{"gene":"TG_207680","gene_symbol":"SKP1","run_date":"2026-09-08T11:06:18","timeline":{"discoveries":[{"year":2012,"finding":"TgPhyA (prolyl 4-hydroxylase) is required for Skp1 glycosylation in Toxoplasma; disruption of the TgphyA locus abolishes Skp1 modification and causes slow growth at physiological O2 levels. Bacterially expressed TgPhyA can prolyl-hydroxylate both Toxoplasma and Dictyostelium Skp1 in vitro, and TgPhyA shows a higher apparent affinity for O2 than DdPhyA.","method":"Genetic locus disruption, in vitro prolyl hydroxylase assay with recombinant TgPhyA, kinetic analysis of O2/α-ketoglutarate dependence, complementation in DdphyA-null cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro enzymatic assay with recombinant protein, genetic disruption with growth phenotype, complementation rescue, multiple orthogonal methods in one study","pmids":["22648409"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Dictyostelium discoideum","strain":""}]},{"year":2015,"finding":"TgSkp1 hydroxyproline is modified by a pentasaccharide assembled sequentially; the first three sugars (GlcNAc, Gal, Fuc) are added by Toxoplasma homologs of Gnt1 and PgtA. Disruption of gnt1 or pgtA reduces parasite growth, rescued by genetic complementation restoring normal glycan and growth.","method":"Mass spectrometry of glycoforms, radioactive sugar nucleotide glycosyltransferase assays in cell extracts, acid hydrolysate chromatography, genetic knockout and complementation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — biochemical reconstitution of glycosyltransferase reactions, MS characterization, genetic KO with complementation, multiple orthogonal methods","pmids":["26719340"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"NMR and molecular dynamics simulations show that the pentasaccharide on Skp1 HyPro-143 interacts with the loop connecting two α-helices of the F-box-combining site, causing the helices to separate and create a more accessible, dynamic F-box interface. This conformational opening explains how glycosylation enhances Skp1 interactions with F-box proteins.","method":"NMR spectroscopy, mass spectrometry, all-atom molecular dynamics simulations with calculated monosaccharide relaxation kinetics validated against NMR data","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure with MD simulations validated by experimental relaxation kinetics; multiple orthogonal biophysical methods in one study","pmids":["28928219"],"is_preprint":false,"attribution":[{"species":"Dictyostelium discoideum","strain":""},{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"The cytoplasmic glycosyltransferase Glt1 (CAZy family GT32) transfers the fourth sugar (glucose) to TgSkp1, forming a Glcα1,3Fuc linkage. Recombinant Glt1 catalyzes UDP-glucose transfer specifically to the trisaccharide form of Skp1; glt1-knockout parasites exhibit a growth defect not rescued by catalytically inactive Glt1.","method":"In vitro glycosyltransferase assay with recombinant Glt1 and [3H]UDP-glucose, NMR linkage determination, glt1 knockout, catalytic-dead Glt1 rescue experiment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — recombinant enzyme assay, NMR, genetic KO with catalytic-dead rescue, multiple orthogonal methods in one study","pmids":["28928220"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"TgFBXO1 interacts with Toxoplasma SKP1 (confirming it as a bona fide F-box protein), localizes to the Inner Membrane Complex in interphase parasites and to the daughter cell scaffold during replication. This localization requires N-myristoylation of TgFBXO1 and depends on TgRab11b. TgFBXO1 is required for parasite growth through its function as a daughter cell scaffold effector.","method":"Co-immunoprecipitation (SKP1–FBXO1 interaction), live imaging and immunofluorescence for localization, myristoylation mutant analysis, TgRab11b-dependent localization assay, conditional knockdown with growth phenotype readout","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, localization by imaging with functional consequence, multiple mutants tested, clean KO growth phenotype, multiple orthogonal methods","pmids":["31348812"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"The terminal (fifth) sugar of TgSkp1 is added by Gat1, a glycogenin-related glycosyltransferase that forms a Galα1,3Glcα linkage rather than the Glcα1,4Glcα linkage formed by glycogenin. Crystallographic and kinetic analyses show Gat1 is specific for Skp1. gat1Δ parasites exhibit a slow-growth phenotype, indicating the fifth sugar is functionally important.","method":"Glycosyltransferase biochemical assay, X-ray crystallography, kinetic analysis, gat1 genetic knockout with growth phenotype, computational conformational analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure, in vitro enzymatic assay, genetic KO with phenotype, multiple orthogonal methods in one study","pmids":["32414843"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"Biochemical and biophysical comparison of TgPhyA and DdPhyA shows that TgPhyA is more active at low O2 concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate; no cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. Crystallographic analysis of DdPhyA reveals absence of the C-terminal helix present in animal PHDs.","method":"In vitro prolyl hydroxylase kinetic assays with full-length Skp1 substrates, X-ray crystallography of DdPhyA","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic assays with defined substrates and kinetics, crystal structure, multiple cross-species substrate tests","pmids":["32934009"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Dictyostelium discoideum","strain":""}]},{"year":2024,"finding":"O2-dependent SKP1 glycosylation (mediated by PHYa) regulates the SKP1 interactome in Toxoplasma: loss of PHYa substantially reduces the representation of F-box proteins FBXO13 and FBXO14 in the SKP1 interactome. Their