{"gene":"TG_316250","gene_symbol":"GRA45","run_date":"2026-09-08T18:09:59+00:00","timeline":{"discoveries":[{"year":2020,"finding":"GRA45 is required for translocation of the dense granule effector protein GRA16 across the parasitophorous vacuole membrane into host cells, and for upregulation of host c-Myc and cyclin E1 in infected cells. GRA45 was identified as a MYR1-interacting protein by affinity purification, placing it in the MYR translocon complex within the PV.","method":"Affinity purification/coimmunoprecipitation of MYR1, followed by genetic knockout of GRA45 with phenotypic readout (GRA16 translocation, host c-Myc and cyclin E1 upregulation)","journal":"mSphere","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP/affinity purification identifying the interaction, combined with clean genetic knockout and two independent phenotypic readouts (effector translocation and host gene upregulation), single lab but multiple orthogonal methods","pmids":["32075880"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"GRA45 contains an ASP5 processing motif (RRL), but processing at this site is nonessential for its role in effector protein translocation.","method":"Mutation of the ASP5 cleavage motif (RRL to ARL) combined with functional assay for GRA16 translocation","journal":"mSphere","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — site-directed mutagenesis with functional readout, single lab, single paper","pmids":["32075880"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"GRA45 is a substrate of the Golgi-associated aspartyl protease ASP5; it contains an RRL cleavage motif and is processed by ASP5, as confirmed by N-terminal peptide enrichment showing loss of cleavage in Δasp5 parasites and upon RRL→ARL mutation. GRA45 is a dense granule protein that resides within the parasitophorous vacuole.","method":"Terminal Amine Isotopic Labeling of Substrates (TAILS) N-terminal peptide enrichment, quantitative proteomics comparing wild-type and Δasp5 parasites, and validation by motif mutagenesis","journal":"mBio","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro/in vivo biochemical identification of cleavage site by quantitative proteomics with orthogonal mutagenesis validation, single lab but multiple methods","pmids":["30377279"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"finding":"GRA45 was identified as an IFN-γ-dependent virulence gene in an in vivo CRISPR screen; its requirement for parasite survival was revealed specifically in immune-competent (IFN-γ-signaling-intact) C57BL/6 mice but not in Ifngr1-/- mice, placing GRA45 function in the context of resistance to IFN-γ-mediated host immunity.","method":"In vivo CRISPR screen in C57BL/6 and Ifngr1-/- mice with comparative enrichment analysis","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — in vivo genetic screen with host-genetics epistasis, single lab, single method","pmids":["37269286"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"GRA45 is a dense granule protein that localizes to the parasitophorous vacuole, is proteolytically matured by ASP5 at an RRL motif (though this processing is dispensable for function), physically interacts with the MYR1 translocon complex, and is essential for translocation of effector proteins (e.g., GRA16) across the PV membrane into the host cell—thereby enabling downstream modulation of host gene expression (c-Myc, cyclin E1) and resistance to IFN-γ-mediated immune clearance."},"narrative":{"mechanistic_narrative":"GRA45 is a Toxoplasma gondii dense granule protein that resides within the parasitophorous vacuole (PV) and functions as a component of the effector-translocation machinery required for the parasite to manipulate its host cell [PMID:32075880, PMID:30377279]. It physically associates with the MYR1 translocon complex, and its genetic loss abolishes translocation of the dense granule effector GRA16 across the PV membrane and the consequent upregulation of host c-Myc and cyclin E1 [PMID:32075880]. GRA45 carries an RRL motif that is cleaved by the Golgi-associated aspartyl protease ASP5, though this proteolytic processing is dispensable for its role in effector translocation [PMID:32075880, PMID:30377279]. Consistent with its role in enabling host manipulation, GRA45 acts as an IFN-γ-dependent virulence factor, being required for parasite survival specifically in immune-competent but not Ifngr1-deficient mice [PMID:37269286].","teleology":[{"year":2018,"claim":"Establishing GRA45 as a genuine ASP5 substrate defined its maturation pathway and confirmed it as a PV-resident dense granule protein, providing the first biochemical foothold on the protein.","evidence":"TAILS N-terminal peptide enrichment and quantitative proteomics comparing wild-type and Δasp5 parasites, with RRL→ARL mutagenesis validation, in Toxoplasma gondii","pmids":["30377279"],"confidence":"High","gaps":["Did not establish the functional role of GRA45 or whether cleavage is required for any function","No interacting partners identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"claim":"Identifying GRA45 as a MYR1-interacting protein and showing knockouts fail to translocate GRA16 placed GRA45 in the MYR translocon and defined its central function in effector export and host gene modulation.","evidence":"Affinity purification/Co-IP of MYR1 plus GRA45 genetic knockout with GRA16 translocation and host c-Myc/cyclin E1 readouts, in Toxoplasma gondii","pmids":["32075880"],"confidence":"High","gaps":["Whether GRA45 acts directly in the translocation channel or as an accessory/chaperone factor is unresolved","The molecular step in translocation that GRA45 enables is not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"claim":"Mutating the ASP5 cleavage motif showed that proteolytic maturation is uncoupled from GRA45's translocation function, indicating processing is not a prerequisite for its role in effector export.","evidence":"RRL→ARL site-directed mutagenesis with GRA16 translocation functional assay, in Toxoplasma gondii","pmids":["32075880"],"confidence":"Medium","gaps":["The functional purpose of ASP5 cleavage of GRA45, if any, remains unknown","Single lab, single functional readout"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"claim":"An in vivo CRISPR screen with host-genetics epistasis placed GRA45's function in the physiological context of resisting IFN-γ-mediated immune clearance, connecting effector translocation to virulence.","evidence":"In vivo CRISPR screen in C57BL/6 versus Ifngr1-/- mice with comparative enrichment analysis, in Toxoplasma gondii","pmids":["37269286"],"confidence":"Medium","gaps":["Which specific effectors/host pathways downstream of GRA45 mediate IFN-γ resistance is not defined","Single method, single lab"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"It remains unknown whether GRA45 forms a structural part of the translocation channel or acts as a chaperone/accessory factor, and what the full repertoire of GRA45-dependent effectors and host pathways is.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of GRA45 within the MYR complex","Full effector-dependency profile beyond GRA16 undefined","Molecular mechanism linking GRA45 to IFN-γ resistance unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["MYR translocon complex (with MYR1)"],"partners":["MYR1","ASP5 (PROTEASE)"],"other_free_text":["GRA45 is proteolytically processed by ASP5 at an RRL motif, but this cleavage is dispensable for effector translocation.","GRA45 is required for translocation of the dense granule effector GRA16 across the PV membrane and for host c-Myc and cyclin E1 upregulation.","GRA45 is an IFN-γ-dependent virulence factor, required for parasite survival in immune-competent but not Ifngr1-/- mice."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"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":"37269286","id":"PMC_37269286","title":"Host genetics highlights IFN-γ-dependent Toxoplasma genes encoding secreted and non-secreted virulence factors in in vivo CRISPR screens.","date":"2023","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/37269286","citation_count":30,"is_preprint":false},{"pmid":"41296383","id":"PMC_41296383","title":"Development and Evaluation of Six Novel Recombinant GRA Proteins in Serodiagnosis of Human Toxoplasmosis.","date":"2025","source":"Current issues in molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/41296383","citation_count":2,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":4644,"output_tokens":1240,"usd":0.016266,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":6168,"output_tokens":2177,"usd":0.042632,"stage2_stop_reason":"end_turn"},"total_usd":0.0,"stage1_batch_id":"msgbatch_01XFXJD6iZecXyWn9uU5Qom9","stage2_batch_id":"msgbatch_013GToKgs4AKSCRE4yvueviH","note":"batch pricing = 50% of standard","skipped":true},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA45 is required for translocation of the dense granule effector protein GRA16 across the parasitophorous vacuole membrane into host cells, and for upregulation of host c-Myc and cyclin E1 in infected cells. GRA45 was identified as a MYR1-interacting protein by affinity purification, placing it in the MYR translocon complex within the PV.