{"gene":"TG_208830","gene_symbol":"GRA16","run_date":"2026-09-05T22:31:42","timeline":{"discoveries":[{"year":2013,"finding":"GRA16 is a dense granule protein that is exported through the parasitophorous vacuole membrane (PVM) and traffics to the host cell nucleus, where it positively modulates host genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 physically binds two host enzymes: the deubiquitinase HAUSP and PP2A phosphatase. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme.","method":"Co-immunoprecipitation (binding to HAUSP and PP2A), fluorescence/subcellular fractionation (nuclear localization), gene expression analysis, GRA16-deficient strain phenotyping (attenuated virulence)","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP for host binding partners, direct localization by imaging/fractionation, loss-of-function phenotype, multiple orthogonal methods in a single rigorous study","pmids":["23601110"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"TgASP5, a Golgi-resident aspartyl protease homologous to Plasmodium Plasmepsin V, cleaves GRA16 at its PEXEL-like (RRLAE) motif in the N-terminal region, and this cleavage is essential for GRA16 export beyond the PVM and trafficking to the host cell nucleus. In the absence of ASP5, GRA16 is retained within the vacuolar space.","method":"N- and C-terminal epitope tagging demonstrating proteolysis; asp5 knockout/conditional mutants showing retention of GRA16 in vacuole; immunofluorescence localization; genetic complementation","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct proteolysis shown by dual-tag approach, replicated in two independent papers (PMID 26270241 and 26473595) with asp5 genetic knockouts","pmids":["26270241","26473595"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"GRA16 export requires two protein elements in its N-terminal region: a PEXEL-like motif (RRLAE) required for TgASP5-mediated cleavage, and a second element also necessary for export. TgASP5 directly mediates GRA16 maturation and export in a PEXEL-dependent manner.","method":"N- and C-terminal epitope-tag proteolysis assays; asp5 deletion mutants; domain deletion/mutation of GRA16 N-terminus; immunofluorescence","journal":"Cellular microbiology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct proteolysis assay, genetic deletion of protease, domain mutagenesis; single lab but multiple orthogonal methods","pmids":["26270241"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"GRA16 export from the parasitophorous vacuole into the host cytosol/nucleus is dependent on MYR1, a protein at the PVM that forms part of an effector translocation machinery. In myr1 deletion mutants, GRA16 fails to reach the host cell nucleus.","method":"myr1 genetic deletion; immunofluorescence tracking of GRA16 nuclear localization in infected cells; flow cytometry-based screen for host transcriptome reprogramming","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic KO with defined localization phenotype for GRA16; replicated across multiple subsequent studies","pmids":["26838724"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"TGGT1"}]},{"year":2018,"finding":"GRA16 translocation across the PVM into the host cell requires MYR2 and MYR3 in addition to MYR1. MYR2 and MYR3 co-localize with MYR1 at the PVM and are membrane-associated. MYR3, but not MYR2, stably associates with MYR1. Fusing a structured DHFR domain to intrinsically disordered effectors blocks their transport, consistent with a translocon-based mechanism.","method":"Genetic deletion of MYR2/MYR3; immunofluorescence of GRA16 nuclear localization; co-immunoprecipitation of MYR1/MYR3; DHFR-fusion effector transport block assay","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, multiple genetic knockouts, DHFR structural block assay; multiple orthogonal methods","pmids":["29357375"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"TGGT1"}]},{"year":2020,"finding":"Three additional MYR1-interacting proteins—GRA44, GRA45, and MYR4—are essential for translocation of GRA16 into the host cell and for GRA16-dependent upregulation of human c-Myc and cyclin E1 in infected cells. These proteins were identified by affinity purification of MYR1.","method":"MYR1 affinity purification/co-immunoprecipitation; genetic knockout of GRA44, GRA45, MYR4; immunofluorescence of GRA16 nuclear localization; host c-Myc/cyclin E1 upregulation assay","journal":"mSphere","confidence":"High","confidence_rationale":"Tier 2 / Strong — affinity purification plus multiple independent KOs with defined GRA16 translocation phenotype; multiple orthogonal methods","pmids":["32075880"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"TGGT1"}]},{"year":2020,"finding":"GRA16 is responsible for Toxoplasma's upregulation of host c-Myc. Expression of GRA16 in Neospora caninum (which does not upregulate host c-Myc) conferred this ability. Deletion of GRA16 from Toxoplasma severely diminished host c-Myc upregulation.","method":"Heterologous expression of GRA16 in Neospora caninum; gra16 genetic deletion in Toxoplasma; flow cytometry/immunofluorescence for c-Myc levels","journal":"mSphere","confidence":"High","confidence_rationale":"Tier 2 / Strong — gain-of-function in heterologous organism plus loss-of-function in native parasite; two orthogonal genetic approaches","pmids":["32581080"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Neospora caninum","strain":""}]},{"year":2020,"finding":"The secreted parasite phosphatase TgPPM3C regulates GRA16 export by dephosphorylating GRA16; phosphomimetic mutation of two GRA16 serine residues causes export defects, demonstrating that dephosphorylation of GRA16 is a critical step in its export process. Loss of TgPPM3C results in defective GRA16 export but not export of other MYR1-dependent effectors.","method":"pppm3c genetic deletion; phosphoproteomics of vacuolar proteins; phosphomimetic mutagenesis of GRA16 serine residues; immunofluorescence tracking of GRA16 nuclear localization","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — phosphoproteomics plus site-directed phosphomimetic mutagenesis plus genetic KO phenotype; single lab, multiple orthogonal methods","pmids":["33370417"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"GRA16 and GRA24 are not secreted beyond the cyst wall membrane in bradyzoite (tissue cyst) stage Toxoplasma; using tetracycline-inducible HA-tagged expression after stage conversion, these proteins remained within cysts. This establishes that trans-PVM effector export by GRA16 is stage-specific and does not occur in tissue cysts.","method":"Tetracycline-inducible expression of HA-tagged GRA16 after bradyzoite induction; immunofluorescence localization","journal":"Frontiers in cellular and infection microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiment with inducible tagged protein, single lab, single method","pmids":["30406043"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"GRA16 