reduced abundance is rescued by proteasomal inhibitors and is phenocopied at 0.5% O2, indicating SCF-mediated degradation when SKP1 is hypoglycosylated. FBXO1 abundance is not affected by PHYa loss.","method":"Interactome/co-immunoprecipitation-MS in PHYaΔ vs. wild-type parasites, proteasomal inhibitor rescue, epitope-tagging at genetic locus, transcript-level controls, low-O2 phenocopy experiment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interactome MS, multiple genetic mutants, proteasome inhibitor rescue, O2-phenocopy, multiple orthogonal methods","pmids":["39307307"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"finding":"TgPHYa (which hydroxylates SKP1) is required for Toxoplasma virulence and brain cyst formation in mice (ME49 strain). PHYa-knockout parasites fail to disseminate to peripheral tissues because they cannot survive within recruited immune cells; this survival defect requires IFNγ. TgPHYa is specifically required for tryptophan scavenging when IDO is induced by IFNγ, providing the first parasite mechanism for evading IFNγ-induced nutritional immunity via the O2-sensing/SKP1 pathway.","method":"TgPHYa genetic knockout in ME49, mouse infection model, IFNγ-KO mouse experiments, IDO-KO mouse experiments, intracellular survival assays in IFNγ-treated cells","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic KO, multiple mouse genetic backgrounds, mechanistic pathway placement via IFNγ/IDO axis, multiple orthogonal in vivo and in vitro assays","pmids":["38857298"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2024,"finding":"TgFBXL2 localizes to perinucleolar sites and is stably engaged in an SCF-E3 complex that is also associated with the COP9 signalosome (a negative regulator of SCF function). Depletion of TgFBXL2 causes centrosome replication defects, daughter cell development failure, and induction of stage-specific (sexual commitment) genes. TgFBXL2 interacts with SKP1 as part of this complex.","method":"Co-immunoprecipitation/complex characterization, conditional depletion, RNAseq, live-cell imaging for centrosome/daughter cell phenotype","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP identifying complex components, conditional KD with multiple defined phenotypic readouts, RNAseq, multiple orthogonal methods","pmids":["38814984"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"finding":"Glycosylation of TgSkp1's disordered C-terminal region (CTR) weakens Skp1 homodimerization. The CTR promotes homodimerization via electrostatic (charge cluster) interactions that are disrupted by glycosylation or CTR deletion, freeing Skp1 for F-box protein binding. Replacement with poly-serine (but not a randomized sequence) recapitulates the weakening, indicating composition-dependent electrostatic dependence. Molecular dynamics simulations support the mechanism.","method":"Sedimentation velocity analytical ultracentrifugation, CTR deletion and substitution mutants, high-salt experiments, all-atom molecular dynamics simulations","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — quantitative biophysical assay (AUC), multiple mutant constructs, MD simulations, multiple orthogonal methods in one study","pmids":["40296701"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2026,"finding":"The terminal SKP1 glycosyltransferase GAT1 forms a native homodimer that complexes with SKP1 monomers with affinity and stoichiometry dictated by SKP1 glycosylation state. GAT1 competes with the same core hydrophobic interface used by F-box proteins and the SKP1 homodimer. Substoichiometric GAT1 can promote SKP1 monomerization in a CTR-dependent manner, suggesting a non-enzymatic role for GAT1 in controlling SKP1 FBP-binding preference.","method":"Sedimentation velocity analytical ultracentrifugation, computational modeling, mutational probing of the hydrophobic interface, gat1-knockout cell interactome analysis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — AUC and mutational probing with computational modeling, preprint, single lab, novel non-enzymatic mechanism proposed but supported by experimental data","pmids":["42327091"],"is_preprint":true,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"Toxoplasma SKP1 is an adaptor subunit of SCF-class E3 ubiquitin ligases that is uniquely regulated by O2-dependent prolyl hydroxylation (by TgPHYa/PhyA at Pro-154) followed by sequential cytoplasmic glycosylation with a pentasaccharide (assembled by Gnt1, PgtA, Glt1, and Gat1); this glycan promotes an open, accessible conformation of the F-box-binding domain by disrupting both SKP1 homodimerization (via the disordered C-terminal region) and GAT1 competition at the F-box interface, thereby selectively stabilizing distinct F-box protein substrate receptors (e.g., FBXO13, FBXO14 but not FBXO1) and allowing the parasite to sense and respond to O2 levels, regulate its proteome during replication and stage conversion, and survive IFNγ-mediated host immunity."},"narrative":{"mechanistic_narrative":"Toxoplasma SKP1 is the adaptor subunit of SCF-class E3 ubiquitin ligases whose engagement of F-box protein substrate receptors is gated by an O2-dependent post-translational modification cascade unique to this lineage [PMID:39307307]. A prolyl 4-hydroxylase (TgPhyA/PHYa) hydroxylates a specific SKP1 proline in an O2-dependent reaction, and TgPhyA is tuned for activity at low O2, allowing SKP1 modification to report on ambient oxygen [PMID:22648409, PMID:32934009]. The resulting hydroxyproline is elaborated into a pentasaccharide by sequential cytoplasmic glycosyltransferases — Gnt1 and PgtA add the first three sugars, Glt1 (GT32) adds a fourth glucose in a Glcα1,3Fuc linkage, and the glycogenin-related Gat1 caps the chain with a Galα1,3Glcα linkage — each step being functionally important for normal growth [PMID:26719340, PMID:28928220, PMID:32414843]. This glycan controls F-box binding through two coupled conformational