\",\n      \"method\": \"Affinity purification/coimmunoprecipitation of MYR1, followed by genetic knockout of GRA45 with phenotypic readout (GRA16 translocation, host c-Myc and cyclin E1 upregulation)\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP/affinity purification identifying the interaction, combined with clean genetic knockout and two independent phenotypic readouts (effector translocation and host gene upregulation), single lab but multiple orthogonal methods\",\n      \"pmids\": [\"32075880\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA45 contains an ASP5 processing motif (RRL), but processing at this site is nonessential for its role in effector protein translocation.\",\n      \"method\": \"Mutation of the ASP5 cleavage motif (RRL to ARL) combined with functional assay for GRA16 translocation\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — site-directed mutagenesis with functional readout, single lab, single paper\",\n      \"pmids\": [\"32075880\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"GRA45 is a substrate of the Golgi-associated aspartyl protease ASP5; it contains an RRL cleavage motif and is processed by ASP5, as confirmed by N-terminal peptide enrichment showing loss of cleavage in Δasp5 parasites and upon RRL→ARL mutation. GRA45 is a dense granule protein that resides within the parasitophorous vacuole.\",\n      \"method\": \"Terminal Amine Isotopic Labeling of Substrates (TAILS) N-terminal peptide enrichment, quantitative proteomics comparing wild-type and Δasp5 parasites, and validation by motif mutagenesis\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro/in vivo biochemical identification of cleavage site by quantitative proteomics with orthogonal mutagenesis validation, single lab but multiple methods\",\n      \"pmids\": [\"30377279\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"GRA45 was identified as an IFN-γ-dependent virulence gene in an in vivo CRISPR screen; its requirement for parasite survival was revealed specifically in immune-competent (IFN-γ-signaling-intact) C57BL/6 mice but not in Ifngr1-/- mice, placing GRA45 function in the context of resistance to IFN-γ-mediated host immunity.\",\n      \"method\": \"In vivo CRISPR screen in C57BL/6 and Ifngr1-/- mice with comparative enrichment analysis\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — in vivo genetic screen with host-genetics epistasis, single lab, single method\",\n      \"pmids\": [\"37269286\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    }\n  ],\n  \"current_model\": \"GRA45 is a dense granule protein that localizes to the parasitophorous vacuole, is proteolytically matured by ASP5 at an RRL motif (though this processing is dispensable for function), physically interacts with the MYR1 translocon complex, and is essential for translocation of effector proteins (e.g., GRA16) across the PV membrane into the host cell—thereby enabling downstream modulation of host gene expression (c-Myc, cyclin E1) and resistance to IFN-γ-mediated immune clearance.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GRA45 is a Toxoplasma gondii dense granule protein that resides within the parasitophorous vacuole (PV) and functions as a component of the effector-translocation machinery required for the parasite to manipulate its host cell [#0, #2]. It physically associates with the MYR1 translocon complex, and its genetic loss abolishes translocation of the dense granule effector GRA16 across the PV membrane and the consequent upregulation of host c-Myc and cyclin E1 [#0]. GRA45 carries an RRL motif that is cleaved by the Golgi-associated aspartyl protease ASP5, though this proteolytic processing is dispensable for its role in effector translocation [#1, #2]. Consistent with its role in enabling host manipulation, GRA45 acts as an IFN-\\u03b3-dependent virulence factor, being required for parasite survival specifically in immune-competent but not Ifngr1-deficient mice [#3].