inhibits NF-κB activation through upregulation of PP2A-B55 regulatory subunit in host cells. GRA16 expression directly elevated PP2A-B55 levels, decreased GWL protein levels and ENSA phosphorylation, activated PP2A-B55, and suppressed AKT/ERK phosphorylation, leading to NF-κB inactivation via reduced IKKβ, de-phosphorylation of IκBα, and reduced nuclear transit of NF-κB p65.","method":"Stable transfection of GRA16 in H1299 NSCLC cells; western blotting for PP2A-B55, GWL, ENSA, AKT, ERK, IKKβ, IκBα, NF-κB p65; xenograft mouse model; pharmacological and siRNA pathway dissection","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — gain-of-function in cancer cell line with multiple pathway readouts; single lab, no native parasite infection context for most experiments","pmids":["32927892"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"HAUSP-bound GRA16 preferentially increases p53 stabilization via nuclear localization of PTEN rather than MDM2-dependent mechanisms in p53-wild-type HepG2 cells. GRA16 expression increased PTEN and p53 levels and induced apoptosis in a p53-dependent manner (absent in p53-null Hep3B cells).","method":"Stable GRA16 expression via retrovirus in HepG2 (p53-WT) and Hep3B (p53-null) cells; western blotting for PTEN, p53, MDM2, AKT; nuclear fractionation; xenograft mouse model; co-immunoprecipitation with HAUSP","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — gain-of-function with p53-null negative control, co-IP with HAUSP, multiple readouts; single lab, no mutagenesis of key residues","pmids":["30834688"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"finding":"GRA16, together with TgIST, GRA24, and GRA28, is necessary for a complete block of IFNγ response in human cells post-infection. Loss of GRA16 (among these four effectors) results in parasite premature egress and host cell death in IFNγ-stimulated human cells, a phenotype not observed in murine cells. This protection requires MYR translocon-dependent export of GRA16.","method":"Genome-wide loss-of-function screens in IFNγ-primed human fibroblasts; genetic KO of individual GRA effectors; parasite egress and host cell death assays across multiple human cell lines; comparison with murine cells","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide screen plus individual KO validation with defined cellular phenotype; human-specific phenotype established; single study","pmids":["39292011"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"finding":"GRA16 (alongside GRA18) contributes to CCR7-mediated macrophage hypermigration and dissemination of Toxoplasma, acting in concert with GRA15 (NF-κB pathway) and GRA24 (p38 MAPK pathway). GRA16/18 contributions were identified by effector-deletion experiments in macrophage chemotaxis assays.","method":"GRA effector deletion mutants; CCR7 expression and macrophage chemotaxis assays; in vivo adoptive transfer migration assay in mice","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with defined migration phenotype, in vivo validation; GRA16 role described as contributory, not the primary driver","pmids":["39207098"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"type II"}]},{"year":2024,"finding":"GRA16 can be fused to large therapeutic proteins (>100 kDa) and used as a delivery vehicle to transport them into host cell nuclei, including neurons in vivo. As a proof of concept, GRA16-MeCP2 fusion was delivered to neurons via intraperitoneal administration in mice and distributed throughout the brain.","method":"Translational fusions to GRA16; immunofluorescence and pull-down assays in cultured cells and brain organoids; scRNA-seq; in vivo intraperitoneal administration and 3D brain distribution imaging in mice","journal":"Nature microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal delivery assays in vitro and in vivo; single study demonstrating functional cargo delivery via GRA16 secretion system","pmids":["39075233"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2026,"finding":"GRA16 is one of eight MYR1-dependent effectors (alongside IST, NSM, HCE1/TEEGR, GRA18, GRA24, GRA28, GRA84) that cooperate to activate the non-canonical NF-κB pathway in host cells. T. gondii infection drives depletion of TRAF3, stabilization of NIK, phosphorylation and processing of p100 to p52, and nuclear accumulation of RelB/p52. No single effector alone is responsible; the eight effectors act additively.","method":"Combinatorial knockout panels of GRA effectors; immunofluorescence and western blotting for RelB, p52, TRAF3, NIK, p100 in infected human and murine cells","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, combinatorial KO approach with defined pathway readouts, but GRA16's individual contribution is inferred additively rather than shown in isolation","pmids":["41959509"],"is_preprint":true,"attribution":[{"species":"Toxoplasma gondii","strain":"type I"},{"species":"Toxoplasma gondii","strain":"type II"}]}],"current_model":"GRA16 is a Toxoplasma gondii dense granule protein that is cleaved at its N-terminal PEXEL-like motif by the Golgi protease TgASP5, dephosphorylated by TgPPM3C, and then exported across the parasitophorous vacuole membrane via a translocon complex (MYR1/MYR2/MYR3/MYR4/GRA44/GRA45/ROP17/ASP5) to reach the host cell nucleus, where it binds the host deubiquitinase HAUSP and PP2A phosphatase to modulate p53 stability, cell-cycle gene expression, c-Myc upregulation, and NF-κB signaling, while also contributing to macrophage hypermigration and protection against IFNγ-driven host cell death—collectively enabling parasite virulence and immune evasion."},"narrative":{"mechanistic_narrative":"GRA16 is a Toxoplasma gondii dense granule effector that is exported across the parasitophorous vacuole membrane (PVM) into the host cell, where it reaches the nucleus and reprograms host-cell physiology to favor parasite virulence and immune evasion [PMID:23601110]. Export is a multistep, tightly gated process: GRA16 must first be cleaved at an N-terminal PEXEL-like (RRLAE) motif by the Golgi aspartyl protease TgASP5, without which it is retained in the vacuolar space, and export additionally requires a second N-terminal element [PMID:26270241, PMID:26473595]; it must also be dephosphorylated by the secreted parasite phosphatase TgPPM3C, since phosphomimetic mutation of two serine residues blocks export [PMID:33370417]. Trans-PVM translocation then proceeds through the MYR translocon, requiring MYR1, MYR2, MYR3, MYR4, GRA44, and GRA45, consistent with passage of the unfolded effector through a channel [PMID:26838724, PMID:29357375, PMID:32075880]. In the host nucleus, GRA16 binds the deubiquitinase HAUSP and PP2A phosphatase to modulate the p53 pathway and cell-cycle gene expression, and it is the effector responsible for Toxoplasma's upregulation of host c-Myc [PMID:23601110, PMID:32581080]. Through these host targets GRA16 