effects: it remodels the loop between the two α-helices of the F-box-combining site to open a more accessible interface [PMID:28928219], and it weakens SKP1 homodimerization mediated by electrostatic charge clusters in the disordered C-terminal region, freeing SKP1 monomers for F-box protein capture [PMID:40296701]. The terminal enzyme GAT1 additionally acts non-enzymatically as a homodimer that competes for the same hydrophobic interface used by F-box proteins and the SKP1 homodimer, further shaping F-box-binding preference in a glycosylation- and CTR-dependent manner [PMID:42327091]. Functionally, loss of PHYa-dependent glycosylation selectively depletes the F-box receptors FBXO13 and FBXO14 from the SKP1 interactome via proteasomal degradation while sparing FBXO1, linking O2 sensing to selective stabilization of distinct SCF substrate receptors [PMID:39307307]. SKP1-associated SCF complexes carry out defined cellular roles: TgFBXO1 is an Inner Membrane Complex/daughter-cell scaffold effector required for replication [PMID:31348812], and TgFBXL2 forms an SCF complex associated with the COP9 signalosome that controls centrosome replication, daughter-cell development, and repression of sexual-commitment genes [PMID:38814984]. In vivo, PHYa-dependent SKP1 modification is required for virulence and brain cyst formation and enables the parasite to survive IFNγ-mediated nutritional immunity through tryptophan scavenging under IDO induction [PMID:38857298].","teleology":[{"year":2012,"claim":"Establishing that an O2-dependent prolyl hydroxylase initiates SKP1 modification connected SKP1 processing to oxygen sensing and to a measurable growth requirement.","evidence":"Genetic disruption of TgphyA with growth phenotype, recombinant TgPhyA prolyl-hydroxylase assay, and cross-species complementation in Dictyostelium","pmids":["22648409"],"confidence":"High","gaps":["Downstream glycan structure not yet defined at this stage","Consequence for SCF F-box binding not addressed"],"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Dictyostelium discoideum","strain":""}]},{"year":2015,"claim":"Identifying the first three sugars and their transferases (Gnt1, PgtA) defined the pentasaccharide assembly pathway and showed the glycan is needed for growth.","evidence":"MS of glycoforms, glycosyltransferase assays in extracts, and knockout/complementation of gnt1 and pgtA","pmids":["26719340"],"confidence":"High","gaps":["Terminal sugars and their enzymes not yet identified","Structural basis for growth requirement unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"Structural and biophysical analysis revealed how the glycan opens the F-box-combining site, providing the mechanistic link between glycosylation and enhanced F-box protein binding.","evidence":"NMR spectroscopy and MD simulations validated by relaxation kinetics of the glycosylated F-box interface","pmids":["28928219"],"confidence":"High","gaps":["Effect on specific endogenous F-box proteins not tested here","Role of homodimerization not yet integrated"],"attribution":[{"species":"Dictyostelium discoideum","strain":""},{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"Characterizing Glt1 as the fourth-sugar transferase extended the assembly pathway and, via catalytic-dead rescue failure, showed the enzymatic product itself is required for normal growth.","evidence":"Recombinant Glt1 assay with [3H]UDP-glucose, NMR linkage determination, glt1 knockout, and catalytic-dead rescue","pmids":["28928220"],"confidence":"High","gaps":["Terminal fifth sugar still uncharacterized","Substrate-receptor consequences not addressed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"Confirming TgFBXO1 as a SKP1-binding F-box protein assigned an SCF complex a concrete cellular role in daughter-cell scaffolding during replication.","evidence":"SKP1–FBXO1 Co-IP, live imaging/IFA localization, myristoylation and TgRab11b-dependence tests, and conditional knockdown growth phenotype","pmids":["31348812"],"confidence":"High","gaps":["Ubiquitination substrates of the FBXO1 SCF not identified","Whether FBXO1 binding depends on SKP1 glycosylation not tested here"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"claim":"Solving Gat1 as the glycogenin-related terminal transferase with a distinct linkage completed the pentasaccharide pathway and showed the capping sugar is functionally important.","evidence":"Glycosyltransferase assay, X-ray crystallography, kinetics, and gat1 knockout growth phenotype","pmids":["32414843"],"confidence":"High","gaps":["Non-catalytic roles of Gat1 not yet examined at this stage","Link to specific F-box selection undefined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"claim":"Comparative enzymology established that TgPhyA is tuned to low O2 and is functionally distinct from animal PHDs, positioning the SKP1 pathway as a bespoke oxygen sensor.","evidence":"In vitro prolyl hydroxylase kinetics with full-length Skp1 substrates, cross-species substrate tests, and DdPhyA crystallography","pmids":["32934009"],"confidence":"High","gaps":["In vivo O2 thresholds in the parasite not defined here","Downstream interactome consequences of O2 changes not yet measured"],"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Dictyostelium discoideum","strain":""}]},{"year":2024,"claim":"Interactome profiling in PHYa-null parasites showed O2-dependent glycosylation selectively stabilizes specific F-box receptors, linking oxygen sensing to differential SCF assembly.","evidence":"Reciprocal interactome MS in PHYaΔ vs wild-type, proteasome inhibitor rescue, low-O2 phenocopy, and transcript controls","pmids":["39307307"],"confidence":"High","gaps":["Why FBXO13/FBXO14 but not FBXO1 are affected not fully resolved","Substrates ubiquitinated by the stabilized SCFs not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"claim":"Genetic and infection studies placed the PHYa/SKP1 O2-sensing