":"Established that TgPHYb is a functional 2-oxoglutarate-dependent prolyl hydroxylase whose requirement is oxygen-dependent, defining it as an oxygen sensor rather than a constitutive housekeeping enzyme.","evidence":"Conditional knockdown strain assayed for growth, invasion, and protein synthesis under varying oxygen tensions in T. gondii","pmids":["30914506"],"confidence":"Medium","gaps":["Direct prolyl hydroxylation substrate of PHYb not identified","Molecular mechanism of oxygen sensing not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"Connected PHYb to the translation machinery by showing it promotes eEF2 activity, linking oxygen sensing to control of translational elongation and adhesin production.","evidence":"Conditional knockdown with eEF2 phosphorylation, elongation-rate, and adhesin-abundance readouts in T. gondii","pmids":["30914506"],"confidence":"Medium","gaps":["Whether eEF2 or an upstream factor is the direct hydroxylation target unknown","Kinase responsible for eEF2 hyper-phosphorylation not yet identified at this stage"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"claim":"Placed PHYb in a defined stress-response pathway upstream of the kinase CDPK3, showing PHYb prevents CDPK3-mediated eEF2 hyper-phosphorylation that otherwise blocks eEF2-ribosome engagement and elongation.","evidence":"Genetic epistasis with CDPK3 loss-of-function in extracellularly stressed parasites, eEF2 phosphorylation assays, and CDPK3-eEF2 co-immunoprecipitation in T. gondii","pmids":["37272703"],"confidence":"Medium","gaps":["Biochemical link between PHYb hydroxylase activity and CDPK3 suppression not established","Direct PHYb substrate connecting oxygen sensing to CDPK3 regulation unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The direct hydroxylation substrate of PHYb and the biochemical mechanism coupling its enzymatic activity to suppression of CDPK3-mediated eEF2 phosphorylation remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No direct prolyl hydroxylation substrate identified","No structural model of PHYb","Mechanism linking hydroxylase activity to CDPK3 inhibition undefined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["EEF2 (TRANSLATION ELONGATION FACTOR, FUNCTIONAL TARGET)","CDPK3 (CALCIUM-DEPENDENT PROTEIN KINASE, DOWNSTREAM EFFECTOR ON EEF2)"],"other_free_text":["PHYb functions as a sensor of elevated oxygen and is required for optimal growth and protein synthesis at high but not low O2 tensions [#0].","PHYb sustains translational elongation by keeping eEF2 dephosphorylated and ribosome-engaged; its loss reduces elongation and parasite adhesin levels [#1].","PHYb acts upstream of CDPK3, preventing CDPK3-mediated hyper-phosphorylation of eEF2 during extracellular stress [#2]."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"30914506","id":"PMC_30914506","title":"A Toxoplasma Prolyl Hydroxylase Mediates Oxygen Stress Responses by Regulating Translation Elongation.","date":"2019","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/30914506","citation_count":15,"is_preprint":false},{"pmid":"37272703","id":"PMC_37272703","title":"Identification of Toxoplasma calcium-dependent protein kinase 3 as a stress-activated elongation factor 2 kinase.","date":"2023","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/37272703","citation_count":5,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":3904,"output_tokens":856,"usd":0.012276,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":5684,"output_tokens":2120,"usd":0.04071,"stage2_stop_reason":"end_turn"},"total_usd":0.052986,"stage1_batch_id":"msgbatch_01WokvBYTB6JLnYrpD4rXnyV","stage2_batch_id":"msgbatch_01M7hsQDvz6AqVC9DLVjQzzc","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2019,\n      \"finding\": \"TgPHYb is a 2-oxoglutarate-dependent prolyl hydroxylase required for optimal parasite growth and protein synthesis at high, but not low, O2 tensions, functioning as a sensor of elevated oxygen levels.\",\n      \"method\": \"Conditional expression strain (knockdown) with growth and invasion assays under varying oxygen conditions\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean conditional knockdown with defined cellular phenotypes (growth, invasion, protein synthesis) under multiple oxygen conditions in a single lab study\",\n      \"pmids\": [\"30914506\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TgPHYb promotes the activity of translation elongation factor eEF2; depletion of TgPHYb leads to hyper-phosphorylation and inactivation of eEF2, decreased rates of translational elongation, and reduced abundance of parasite adhesins.\",\n      \"method\": \"Conditional knockdown of TgPHYb with measurement of eEF2 phosphorylation status, translational elongation rates, and adhesin protein levels\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KD with multiple orthogonal readouts (phosphorylation, translation rate, protein abundance), single lab\",\n      \"pmids\": [\"30914506\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PHYb acts upstream of CDPK3 in a stress-response pathway: PHYb promotes eEF2 activity by preventing CDPK3-mediated hyper-phosphorylation of eEF2; in the absence of PHYb, eEF2 is hyper-phosphorylated by CDPK3, inhibiting eEF2 interaction with the ribosome and blocking translational elongation.