also inhibits NF-κB activation via PP2A-B55 and contributes, together with other MYR-dependent effectors, to blocking the IFNγ-driven host response and to CCR7-mediated macrophage hypermigration [PMID:32927892, PMID:39292011, PMID:39207098]. Export is stage-specific, occurring in tachyzoites but not in bradyzoite tissue cysts [PMID:30406043].","teleology":[{"year":2013,"claim":"Established that a dense granule protein can act as a nuclear-targeting host effector, defining GRA16 as an exported modulator of host cell-cycle and p53 pathways with defined host-enzyme partners.","evidence":"Reciprocal co-IP for HAUSP and PP2A binding, imaging/fractionation for nuclear localization, and GRA16-deficient strain phenotyping","pmids":["23601110"],"confidence":"High","gaps":["Export machinery not yet identified","Structural basis of HAUSP/PP2A binding unresolved","Direct link between p53 modulation and virulence not dissected"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"Identified the proteolytic gate for export, showing GRA16 is matured by TgASP5 cleavage at a PEXEL-like motif and that this cleavage plus a second N-terminal element are required to escape the vacuole.","evidence":"Dual N-/C-terminal epitope-tag proteolysis assays, asp5 knockout/complementation, and N-terminal domain mutagenesis with immunofluorescence","pmids":["26270241","26473595"],"confidence":"High","gaps":["Identity/function of the second required N-terminal element unresolved","How cleavage licenses translocation mechanistically unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"Defined the trans-PVM translocation requirement, establishing that GRA16 nuclear delivery depends on MYR1 at the PVM.","evidence":"myr1 genetic deletion with immunofluorescence tracking of GRA16 nuclear localization and transcriptome screening","pmids":["26838724"],"confidence":"High","gaps":["Composition of the full translocon unknown at this stage","Whether MYR1 forms a pore not established"],"attribution":[{"species":"Toxoplasma gondii","strain":"TGGT1"}]},{"year":2018,"claim":"Expanded the translocon to a multi-subunit machinery and provided evidence for a channel-based mechanism requiring effector unfolding.","evidence":"MYR2/MYR3 knockouts, MYR1/MYR3 co-IP, and a DHFR structural-fusion transport-block assay","pmids":["29357375"],"confidence":"High","gaps":["Direct demonstration of a pore structure lacking","Energetics/driving force of translocation unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":"TGGT1"}]},{"year":2018,"claim":"Showed that GRA16 export is developmentally restricted, occurring in tachyzoites but not bradyzoite tissue cysts, implying stage-specific host manipulation.","evidence":"Tetracycline-inducible HA-tagged GRA16 after bradyzoite conversion with immunofluorescence localization","pmids":["30406043"],"confidence":"Medium","gaps":["Single-method, single-lab observation","Mechanism restricting export in cysts unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"Refined the p53 mechanism, showing HAUSP-bound GRA16 stabilizes p53 via nuclear PTEN rather than MDM2, driving p53-dependent apoptosis.","evidence":"Stable GRA16 expression in p53-WT HepG2 vs p53-null Hep3B cells, HAUSP co-IP, nuclear fractionation, and xenograft models","pmids":["30834688"],"confidence":"Medium","gaps":["No mutagenesis of GRA16 residues required for HAUSP binding","Relevance to natural infection context untested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"claim":"Completed the translocon parts list and linked it to a defined GRA16-driven host phenotype, showing GRA44/GRA45/MYR4 are needed for c-Myc/cyclin E1 upregulation.","evidence":"MYR1 affinity purification, knockouts of GRA44/GRA45/MYR4, GRA16 localization, and host c-Myc/cyclin E1 assays","pmids":["32075880"],"confidence":"High","gaps":["Stoichiometry/architecture of the assembled translocon unresolved","Direct GRA16 role in c-Myc induction not separated from translocation defect"],"attribution":[{"species":"Toxoplasma gondii","strain":"TGGT1"}]},{"year":2020,"claim":"Assigned host c-Myc upregulation specifically to GRA16 via gain- and loss-of-function, establishing it as the responsible effector.","evidence":"Heterologous GRA16 expression in Neospora caninum and gra16 deletion in Toxoplasma with c-Myc readouts","pmids":["32581080"],"confidence":"High","gaps":["Molecular route from GRA16 to c-Myc transcription undefined","Whether HAUSP/PP2A binding mediates c-Myc effect unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Neospora caninum","strain":""}]},{"year":2020,"claim":"Added a dephosphorylation checkpoint to export, showing TgPPM3C dephosphorylates GRA16 and that this is selectively required for its export.","evidence":"pppm3c knockout, vacuolar phosphoproteomics, and GRA16 serine phosphomimetic mutagenesis with localization tracking","pmids":["33370417"],"confidence":"High","gaps":["Why GRA16 specifically requires dephosphorylation while other effectors do not is unclear","Kinase that phosphorylates these sites not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"claim":"Connected GRA16 to NF-κB suppression via a PP2A-B55/GWL/ENSA axis dampening AKT/ERK and IKKβ signaling.","evidence":"Stable GRA16 expression in H1299 cells with western-blot pathway dissection, siRNA/pharmacology, and xenograft model","pmids":["32927892"],"confidence":"Medium","gaps":["Performed largely outside native infection context","Direct GRA16-PP2A-B55 interaction not shown at residue level"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"claim":"Placed GRA16 within a redundant effector set required to fully block the IFNγ response in human cells, protecting against premature egress and host cell death.","evidence":"Genome-wide loss-of-function screens in IFNγ-primed human fibroblasts, individual GRA knockouts, and egress/death assays with murine comparison","pmids":["39292011"],"confidence":"Medium","gaps":["GRA16's isolated mechanistic contribution not separated from the other three effectors","Human-vs-murine specificity mechanism unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"claim":"Implicated GRA16 as a contributor to CCR7-driven macrophage hypermigration and dissemination alongside other effectors.","evidence":"GRA effector deletion mutants with CCR7 and macrophage chemotaxis assays and in vivo adoptive-transfer migration","pmids":["39207098"],"confidence":"Medium","gaps":["GRA16 described as contributory, not primary driver","Downstream target linking GRA16 to CCR7 unidentified"],"attribution":[{"species":"Toxoplasma gondii","strain":"type II"}]},{"year":2024,"claim":"Repurposed the GRA16 export system as a biotechnology tool, demonstrating nuclear delivery of large therapeutic cargo including into neurons in vivo.","evidence":"GRA16 translational fusions (e.g. GRA16-MeCP2), pull-downs, brain organoids, scRNA-seq, and in vivo intraperitoneal delivery with brain