pathway in host immune evasion, showing it is required for virulence, cyst formation, and survival of IFNγ-induced nutritional immunity via tryptophan scavenging.","evidence":"TgPHYa knockout in ME49, mouse infection with IFNγ-KO and IDO-KO backgrounds, and intracellular survival assays in IFNγ-treated cells","pmids":["38857298"],"confidence":"High","gaps":["The specific SCF substrates mediating tryptophan scavenging not identified","Whether other strains behave identically not tested"],"attribution":[{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2024,"claim":"Characterizing the TgFBXL2 SCF complex, its COP9-signalosome association, and depletion phenotypes assigned SKP1-based SCF ligases roles in centrosome/daughter-cell cycle control and repression of sexual commitment.","evidence":"Co-IP complex characterization, conditional depletion with centrosome/daughter-cell imaging, and RNAseq","pmids":["38814984"],"confidence":"High","gaps":["Ubiquitination substrates of FBXL2 SCF not identified","Whether FBXL2 engagement depends on SKP1 glycosylation not tested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"claim":"Biophysical dissection of the SKP1 C-terminal region revealed that glycosylation frees SKP1 for F-box binding by weakening electrostatically driven homodimerization, adding a second conformational control mechanism.","evidence":"Sedimentation velocity AUC, CTR deletion/poly-serine substitution mutants, high-salt tests, and MD simulations","pmids":["40296701"],"confidence":"High","gaps":["Direct competition with individual F-box proteins not measured","In vivo contribution of dimerization to F-box selection not established"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2026,"claim":"AUC and interface mutagenesis proposed a non-enzymatic role for GAT1 as a glycosylation-sensitive competitor at the F-box interface, refining how SKP1's binding preference is set.","evidence":"Sedimentation velocity AUC, computational modeling, hydrophobic-interface mutants, and gat1-knockout interactome (preprint)","pmids":["42327091"],"confidence":"Medium","gaps":["Preprint, single lab, not yet peer-reviewed","In vivo relevance of GAT1-driven monomerization to specific F-box selection not demonstrated"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The direct ubiquitination substrates of the SKP1-based SCF complexes and how O2-tuned F-box selection reshapes the parasite proteome during replication and stage conversion remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No SCF substrate targets identified for any F-box receptor in the corpus","Molecular basis for differential FBXO13/14 vs FBXO1 sensitivity to glycosylation state incomplete"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["SCF (SKP1-Cullin-F-box) E3 ubiquitin ligase","SCF-FBXO1 complex (daughter-cell scaffold)","SCF-FBXL2 complex associated with the COP9 signalosome","SKP1 homodimer","SKP1-GAT1 complex"],"partners":["TGPHYA/PHYA (PROLYL 4-HYDROXYLASE)","GNT1","PGTA","GLT1","GAT1/GAT1","TGFBXO1","TGFBXO13","TGFBXO14","TGFBXL2","COP9 SIGNALOSOME"],"other_free_text":["SKP1 is modified at a specific hydroxyproline (Pro-143/Pro-154) by an O2-dependent prolyl hydroxylation followed by sequential assembly of a pentasaccharide (GlcNAc-Gal-Fuc-Glc-Gal).","Glycosylation opens the F-box-combining site (helix separation) and weakens electrostatically driven homodimerization of the disordered C-terminal region, promoting F-box protein binding.","PHYa-dependent glycosylation selectively stabilizes FBXO13 and FBXO14 (proteasomally degraded when SKP1 is hypoglycosylated) but not FBXO1.","The O2-sensing/SKP1 pathway is required for virulence, brain cyst formation, and survival of IFNγ-induced nutritional immunity via tryptophan scavenging under IDO induction (ME49)."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"22648409","id":"PMC_22648409","title":"The Skp1 protein from Toxoplasma is modified by a cytoplasmic prolyl 4-hydroxylase associated with oxygen sensing in the social amoeba Dictyostelium.","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22648409","citation_count":41,"is_preprint":false},{"pmid":"26719340","id":"PMC_26719340","title":"The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development.","date":"2015","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26719340","citation_count":37,"is_preprint":false},{"pmid":"19914348","id":"PMC_19914348","title":"A cytoplasmic prolyl hydroxylation and glycosylation pathway modifies Skp1 and regulates O2-dependent development in Dictyostelium.","date":"2009","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/19914348","citation_count":36,"is_preprint":false},{"pmid":"15238247","id":"PMC_15238247","title":"Cytoplasmic glycosylation of protein-hydroxyproline and its relationship to other glycosylation pathways.","date":"2004","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/15238247","citation_count":36,"is_preprint":false},{"pmid":"31348812","id":"PMC_31348812","title":"Toxoplasma F-box protein 1 is required for daughter cell scaffold function during parasite replication.","date":"2019","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/31348812","citation_count":36,"is_preprint":false},{"pmid":"28928219","id":"PMC_28928219","title":"O2 sensing-associated glycosylation exposes the F-box-combining site of the Dictyostelium Skp1 subunit in E3 ubiquitin ligases.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28928219","citation_count":26,"is_preprint":false},{"pmid":"23098648","id":"PMC_23098648","title":"Role of the Skp1 prolyl-hydroxylation/glycosylation pathway in oxygen dependent submerged development of Dictyostelium.","date":"2012","source":"BMC developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/23098648","citation_count":21,"is_preprint":false},{"pmid":"28928220","id":"PMC_28928220","title":"Characterization of a cytoplasmic glucosyltransferase that extends the core trisaccharide of the Toxoplasma Skp1 E3 ubiquitin ligase subunit.