\",\n      \"method\": \"Genetic epistasis (CDPK3 loss-of-function in extracellularly stressed parasites), eEF2 phosphorylation assays, and co-immunoprecipitation of CDPK3 with eEF2\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis plus co-IP and phosphorylation assays, single lab, two orthogonal methods placing PHYb in the pathway\",\n      \"pmids\": [\"37272703\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgPHYb is a 2-oxoglutarate-dependent prolyl hydroxylase that senses high oxygen levels in Toxoplasma gondii and supports translational elongation by suppressing CDPK3-mediated hyper-phosphorylation of eEF2, thereby maintaining eEF2 ribosome engagement and parasite protein synthesis during extracellular stress.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TgPHYb is a 2-oxoglutarate-dependent prolyl hydroxylase in Toxoplasma gondii that acts as a sensor of elevated oxygen levels, supporting parasite growth, invasion, and protein synthesis specifically at high but not low O2 tensions [#0]. Its mechanistic role is to sustain translational elongation: PHYb maintains translation elongation factor eEF2 in an active, ribosome-engaged state, and its depletion causes eEF2 hyper-phosphorylation, reduced elongation rates, and lower abundance of parasite adhesins [#1]. This regulation operates through a stress-response pathway in which PHYb acts upstream of CDPK3, preventing CDPK3-mediated hyper-phosphorylation of eEF2 that would otherwise block eEF2-ribosome interaction and halt elongation [#2]. Beyond this oxygen-sensing/eEF2-CDPK3 axis, the direct hydroxylation substrate of PHYb has not been characterized in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2019,\n      \"claim\": \"Established that TgPHYb is a functional 2-oxoglutarate-dependent prolyl hydroxylase whose requirement is oxygen-dependent, defining it as an oxygen sensor rather than a constitutive housekeeping enzyme.\",\n      \"evidence\": \"Conditional knockdown strain assayed for growth, invasion, and protein synthesis under varying oxygen tensions in T. gondii\",\n      \"pmids\": [\"30914506\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct prolyl hydroxylation substrate of PHYb not identified\", \"Molecular mechanism of oxygen sensing not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected PHYb to the translation machinery by showing it promotes eEF2 activity, linking oxygen sensing to control of translational elongation and adhesin production.\",\n      \"evidence\": \"Conditional knockdown with eEF2 phosphorylation, elongation-rate, and adhesin-abundance readouts in T. gondii\",\n      \"pmids\": [\"30914506\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether eEF2 or an upstream factor is the direct hydroxylation target unknown\", \"Kinase responsible for eEF2 hyper-phosphorylation not yet identified at this stage\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Placed PHYb in a defined stress-response pathway upstream of the kinase CDPK3, showing PHYb prevents CDPK3-mediated eEF2 hyper-phosphorylation that otherwise blocks eEF2-ribosome engagement and elongation.\",\n      \"evidence\": \"Genetic epistasis with CDPK3 loss-of-function in extracellularly stressed parasites, eEF2 phosphorylation assays, and CDPK3-eEF2 co-immunoprecipitation in T. gondii\",\n      \"pmids\": [\"37272703\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Biochemical link between PHYb hydroxylase activity and CDPK3 suppression not established\", \"Direct PHYb substrate connecting oxygen sensing to CDPK3 regulation unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct hydroxylation substrate of PHYb and the biochemical mechanism coupling its enzymatic activity to suppression of CDPK3-mediated eEF2 phosphorylation remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct prolyl hydroxylation substrate identified\", \"No structural model of PHYb\", \"Mechanism linking hydroxylase activity to CDPK3 inhibition undefined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0031418\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0004656\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006414\", \"supporting_discovery_ids\": [1, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0071456\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006979\", \"supporting_discovery_ids\": [2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"eEF2 (translation elongation factor, functional target)\", \"CDPK3 (calcium-dependent protein kinase, downstream effector on eEF2)\"],\n    \"other_free_text\": [\n      \"PHYb functions as a sensor of elevated oxygen and is required for optimal growth and protein synthesis at high but not low O2 tensions [#0].\",\n      \"PHYb sustains translational elongation by keeping eEF2 dephosphorylated and ribosome-engaged; its loss reduces elongation and parasite adhesin levels [#1].\",\n      \"PHYb acts upstream of CDPK3, preventing CDPK3-mediated hyper-phosphorylation of eEF2 during extracellular stress [#2].\"\n    ]\n  },\n  \"strain_conflicts\": []\n}\n```","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_214620","name":"PHYb","product":"prolyl hydroxylase PHYb","uniprot_acc":"S8EZF0","chromosome":"TGME49_chrX","start_pos":6438227},{"strain":"GT1","gene_id":"TGGT1_214620","name":null,"product":"putative hypoxia- inducible factor prolyl hydroxylase (phd2)","uniprot_acc":null,"chromosome":"TGGT1_chrX","start_pos":6329330},{"strain":"RH88","gene_id":"TGRH88_039890","name":null,"product":"putative hypoxia- inducible factor prolyl hydroxylase (phd2)","uniprot_acc":null,"chromosome":"CM023091","start_pos":1091474},{"strain":"VEG","gene_id":"TGVEG_214620","name":null,"product":"putative hypoxia- inducible factor prolyl hydroxylase (phd2)","uniprot_acc":"V4ZB09","chromosome":"TGVEG_chrX","start_pos":6367268}]}