imaging","pmids":["39075233"],"confidence":"Medium","gaps":["Efficiency and safety of delivery not fully characterized","Whether native export determinants limit cargo size undefined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2026,"claim":"Positioned GRA16 as one of eight MYR1-dependent effectors that additively activate the non-canonical NF-κB pathway via TRAF3 depletion and NIK/p100 processing.","evidence":"Combinatorial GRA effector knockout panels with RelB/p52/TRAF3/NIK/p100 readouts in human and murine cells (preprint)","pmids":["41959509"],"confidence":"Low","gaps":["Preprint; GRA16's individual contribution inferred additively, not shown in isolation","Apparent tension with reported GRA16-mediated NF-κB suppression not reconciled"],"attribution":[{"species":"Toxoplasma gondii","strain":"type I"},{"species":"Toxoplasma gondii","strain":"type II"}]},{"year":null,"claim":"How GRA16's host targets (HAUSP, PP2A) mechanistically integrate its multiple downstream effects (p53, c-Myc, NF-κB, IFNγ protection, migration) into a single coherent virulence program remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of GRA16 or its host-partner interfaces","Residue-level mapping of HAUSP/PP2A binding lacking","Divergent NF-κB outcomes (suppression vs non-canonical activation) not reconciled"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["MYR translocon (MYR1/MYR2/MYR3/MYR4/GRA44/GRA45) mediating trans-PVM effector export"],"partners":["HAUSP (HOST DEUBIQUITINASE)","PP2A HOLOENZYME / PP2A-B55 REGULATORY SUBUNIT (HOST PHOSPHATASE)","TGASP5 (PARASITE GOLGI ASPARTYL PROTEASE, PROCESSING)","TGPPM3C (PARASITE PHOSPHATASE, EXPORT REGULATION)","MYR1 (TRANSLOCON COMPONENT, EXPORT)"],"other_free_text":["Cleaved at N-terminal PEXEL-like RRLAE motif by TgASP5; a second N-terminal element is also required for export [#1, #2]","Export gated by TgPPM3C-mediated dephosphorylation of two serine residues [#7]","Export is stage-specific: occurs in tachyzoites, not in bradyzoite tissue cysts [#8]","Identified as the effector responsible for host c-Myc upregulation via heterologous expression in Neospora caninum [#6]","Repurposed as a delivery vehicle for large therapeutic proteins into host nuclei, including neurons in vivo [#13]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"23601110","id":"PMC_23601110","title":"Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression.","date":"2013","source":"Cell host & microbe","url":"https://pubmed.ncbi.nlm.nih.gov/23601110","citation_count":195,"is_preprint":false},{"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":"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":"24373221","id":"PMC_24373221","title":"Toxoplasma exports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression.","date":"2014","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/24373221","citation_count":54,"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":"27447180","id":"PMC_27447180","title":"Secretion of Rhoptry and Dense Granule Effector Proteins by Nonreplicating Toxoplasma gondii Uracil Auxotrophs Controls the Development of Antitumor Immunity.","date":"2016","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/27447180","citation_count":47,"is_preprint":false},{"pmid":"10072322","id":"PMC_10072322","title":"The expression and distribution of dense granule proteins in the enteric (Coccidian) forms of Toxoplasma gondii in the small intestine of the cat.","date":"1999","source":"Experimental parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/10072322","citation_count":46,"is_preprint":false},{"pmid":"32927892","id":"PMC_32927892","title":"Toxoplasma GRA16 Inhibits NF-κB Activation through PP2A-B55 Upregulation in Non-Small-Cell Lung Carcinoma Cells.","date":"2020","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/32927892","citation_count":37,"is_preprint":false},{"pmid":"30834688","id":"PMC_30834688","title":"Increase in the nuclear localization of PTEN by the Toxoplasma GRA16 protein and subsequent induction of p53-dependent apoptosis and anticancer effect.","date":"2019","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/30834688","citation_count":26,"is_preprint":false},{"pmid":"10503251","id":"PMC_10503251","title":"In vivo expression and distribution of dense granule protein 7 (GRA7) in the exoenteric (tachyzoite, bradyzoite) and enteric (coccidian) forms of Toxoplasma gondii.","date":"1999","source":"Parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/10503251","citation_count":26,"is_preprint":false},{"pmid":"30406043","id":"PMC_30406043","title":"Toxoplasma Does Not Secrete the GRA16 and GRA24 Effectors Beyond the Parasitophorous Vacuole Membrane of Tissue Cysts.","date":"2018","source":"Frontiers in cellular and infection microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/30406043","citation_count":21,"is_preprint":false},{"pmid":"39075233","id":"PMC_39075233","title":"Engineering Toxoplasma gondii secretion systems for intracellular delivery of multiple large therapeutic proteins to neurons.","date":"2024","source":"Nature microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/39075233","citation_count":20,"is_preprint":false},{"pmid":"33370417","id":"PMC_33370417","title":"Toxoplasma gondii PPM3C, a secreted protein phosphatase, affects parasitophorous vacuole effector export.","date":"2020","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/33370417","citation_count":20,"is_preprint":false},{"pmid":"32581080","id":"PMC_32581080","title":"Toxoplasma Uses GRA16 To Upregulate Host c-Myc.","date":"2020","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/32581080","citation_count":19,"is_preprint":false},{"pmid":"35810694","id":"PMC_35810694","title":"PTEN/AKT signaling pathway related to hTERT downregulation and telomere shortening induced in Toxoplasma GRA16-expressing colorectal cancer cells.","date":"2022","source":"Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie","url":"https://pubmed.ncbi.nlm.nih.gov/35810694","citation_count":16,"is_preprint":false},{"pmid":"39207098","id":"PMC_39207098","title":"Hypermigration of macrophages through the concerted action of GRA effectors on NF-κB/p38 signaling and host chromatin accessibility potentiates Toxoplasma dissemination.","date":"2024","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/39207098","citation_count":13,"is_preprint":false},{"pmid":"28875149","id":"PMC_28875149","title":"Resistance to Chronic Toxoplasma gondii Infection Induced by a DNA Vaccine Expressing GRA16.","date":"2017","source":"BioMed research international","url":"https://pubmed.ncbi.nlm.nih.gov/28875149","citation_count":13,"is_preprint":false},{"pmid":"39292011","id":"PMC_39292011","title":"A combination of four Toxoplasma gondii nuclear-targeted effectors protects against interferon gamma-driven human host cell death.","date":"2024","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/39292011","citation_count":8,"is_preprint":false},{"pmid":"40556512","id":"PMC_40556512","title":"Toxoplasma gondii GRA16 Suppresses Aerobic Glycolysis by Downregulating c-Myc and TERT Expressions in Colorectal Cancer Cells.","date":"2025","source":"Biomolecules & therapeutics","url":"https://pubmed.ncbi.nlm.nih.gov/40556512","citation_count":4,"is_preprint":false},{"pmid":"40389494","id":"PMC_40389494","title":"Toxoplasma GRA16 attenuates Tau hyperphosphorylation and enhances autophagy in thrombin-treated HT-22 hippocampal neuronal cells.