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28928220","citation_count":18,"is_preprint":false},{"pmid":"32414843","id":"PMC_32414843","title":"A terminal α3-galactose modification regulates an E3 ubiquitin ligase subunit in Toxoplasma gondii.","date":"2020","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/32414843","citation_count":16,"is_preprint":false},{"pmid":"32934009","id":"PMC_32934009","title":"Biochemical and biophysical analyses of hypoxia sensing prolyl hydroxylases from Dictyostelium discoideum and Toxoplasma gondii.","date":"2020","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/32934009","citation_count":15,"is_preprint":false},{"pmid":"17132515","id":"PMC_17132515","title":"Detection of cytoplasmic glycosylation associated with hydroxyproline.","date":"2006","source":"Methods in enzymology","url":"https://pubmed.ncbi.nlm.nih.gov/17132515","citation_count":13,"is_preprint":false},{"pmid":"39307307","id":"PMC_39307307","title":"Oxygen-dependent regulation of F-box proteins in Toxoplasma gondii is mediated by Skp1 glycosylation.","date":"2024","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/39307307","citation_count":12,"is_preprint":false},{"pmid":"31281925","id":"PMC_31281925","title":"Skp1 isoforms are differentially modified by a dual function prolyl 4-hydroxylase/N-acety lglucosaminyltransferase in a plant pathogen.","date":"2019","source":"Glycobiology","url":"https://pubmed.ncbi.nlm.nih.gov/31281925","citation_count":11,"is_preprint":false},{"pmid":"38857298","id":"PMC_38857298","title":"The Toxoplasma oxygen-sensing protein, TgPhyA, is required for resistance to interferon gamma-mediated nutritional immunity in mice.","date":"2024","source":"PLoS biology","url":"https://pubmed.ncbi.nlm.nih.gov/38857298","citation_count":6,"is_preprint":false},{"pmid":"38814984","id":"PMC_38814984","title":"The Toxoplasma gondii F-Box Protein L2 Functions as a Repressor of Stage Specific Gene Expression.","date":"2024","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/38814984","citation_count":6,"is_preprint":false},{"pmid":"40296701","id":"PMC_40296701","title":"Glycosylation Weakens Skp1 Homodimerization in Toxoplasma gondii by Interrupting a Fuzzy Interaction.","date":"2025","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40296701","citation_count":3,"is_preprint":false},{"pmid":"38187549","id":"PMC_38187549","title":"Toxoplasma gondii F-Box Protein L2 Silences Feline-Restricted Genes Necessary for Sexual Commitment.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/38187549","citation_count":2,"is_preprint":false},{"pmid":"41269234","id":"PMC_41269234","title":"Glycoregulation of E3(SCF) ubiquitin ligases in unicellular eukaryotes.","date":"2025","source":"Glycobiology","url":"https://pubmed.ncbi.nlm.nih.gov/41269234","citation_count":1,"is_preprint":false},{"pmid":"42327091","id":"PMC_42327091","title":"Potential for the Terminal SKP1 Glycosyltransferase to Exert Non-Enzymatic Control of SKP1 in Toxoplasma gondii.","date":"2026","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/42327091","citation_count":0,"is_preprint":false},{"pmid":"41796871","id":"PMC_41796871","title":"Protist oxygen-sensing and the role of prolyl hydroxylase-regulation of E3(SCF) ubiquitin ligases.","date":"2026","source":"Biochimica et biophysica acta. General subjects","url":"https://pubmed.ncbi.nlm.nih.gov/41796871","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13951,"output_tokens":4046,"usd":0.051271,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10240,"output_tokens":5561,"usd":0.095113,"stage2_stop_reason":"end_turn"},"total_usd":0.146384,"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\": 2012,\n      \"finding\": \"TgPhyA (prolyl 4-hydroxylase) is required for Skp1 glycosylation in Toxoplasma; disruption of the TgphyA locus abolishes Skp1 modification and causes slow growth at physiological O2 levels. Bacterially expressed TgPhyA can prolyl-hydroxylate both Toxoplasma and Dictyostelium Skp1 in vitro, and TgPhyA shows a higher apparent affinity for O2 than DdPhyA.\",\n      \"method\": \"Genetic locus disruption, in vitro prolyl hydroxylase assay with recombinant TgPhyA, kinetic analysis of O2/α-ketoglutarate dependence, complementation in DdphyA-null cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro enzymatic assay with recombinant protein, genetic disruption with growth phenotype, complementation rescue, multiple orthogonal methods in one study\",\n      \"pmids\": [\"22648409\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"},\n        {\"species\": \"Dictyostelium discoideum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TgSkp1 hydroxyproline is modified by a pentasaccharide assembled sequentially; the first three sugars (GlcNAc, Gal, Fuc) are added by Toxoplasma homologs of Gnt1 and PgtA. Disruption of gnt1 or pgtA reduces parasite growth, rescued by genetic complementation restoring normal glycan and growth.\",\n      \"method\": \"Mass spectrometry of glycoforms, radioactive sugar nucleotide glycosyltransferase assays in cell extracts, acid hydrolysate chromatography, genetic knockout and complementation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — biochemical reconstitution of glycosyltransferase reactions, MS characterization, genetic KO with complementation, multiple orthogonal methods\",\n      \"pmids\": [\"26719340\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NMR and molecular dynamics simulations show that the pentasaccharide on Skp1 HyPro-143 interacts with the loop connecting two α-helices of the F-box-combining site, causing the helices to separate and create a more accessible, dynamic F-box interface. This conformational opening explains how glycosylation enhances Skp1 interactions with F-box proteins.