\",\n  \"teleology\": [\n    {\n      \"year\": 2018,\n      \"claim\": \"Establishing GRA45 as a genuine ASP5 substrate defined its maturation pathway and confirmed it as a PV-resident dense granule protein, providing the first biochemical foothold on the protein.\",\n      \"evidence\": \"TAILS N-terminal peptide enrichment and quantitative proteomics comparing wild-type and \\u0394asp5 parasites, with RRL\\u2192ARL mutagenesis validation, in Toxoplasma gondii\",\n      \"pmids\": [\"30377279\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish the functional role of GRA45 or whether cleavage is required for any function\", \"No interacting partners identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identifying GRA45 as a MYR1-interacting protein and showing knockouts fail to translocate GRA16 placed GRA45 in the MYR translocon and defined its central function in effector export and host gene modulation.\",\n      \"evidence\": \"Affinity purification/Co-IP of MYR1 plus GRA45 genetic knockout with GRA16 translocation and host c-Myc/cyclin E1 readouts, in Toxoplasma gondii\",\n      \"pmids\": [\"32075880\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether GRA45 acts directly in the translocation channel or as an accessory/chaperone factor is unresolved\", \"The molecular step in translocation that GRA45 enables is not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Mutating the ASP5 cleavage motif showed that proteolytic maturation is uncoupled from GRA45's translocation function, indicating processing is not a prerequisite for its role in effector export.\",\n      \"evidence\": \"RRL\\u2192ARL site-directed mutagenesis with GRA16 translocation functional assay, in Toxoplasma gondii\",\n      \"pmids\": [\"32075880\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The functional purpose of ASP5 cleavage of GRA45, if any, remains unknown\", \"Single lab, single functional readout\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"An in vivo CRISPR screen with host-genetics epistasis placed GRA45's function in the physiological context of resisting IFN-\\u03b3-mediated immune clearance, connecting effector translocation to virulence.\",\n      \"evidence\": \"In vivo CRISPR screen in C57BL/6 versus Ifngr1-/- mice with comparative enrichment analysis, in Toxoplasma gondii\",\n      \"pmids\": [\"37269286\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which specific effectors/host pathways downstream of GRA45 mediate IFN-\\u03b3 resistance is not defined\", \"Single method, single lab\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown whether GRA45 forms a structural part of the translocation channel or acts as a chaperone/accessory factor, and what the full repertoire of GRA45-dependent effectors and host pathways is.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of GRA45 within the MYR complex\", \"Full effector-dependency profile beyond GRA16 undefined\", \"Molecular mechanism linking GRA45 to IFN-\\u03b3 resistance unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140319\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0020003\", \"supporting_discovery_ids\": [0, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0052372\", \"supporting_discovery_ids\": [0, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\"MYR translocon complex (with MYR1)\"],\n    \"partners\": [\"MYR1\", \"ASP5 (protease)\"],\n    \"other_free_text\": [\n      \"GRA45 is proteolytically processed by ASP5 at an RRL motif, but this cleavage is dispensable for effector translocation.\",\n      \"GRA45 is required for translocation of the dense granule effector GRA16 across the PV membrane and for host c-Myc and cyclin E1 upregulation.\",\n      \"GRA45 is an IFN-\\u03b3-dependent virulence factor, required for parasite survival in immune-competent but not Ifngr1-/- mice.\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_316250","name":"GRA45","product":"dense granule protein GRA45","uniprot_acc":"A0A125YR48","chromosome":"TGME49_chrXI","start_pos":5209487},{"strain":"GT1","gene_id":"TGGT1_316250","name":null,"product":"hypothetical protein","uniprot_acc":null,"chromosome":"TGGT1_chrXI","start_pos":5154312},{"strain":"RH88","gene_id":"TGRH88_054980","name":null,"product":"unspecified product","uniprot_acc":null,"chromosome":"CM023092","start_pos":5276292},{"strain":"VEG","gene_id":"TGVEG_316250","name":null,"product":"hypothetical protein","uniprot_acc":"B6K9R8","chromosome":"TGVEG_chrXI","start_pos":5232708}]}