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/40389494","citation_count":2,"is_preprint":false},{"pmid":"41036225","id":"PMC_41036225","title":"Proteome changes during in vitro culture adaptation of Toxoplasma gondii archetypal II and III field isolates.","date":"2025","source":"Frontiers in cellular and infection microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/41036225","citation_count":1,"is_preprint":false},{"pmid":"38370679","id":"PMC_38370679","title":"Hypermigration of macrophages through the concerted action of GRA effectors on NF-κB/p38 signaling and host chromatin accessibility potentiates Toxoplasma dissemination.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/38370679","citation_count":1,"is_preprint":false},{"pmid":"38234811","id":"PMC_38234811","title":"A Combination of Four Nuclear Targeted Effectors Protects Toxoplasma Against Interferon Gamma Driven Human Host Cell Death During Acute Infection.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/38234811","citation_count":1,"is_preprint":false},{"pmid":"26695219","id":"PMC_26695219","title":"Sequence variation and bioinformatics analysis of Toxoplasma gondii GRA16 Gene.","date":"2015","source":"Tropical biomedicine","url":"https://pubmed.ncbi.nlm.nih.gov/26695219","citation_count":0,"is_preprint":false},{"pmid":"42108599","id":"PMC_42108599","title":"The Potential Function of Toxoplasma gondii in Tumour Immunotherapy.","date":"2026","source":"Immunology","url":"https://pubmed.ncbi.nlm.nih.gov/42108599","citation_count":0,"is_preprint":false},{"pmid":"41959509","id":"PMC_41959509","title":"A Suite of Eight Toxoplasma gondii Effectors Cooperates to Activate the Non-canonical NF-κB Pathway.","date":"2026","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41959509","citation_count":0,"is_preprint":false},{"pmid":"31366706","id":"PMC_31366706","title":"Neighbors Working Together: a Toxoplasma Rhoptry Protein That Facilitates Dense Granule Protein Translocation into the Host 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GRA16 physically binds two host enzymes: the deubiquitinase HAUSP and PP2A phosphatase. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme.\",\n      \"method\": \"Co-immunoprecipitation (binding to HAUSP and PP2A), fluorescence/subcellular fractionation (nuclear localization), gene expression analysis, GRA16-deficient strain phenotyping (attenuated virulence)\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP for host binding partners, direct localization by imaging/fractionation, loss-of-function phenotype, multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"23601110\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TgASP5, a Golgi-resident aspartyl protease homologous to Plasmodium Plasmepsin V, cleaves GRA16 at its PEXEL-like (RRLAE) motif in the N-terminal region, and this cleavage is essential for GRA16 export beyond the PVM and trafficking to the host cell nucleus. In the absence of ASP5, GRA16 is retained within the vacuolar space.\",\n      \"method\": \"N- and C-terminal epitope tagging demonstrating proteolysis; asp5 knockout/conditional mutants showing retention of GRA16 in vacuole; immunofluorescence localization; genetic complementation\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct proteolysis shown by dual-tag approach, replicated in two independent papers (PMID 26270241 and 26473595) with asp5 genetic knockouts\",\n      \"pmids\": [\"26270241\", \"26473595\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"GRA16 export requires two protein elements in its N-terminal region: a PEXEL-like motif (RRLAE) required for TgASP5-mediated cleavage, and a second element also necessary for export. TgASP5 directly mediates GRA16 maturation and export in a PEXEL-dependent manner.\",\n      \"method\": \"N- and C-terminal epitope-tag proteolysis assays; asp5 deletion mutants; domain deletion/mutation of GRA16 N-terminus; immunofluorescence\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct proteolysis assay, genetic deletion of protease, domain mutagenesis; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"26270241\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"GRA16 export from the parasitophorous vacuole into the host cytosol/nucleus is dependent on MYR1, a protein at the PVM that forms part of an effector translocation machinery. In myr1 deletion mutants, GRA16 fails to reach the host cell nucleus.\",\n      \"method\": \"myr1 genetic deletion; immunofluorescence tracking of GRA16 nuclear localization in infected cells; flow cytometry-based screen for host transcriptome reprogramming\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic KO with defined localization phenotype for GRA16; replicated across multiple subsequent studies\",\n      \"pmids\": [\"26838724\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"TGGT1\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"GRA16 translocation across the PVM into the host cell requires MYR2 and MYR3 in addition to MYR1. MYR2 and MYR3 co-localize with MYR1 at the PVM and are membrane-associated. MYR3, but not MYR2, stably associates with MYR1. Fusing a structured DHFR domain to intrinsically disordered effectors blocks their transport, consistent with a translocon-based mechanism.\",\n      \"method\": \"Genetic deletion of MYR2/MYR3; immunofluorescence of GRA16 nuclear localization; co-immunoprecipitation of MYR1/MYR3; DHFR-fusion effector transport block assay\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, multiple genetic knockouts, DHFR structural block assay; multiple orthogonal methods\",\n      \"pmids\": [\"29357375\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"TGGT1\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Three additional MYR1-interacting proteins—GRA44, GRA45, and MYR4—are essential for translocation of GRA16 into the host cell and for GRA16-dependent upregulation of human c-Myc and cyclin E1 in infected cells. These proteins were identified by affinity purification of MYR1.\",\n      \"method\": \"MYR1 affinity purification/co-immunoprecipitation; genetic knockout of GRA44, GRA45, MYR4; immunofluorescence of GRA16 nuclear localization; host c-Myc/cyclin E1 upregulation assay\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — affinity purification plus multiple independent KOs with defined GRA16 translocation phenotype; multiple orthogonal methods\",\n      \"pmids\": [\"32075880\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"TGGT1\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA16 is responsible for Toxoplasma's upregulation of host c-Myc. Expression of GRA16 in Neospora caninum (which does not upregulate host c-Myc) conferred this ability. Deletion of GRA16 from Toxoplasma severely diminished host c-Myc upregulation.