\",\n      \"method\": \"NMR spectroscopy, mass spectrometry, all-atom molecular dynamics simulations with calculated monosaccharide relaxation kinetics validated against NMR data\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure with MD simulations validated by experimental relaxation kinetics; multiple orthogonal biophysical methods in one study\",\n      \"pmids\": [\"28928219\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Dictyostelium discoideum\", \"strain\": \"\"},\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The cytoplasmic glycosyltransferase Glt1 (CAZy family GT32) transfers the fourth sugar (glucose) to TgSkp1, forming a Glcα1,3Fuc linkage. Recombinant Glt1 catalyzes UDP-glucose transfer specifically to the trisaccharide form of Skp1; glt1-knockout parasites exhibit a growth defect not rescued by catalytically inactive Glt1.\",\n      \"method\": \"In vitro glycosyltransferase assay with recombinant Glt1 and [3H]UDP-glucose, NMR linkage determination, glt1 knockout, catalytic-dead Glt1 rescue experiment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — recombinant enzyme assay, NMR, genetic KO with catalytic-dead rescue, multiple orthogonal methods in one study\",\n      \"pmids\": [\"28928220\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TgFBXO1 interacts with Toxoplasma SKP1 (confirming it as a bona fide F-box protein), localizes to the Inner Membrane Complex in interphase parasites and to the daughter cell scaffold during replication. This localization requires N-myristoylation of TgFBXO1 and depends on TgRab11b. TgFBXO1 is required for parasite growth through its function as a daughter cell scaffold effector.\",\n      \"method\": \"Co-immunoprecipitation (SKP1–FBXO1 interaction), live imaging and immunofluorescence for localization, myristoylation mutant analysis, TgRab11b-dependent localization assay, conditional knockdown with growth phenotype readout\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, localization by imaging with functional consequence, multiple mutants tested, clean KO growth phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"31348812\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The terminal (fifth) sugar of TgSkp1 is added by Gat1, a glycogenin-related glycosyltransferase that forms a Galα1,3Glcα linkage rather than the Glcα1,4Glcα linkage formed by glycogenin. Crystallographic and kinetic analyses show Gat1 is specific for Skp1. gat1Δ parasites exhibit a slow-growth phenotype, indicating the fifth sugar is functionally important.\",\n      \"method\": \"Glycosyltransferase biochemical assay, X-ray crystallography, kinetic analysis, gat1 genetic knockout with growth phenotype, computational conformational analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure, in vitro enzymatic assay, genetic KO with phenotype, multiple orthogonal methods in one study\",\n      \"pmids\": [\"32414843\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Biochemical and biophysical comparison of TgPhyA and DdPhyA shows that TgPhyA is more active at low O2 concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate; no cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. Crystallographic analysis of DdPhyA reveals absence of the C-terminal helix present in animal PHDs.\",\n      \"method\": \"In vitro prolyl hydroxylase kinetic assays with full-length Skp1 substrates, X-ray crystallography of DdPhyA\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic assays with defined substrates and kinetics, crystal structure, multiple cross-species substrate tests\",\n      \"pmids\": [\"32934009\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"},\n        {\"species\": \"Dictyostelium discoideum\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"O2-dependent SKP1 glycosylation (mediated by PHYa) regulates the SKP1 interactome in Toxoplasma: loss of PHYa substantially reduces the representation of F-box proteins FBXO13 and FBXO14 in the SKP1 interactome. Their reduced abundance is rescued by proteasomal inhibitors and is phenocopied at 0.5% O2, indicating SCF-mediated degradation when SKP1 is hypoglycosylated. FBXO1 abundance is not affected by PHYa loss.\",\n      \"method\": \"Interactome/co-immunoprecipitation-MS in PHYaΔ vs. wild-type parasites, proteasomal inhibitor rescue, epitope-tagging at genetic locus, transcript-level controls, low-O2 phenocopy experiment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interactome MS, multiple genetic mutants, proteasome inhibitor rescue, O2-phenocopy, multiple orthogonal methods\",\n      \"pmids\": [\"39307307\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TgPHYa (which hydroxylates SKP1) is required for Toxoplasma virulence and brain cyst formation in mice (ME49 strain). PHYa-knockout parasites fail to disseminate to peripheral tissues because they cannot survive within recruited immune cells; this survival defect requires IFNγ. TgPHYa is specifically required for tryptophan scavenging when IDO is induced by IFNγ, providing the first parasite mechanism for evading IFNγ-induced nutritional immunity via the O2-sensing/SKP1 pathway.\",\n      \"method\": \"TgPHYa genetic knockout in ME49, mouse infection model, IFNγ-KO mouse experiments, IDO-KO mouse experiments, intracellular survival assays in IFNγ-treated cells\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic KO, multiple mouse genetic backgrounds, mechanistic pathway placement via IFNγ/IDO axis, multiple orthogonal in vivo and in vitro assays\",\n      \"pmids\": [\"38857298\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TgFBXL2 localizes to perinucleolar sites and is stably engaged in an SCF-E3 complex that is also associated with the COP9 signalosome (a negative regulator of SCF function). Depletion of TgFBXL2 causes centrosome replication defects, daughter cell development failure, and induction of stage-specific (sexual commitment) genes. TgFBXL2 interacts with SKP1 as part of this complex.