\",\n      \"method\": \"Heterologous expression of GRA16 in Neospora caninum; gra16 genetic deletion in Toxoplasma; flow cytometry/immunofluorescence for c-Myc levels\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — gain-of-function in heterologous organism plus loss-of-function in native parasite; two orthogonal genetic approaches\",\n      \"pmids\": [\"32581080\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Neospora caninum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The secreted parasite phosphatase TgPPM3C regulates GRA16 export by dephosphorylating GRA16; phosphomimetic mutation of two GRA16 serine residues causes export defects, demonstrating that dephosphorylation of GRA16 is a critical step in its export process. Loss of TgPPM3C results in defective GRA16 export but not export of other MYR1-dependent effectors.\",\n      \"method\": \"pppm3c genetic deletion; phosphoproteomics of vacuolar proteins; phosphomimetic mutagenesis of GRA16 serine residues; immunofluorescence tracking of GRA16 nuclear localization\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — phosphoproteomics plus site-directed phosphomimetic mutagenesis plus genetic KO phenotype; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"33370417\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"GRA16 and GRA24 are not secreted beyond the cyst wall membrane in bradyzoite (tissue cyst) stage Toxoplasma; using tetracycline-inducible HA-tagged expression after stage conversion, these proteins remained within cysts. This establishes that trans-PVM effector export by GRA16 is stage-specific and does not occur in tissue cysts.\",\n      \"method\": \"Tetracycline-inducible expression of HA-tagged GRA16 after bradyzoite induction; immunofluorescence localization\",\n      \"journal\": \"Frontiers in cellular and infection microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment with inducible tagged protein, single lab, single method\",\n      \"pmids\": [\"30406043\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GRA16 inhibits NF-κB activation through upregulation of PP2A-B55 regulatory subunit in host cells. GRA16 expression directly elevated PP2A-B55 levels, decreased GWL protein levels and ENSA phosphorylation, activated PP2A-B55, and suppressed AKT/ERK phosphorylation, leading to NF-κB inactivation via reduced IKKβ, de-phosphorylation of IκBα, and reduced nuclear transit of NF-κB p65.\",\n      \"method\": \"Stable transfection of GRA16 in H1299 NSCLC cells; western blotting for PP2A-B55, GWL, ENSA, AKT, ERK, IKKβ, IκBα, NF-κB p65; xenograft mouse model; pharmacological and siRNA pathway dissection\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — gain-of-function in cancer cell line with multiple pathway readouts; single lab, no native parasite infection context for most experiments\",\n      \"pmids\": [\"32927892\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HAUSP-bound GRA16 preferentially increases p53 stabilization via nuclear localization of PTEN rather than MDM2-dependent mechanisms in p53-wild-type HepG2 cells. GRA16 expression increased PTEN and p53 levels and induced apoptosis in a p53-dependent manner (absent in p53-null Hep3B cells).\",\n      \"method\": \"Stable GRA16 expression via retrovirus in HepG2 (p53-WT) and Hep3B (p53-null) cells; western blotting for PTEN, p53, MDM2, AKT; nuclear fractionation; xenograft mouse model; co-immunoprecipitation with HAUSP\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — gain-of-function with p53-null negative control, co-IP with HAUSP, multiple readouts; single lab, no mutagenesis of key residues\",\n      \"pmids\": [\"30834688\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"GRA16, together with TgIST, GRA24, and GRA28, is necessary for a complete block of IFNγ response in human cells post-infection. Loss of GRA16 (among these four effectors) results in parasite premature egress and host cell death in IFNγ-stimulated human cells, a phenotype not observed in murine cells. This protection requires MYR translocon-dependent export of GRA16.\",\n      \"method\": \"Genome-wide loss-of-function screens in IFNγ-primed human fibroblasts; genetic KO of individual GRA effectors; parasite egress and host cell death assays across multiple human cell lines; comparison with murine cells\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide screen plus individual KO validation with defined cellular phenotype; human-specific phenotype established; single study\",\n      \"pmids\": [\"39292011\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"GRA16 (alongside GRA18) contributes to CCR7-mediated macrophage hypermigration and dissemination of Toxoplasma, acting in concert with GRA15 (NF-κB pathway) and GRA24 (p38 MAPK pathway). GRA16/18 contributions were identified by effector-deletion experiments in macrophage chemotaxis assays.\",\n      \"method\": \"GRA effector deletion mutants; CCR7 expression and macrophage chemotaxis assays; in vivo adoptive transfer migration assay in mice\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with defined migration phenotype, in vivo validation; GRA16 role described as contributory, not the primary driver\",\n      \"pmids\": [\"39207098\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"GRA16 can be fused to large therapeutic proteins (>100 kDa) and used as a delivery vehicle to transport them into host cell nuclei, including neurons in vivo. As a proof of concept, GRA16-MeCP2 fusion was delivered to neurons via intraperitoneal administration in mice and distributed throughout the brain.\",\n      \"method\": \"Translational fusions to GRA16; immunofluorescence and pull-down assays in cultured cells and brain organoids; scRNA-seq; in vivo intraperitoneal administration and 3D brain distribution imaging in mice\",\n      \"journal\": \"Nature microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal delivery assays in vitro and in vivo; single study demonstrating functional cargo delivery via GRA16 secretion system\",\n      \"pmids\": [\"39075233\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"GRA16 is one of eight MYR1-dependent effectors (alongside IST, NSM, HCE1/TEEGR, GRA18, GRA24, GRA28, GRA84) that cooperate to activate the non-canonical NF-κB pathway in host cells. T. gondii infection drives depletion of TRAF3, stabilization of NIK, phosphorylation and processing of p100 to p52, and nuclear accumulation of RelB/p52. No single effector alone is responsible; the eight effectors act additively.