\",\n      \"method\": \"Co-immunoprecipitation/complex characterization, conditional depletion, RNAseq, live-cell imaging for centrosome/daughter cell phenotype\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP identifying complex components, conditional KD with multiple defined phenotypic readouts, RNAseq, multiple orthogonal methods\",\n      \"pmids\": [\"38814984\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Glycosylation of TgSkp1's disordered C-terminal region (CTR) weakens Skp1 homodimerization. The CTR promotes homodimerization via electrostatic (charge cluster) interactions that are disrupted by glycosylation or CTR deletion, freeing Skp1 for F-box protein binding. Replacement with poly-serine (but not a randomized sequence) recapitulates the weakening, indicating composition-dependent electrostatic dependence. Molecular dynamics simulations support the mechanism.\",\n      \"method\": \"Sedimentation velocity analytical ultracentrifugation, CTR deletion and substitution mutants, high-salt experiments, all-atom molecular dynamics simulations\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — quantitative biophysical assay (AUC), multiple mutant constructs, MD simulations, multiple orthogonal methods in one study\",\n      \"pmids\": [\"40296701\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"The terminal SKP1 glycosyltransferase GAT1 forms a native homodimer that complexes with SKP1 monomers with affinity and stoichiometry dictated by SKP1 glycosylation state. GAT1 competes with the same core hydrophobic interface used by F-box proteins and the SKP1 homodimer. Substoichiometric GAT1 can promote SKP1 monomerization in a CTR-dependent manner, suggesting a non-enzymatic role for GAT1 in controlling SKP1 FBP-binding preference.\",\n      \"method\": \"Sedimentation velocity analytical ultracentrifugation, computational modeling, mutational probing of the hydrophobic interface, gat1-knockout cell interactome analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — AUC and mutational probing with computational modeling, preprint, single lab, novel non-enzymatic mechanism proposed but supported by experimental data\",\n      \"pmids\": [\"42327091\"],\n      \"is_preprint\": true,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}\n      ]\n    }\n  ],\n  \"current_model\": \"Toxoplasma SKP1 is an adaptor subunit of SCF-class E3 ubiquitin ligases that is uniquely regulated by O2-dependent prolyl hydroxylation (by TgPHYa/PhyA at Pro-154) followed by sequential cytoplasmic glycosylation with a pentasaccharide (assembled by Gnt1, PgtA, Glt1, and Gat1); this glycan promotes an open, accessible conformation of the F-box-binding domain by disrupting both SKP1 homodimerization (via the disordered C-terminal region) and GAT1 competition at the F-box interface, thereby selectively stabilizing distinct F-box protein substrate receptors (e.g., FBXO13, FBXO14 but not FBXO1) and allowing the parasite to sense and respond to O2 levels, regulate its proteome during replication and stage conversion, and survive IFNγ-mediated host immunity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"Toxoplasma SKP1 is the adaptor subunit of SCF-class E3 ubiquitin ligases whose engagement of F-box protein substrate receptors is gated by an O2-dependent post-translational modification cascade unique to this lineage [#7]. A prolyl 4-hydroxylase (TgPhyA/PHYa) hydroxylates a specific SKP1 proline in an O2-dependent reaction, and TgPhyA is tuned for activity at low O2, allowing SKP1 modification to report on ambient oxygen [#0, #6]. The resulting hydroxyproline is elaborated into a pentasaccharide by sequential cytoplasmic glycosyltransferases — Gnt1 and PgtA add the first three sugars, Glt1 (GT32) adds a fourth glucose in a Glc\\u03b11,3Fuc linkage, and the glycogenin-related Gat1 caps the chain with a Gal\\u03b11,3Glc\\u03b1 linkage — each step being functionally important for normal growth [#1, #3, #5]. This glycan controls F-box binding through two coupled conformational effects: it remodels the loop between the two \\u03b1-helices of the F-box-combining site to open a more accessible interface [#2], and it weakens SKP1 homodimerization mediated by electrostatic charge clusters in the disordered C-terminal region, freeing SKP1 monomers for F-box protein capture [#10]. The terminal enzyme GAT1 additionally acts non-enzymatically as a homodimer that competes for the same hydrophobic interface used by F-box proteins and the SKP1 homodimer, further shaping F-box-binding preference in a glycosylation- and CTR-dependent manner [#11]. Functionally, loss of PHYa-dependent glycosylation selectively depletes the F-box receptors FBXO13 and FBXO14 from the SKP1 interactome via proteasomal degradation while sparing FBXO1, linking O2 sensing to selective stabilization of distinct SCF substrate receptors [#7]. SKP1-associated SCF complexes carry out defined cellular roles: TgFBXO1 is an Inner Membrane Complex/daughter-cell scaffold effector required for replication [#4], and TgFBXL2 forms an SCF complex associated with the COP9 signalosome that controls centrosome replication, daughter-cell development, and repression of sexual-commitment genes [#9]. In vivo, PHYa-dependent SKP1 modification is required for virulence and brain cyst formation and enables the parasite to survive IFN\\u03b3-mediated nutritional immunity through tryptophan scavenging under IDO induction [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Establishing that an O2-dependent prolyl hydroxylase initiates SKP1 modification connected SKP1 processing to oxygen sensing and to a measurable growth requirement.