\",\n      \"method\": \"Combinatorial knockout panels of GRA effectors; immunofluorescence and western blotting for RelB, p52, TRAF3, NIK, p100 in infected human and murine cells\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, combinatorial KO approach with defined pathway readouts, but GRA16's individual contribution is inferred additively rather than shown in isolation\",\n      \"pmids\": [\"41959509\"],\n      \"is_preprint\": true,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}]\n    }\n  ],\n  \"current_model\": \"GRA16 is a Toxoplasma gondii dense granule protein that is cleaved at its N-terminal PEXEL-like motif by the Golgi protease TgASP5, dephosphorylated by TgPPM3C, and then exported across the parasitophorous vacuole membrane via a translocon complex (MYR1/MYR2/MYR3/MYR4/GRA44/GRA45/ROP17/ASP5) to reach the host cell nucleus, where it binds the host deubiquitinase HAUSP and PP2A phosphatase to modulate p53 stability, cell-cycle gene expression, c-Myc upregulation, and NF-κB signaling, while also contributing to macrophage hypermigration and protection against IFNγ-driven host cell death—collectively enabling parasite virulence and immune evasion.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GRA16 is a Toxoplasma gondii dense granule effector that is exported across the parasitophorous vacuole membrane (PVM) into the host cell, where it reaches the nucleus and reprograms host-cell physiology to favor parasite virulence and immune evasion [#0]. Export is a multistep, tightly gated process: GRA16 must first be cleaved at an N-terminal PEXEL-like (RRLAE) motif by the Golgi aspartyl protease TgASP5, without which it is retained in the vacuolar space, and export additionally requires a second N-terminal element [#1, #2]; it must also be dephosphorylated by the secreted parasite phosphatase TgPPM3C, since phosphomimetic mutation of two serine residues blocks export [#7]. Trans-PVM translocation then proceeds through the MYR translocon, requiring MYR1, MYR2, MYR3, MYR4, GRA44, and GRA45, consistent with passage of the unfolded effector through a channel [#3, #4, #5]. In the host nucleus, GRA16 binds the deubiquitinase HAUSP and PP2A phosphatase to modulate the p53 pathway and cell-cycle gene expression, and it is the effector responsible for Toxoplasma's upregulation of host c-Myc [#0, #6]. Through these host targets GRA16 also inhibits NF-\\u03baB activation via PP2A-B55 and contributes, together with other MYR-dependent effectors, to blocking the IFN\\u03b3-driven host response and to CCR7-mediated macrophage hypermigration [#9, #11, #12]. Export is stage-specific, occurring in tachyzoites but not in bradyzoite tissue cysts [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2013,\n      \"claim\": \"Established that a dense granule protein can act as a nuclear-targeting host effector, defining GRA16 as an exported modulator of host cell-cycle and p53 pathways with defined host-enzyme partners.\",\n      \"evidence\": \"Reciprocal co-IP for HAUSP and PP2A binding, imaging/fractionation for nuclear localization, and GRA16-deficient strain phenotyping\",\n      \"pmids\": [\"23601110\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Export machinery not yet identified\", \"Structural basis of HAUSP/PP2A binding unresolved\", \"Direct link between p53 modulation and virulence not dissected\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified the proteolytic gate for export, showing GRA16 is matured by TgASP5 cleavage at a PEXEL-like motif and that this cleavage plus a second N-terminal element are required to escape the vacuole.\",\n      \"evidence\": \"Dual N-/C-terminal epitope-tag proteolysis assays, asp5 knockout/complementation, and N-terminal domain mutagenesis with immunofluorescence\",\n      \"pmids\": [\"26270241\", \"26473595\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity/function of the second required N-terminal element unresolved\", \"How cleavage licenses translocation mechanistically unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined the trans-PVM translocation requirement, establishing that GRA16 nuclear delivery depends on MYR1 at the PVM.\",\n      \"evidence\": \"myr1 genetic deletion with immunofluorescence tracking of GRA16 nuclear localization and transcriptome screening\",\n      \"pmids\": [\"26838724\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Composition of the full translocon unknown at this stage\", \"Whether MYR1 forms a pore not established\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"TGGT1\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Expanded the translocon to a multi-subunit machinery and provided evidence for a channel-based mechanism requiring effector unfolding.\",\n      \"evidence\": \"MYR2/MYR3 knockouts, MYR1/MYR3 co-IP, and a DHFR structural-fusion transport-block assay\",\n      \"pmids\": [\"29357375\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct demonstration of a pore structure lacking\", \"Energetics/driving force of translocation unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"TGGT1\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed that GRA16 export is developmentally restricted, occurring in tachyzoites but not bradyzoite tissue cysts, implying stage-specific host manipulation.\",\n      \"evidence\": \"Tetracycline-inducible HA-tagged GRA16 after bradyzoite conversion with immunofluorescence localization\",\n      \"pmids\": [\"30406043\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-method, single-lab observation\", \"Mechanism restricting export in cysts unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Refined the p53 mechanism, showing HAUSP-bound GRA16 stabilizes p53 via nuclear PTEN rather than MDM2, driving p53-dependent apoptosis.\",\n      \"evidence\": \"Stable GRA16 expression in p53-WT HepG2 vs p53-null Hep3B cells, HAUSP co-IP, nuclear fractionation, and xenograft models\",\n      \"pmids\": [\"30834688\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No mutagenesis of GRA16 residues required for HAUSP binding\", \"Relevance to natural infection context untested\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Completed the translocon parts list and linked it to a defined GRA16-driven host phenotype, showing GRA44/GRA45/MYR4 are needed for c-Myc/cyclin E1 upregulation.\",\n      \"evidence\": \"MYR1 affinity purification, knockouts of GRA44/GRA45/MYR4, GRA16 localization, and host c-Myc/cyclin E1 assays\",\n      \"pmids\": [\"32075880\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry/architecture of the assembled translocon unresolved\", \"Direct GRA16 role in c-Myc induction not separated from translocation defect\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"TGGT1\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Assigned host c-Myc upregulation specifically to GRA16 via gain- and loss-of-function, establishing it as the responsible effector.\",\n      \"evidence\": \"Heterologous GRA16 expression in Neospora caninum and gra16 deletion in Toxoplasma with c-Myc readouts\",\n      \"pmids\": [\"32581080\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular route from GRA16 to c-Myc transcription undefined\", \"Whether HAUSP/PP2A binding mediates c-Myc effect unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Neospora caninum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Added a dephosphorylation checkpoint to export, showing TgPPM3C dephosphorylates GRA16 and that this is selectively required for its export.\",\n      \"evidence\": \"pppm3c knockout, vacuolar phosphoproteomics, and GRA16 serine phosphomimetic mutagenesis with localization tracking\",\n      \"pmids\": [\"33370417\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why GRA16 specifically requires dephosphorylation while other effectors do not is unclear\", \"Kinase that phosphorylates these sites not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Connected GRA16 to NF-\\u03baB suppression via a PP2A-B55/GWL/ENSA axis dampening AKT/ERK and IKK\\u03b2 signaling.\",\n      \"evidence\": \"Stable GRA16 expression in H1299 cells with western-blot pathway dissection, siRNA/pharmacology, and xenograft model\",\n      \"pmids\": [\"32927892\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Performed largely outside native infection context\", \"Direct GRA16-PP2A-B55 interaction not shown at residue level\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placed GRA16 within a redundant effector set required to fully block the IFN\\u03b3 response in human cells, protecting against premature egress and host cell death.\",\n      \"evidence\": \"Genome-wide loss-of-function screens in IFN\\u03b3-primed human fibroblasts, individual GRA knockouts, and egress/death assays with murine comparison\",\n      \"pmids\": [\"39292011\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"GRA16's isolated mechanistic contribution not separated from the other three effectors\", \"Human-vs-murine specificity mechanism unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Implicated GRA16 as a contributor to CCR7-driven macrophage hypermigration and dissemination alongside other effectors.\",\n      \"evidence\": \"GRA effector deletion mutants with CCR7 and macrophage chemotaxis assays and in vivo adoptive-transfer migration\",\n      \"pmids\": [\"39207098\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"GRA16 described as contributory, not primary driver\", \"Downstream target linking GRA16 to CCR7 unidentified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Repurposed the GRA16 export system as a biotechnology tool, demonstrating nuclear delivery of large therapeutic cargo including into neurons in vivo.\",\n      \"evidence\": \"GRA16 translational fusions (e.g. GRA16-MeCP2), pull-downs, brain organoids, scRNA-seq, and in vivo intraperitoneal delivery with brain imaging\",\n      \"pmids\": [\"39075233\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Efficiency and safety of delivery not fully characterized\", \"Whether native export determinants limit cargo size undefined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Positioned GRA16 as one of eight MYR1-dependent effectors that additively activate the non-canonical NF-\\u03baB pathway via TRAF3 depletion and NIK/p100 processing.\",\n      \"evidence\": \"Combinatorial GRA effector knockout panels with RelB/p52/TRAF3/NIK/p100 readouts in human and murine cells (preprint)\",\n      \"pmids\": [\"41959509\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Preprint; GRA16's individual contribution inferred additively, not shown in isolation\", \"Apparent tension with reported GRA16-mediated NF-\\u03baB suppression not reconciled\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"type I\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"type II\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How GRA16's host targets (HAUSP, PP2A) mechanistically integrate its multiple downstream effects (p53, c-Myc, NF-\\u03baB, IFN\\u03b3 protection, migration) into a single coherent virulence program remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of GRA16 or its host-partner interfaces\", \"Residue-level mapping of HAUSP/PP2A binding lacking\", \"Divergent NF-\\u03baB outcomes (suppression vs non-canonical activation) not reconciled\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0019904\", \"supporting_discovery_ids\": [0, 10], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0140677\", \"supporting_discovery_ids\": [0, 6, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0042025\", \"supporting_discovery_ids\": [0, 3, 4, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0020003\", \"supporting_discovery_ids\": [1, 2, 8], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0072331\", \"supporting_discovery_ids\": [0, 10], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0043123\", \"supporting_discovery_ids\": [9, 14], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0140546\", \"supporting_discovery_ids\": [11], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0007204\", \"supporting_discovery_ids\": [0, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"MYR translocon (MYR1/MYR2/MYR3/MYR4/GRA44/GRA45) mediating trans-PVM effector export\"\n    ],\n    \"partners\": [\n      \"HAUSP (host deubiquitinase)\",\n      \"PP2A holoenzyme / PP2A-B55 regulatory subunit (host phosphatase)\",\n      \"TgASP5 (parasite Golgi aspartyl protease, processing)\",\n      \"TgPPM3C (parasite phosphatase, export regulation)\",\n      \"MYR1 (translocon component, export)\"\n    ],\n    \"other_free_text\": [\n      \"Cleaved at N-terminal PEXEL-like RRLAE motif by TgASP5; a second N-terminal element is also required for export [#1, #2]\",\n      \"Export gated by TgPPM3C-mediated dephosphorylation of two serine residues [#7]\",\n      \"Export is stage-specific: occurs in tachyzoites, not in bradyzoite tissue cysts [#8]\",\n      \"Identified as the effector responsible for host c-Myc upregulation via heterologous expression in Neospora caninum [#6]\",\n      \"Repurposed as a delivery vehicle for large therapeutic proteins into host nuclei, including neurons in vivo [#13]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_208830","name":"GRA16","product":"dense granule protein GRA16","uniprot_acc":"A0A125YUV5","chromosome":"TGME49_chrIb","start_pos":888008},{"strain":"GT1","gene_id":"TGGT1_208830","name":null,"product":"hypothetical protein","uniprot_acc":null,"chromosome":"TGGT1_chrIb","start_pos":904648},{"strain":"RH88","gene_id":"TGRH88_022180","name":null,"product":"unspecified product","uniprot_acc":null,"chromosome":"CM023082","start_pos":1121203},{"strain":"VEG","gene_id":"TGVEG_208830","name":null,"product":"hypothetical protein","uniprot_acc":"B6KP80","chromosome":"TGVEG_chrIb","start_pos":887633}]}