\",\n      \"evidence\": \"Genetic disruption of TgphyA with growth phenotype, recombinant TgPhyA prolyl-hydroxylase assay, and cross-species complementation in Dictyostelium\",\n      \"pmids\": [\"22648409\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream glycan structure not yet defined at this stage\", \"Consequence for SCF F-box binding not addressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Dictyostelium discoideum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identifying the first three sugars and their transferases (Gnt1, PgtA) defined the pentasaccharide assembly pathway and showed the glycan is needed for growth.\",\n      \"evidence\": \"MS of glycoforms, glycosyltransferase assays in extracts, and knockout/complementation of gnt1 and pgtA\",\n      \"pmids\": [\"26719340\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Terminal sugars and their enzymes not yet identified\", \"Structural basis for growth requirement unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Structural and biophysical analysis revealed how the glycan opens the F-box-combining site, providing the mechanistic link between glycosylation and enhanced F-box protein binding.\",\n      \"evidence\": \"NMR spectroscopy and MD simulations validated by relaxation kinetics of the glycosylated F-box interface\",\n      \"pmids\": [\"28928219\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Effect on specific endogenous F-box proteins not tested here\", \"Role of homodimerization not yet integrated\"],\n      \"attribution\": [{\"species\": \"Dictyostelium discoideum\", \"strain\": \"\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Characterizing Glt1 as the fourth-sugar transferase extended the assembly pathway and, via catalytic-dead rescue failure, showed the enzymatic product itself is required for normal growth.\",\n      \"evidence\": \"Recombinant Glt1 assay with [3H]UDP-glucose, NMR linkage determination, glt1 knockout, and catalytic-dead rescue\",\n      \"pmids\": [\"28928220\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Terminal fifth sugar still uncharacterized\", \"Substrate-receptor consequences not addressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Confirming TgFBXO1 as a SKP1-binding F-box protein assigned an SCF complex a concrete cellular role in daughter-cell scaffolding during replication.\",\n      \"evidence\": \"SKP1\\u2013FBXO1 Co-IP, live imaging/IFA localization, myristoylation and TgRab11b-dependence tests, and conditional knockdown growth phenotype\",\n      \"pmids\": [\"31348812\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitination substrates of the FBXO1 SCF not identified\", \"Whether FBXO1 binding depends on SKP1 glycosylation not tested here\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Solving Gat1 as the glycogenin-related terminal transferase with a distinct linkage completed the pentasaccharide pathway and showed the capping sugar is functionally important.\",\n      \"evidence\": \"Glycosyltransferase assay, X-ray crystallography, kinetics, and gat1 knockout growth phenotype\",\n      \"pmids\": [\"32414843\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Non-catalytic roles of Gat1 not yet examined at this stage\", \"Link to specific F-box selection undefined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Comparative enzymology established that TgPhyA is tuned to low O2 and is functionally distinct from animal PHDs, positioning the SKP1 pathway as a bespoke oxygen sensor.\",\n      \"evidence\": \"In vitro prolyl hydroxylase kinetics with full-length Skp1 substrates, cross-species substrate tests, and DdPhyA crystallography\",\n      \"pmids\": [\"32934009\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo O2 thresholds in the parasite not defined here\", \"Downstream interactome consequences of O2 changes not yet measured\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Dictyostelium discoideum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Interactome profiling in PHYa-null parasites showed O2-dependent glycosylation selectively stabilizes specific F-box receptors, linking oxygen sensing to differential SCF assembly.\",\n      \"evidence\": \"Reciprocal interactome MS in PHYa\\u0394 vs wild-type, proteasome inhibitor rescue, low-O2 phenocopy, and transcript controls\",\n      \"pmids\": [\"39307307\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why FBXO13/FBXO14 but not FBXO1 are affected not fully resolved\", \"Substrates ubiquitinated by the stabilized SCFs not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Genetic and infection studies placed the PHYa/SKP1 O2-sensing pathway in host immune evasion, showing it is required for virulence, cyst formation, and survival of IFN\\u03b3-induced nutritional immunity via tryptophan scavenging.\",\n      \"evidence\": \"TgPHYa knockout in ME49, mouse infection with IFN\\u03b3-KO and IDO-KO backgrounds, and intracellular survival assays in IFN\\u03b3-treated cells\",\n      \"pmids\": [\"38857298\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The specific SCF substrates mediating tryptophan scavenging not identified\", \"Whether other strains behave identically not tested\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Characterizing the TgFBXL2 SCF complex, its COP9-signalosome association, and depletion phenotypes assigned SKP1-based SCF ligases roles in centrosome/daughter-cell cycle control and repression of sexual commitment.\",\n      \"evidence\": \"Co-IP complex characterization, conditional depletion with centrosome/daughter-cell imaging, and RNAseq\",\n      \"pmids\": [\"38814984\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitination substrates of FBXL2 SCF not identified\", \"Whether FBXL2 engagement depends on SKP1 glycosylation not tested\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Biophysical dissection of the SKP1 C-terminal region revealed that glycosylation frees SKP1 for F-box binding by weakening electrostatically driven homodimerization, adding a second conformational control mechanism.\",\n      \"evidence\": \"Sedimentation velocity AUC, CTR deletion/poly-serine substitution mutants, high-salt tests, and MD simulations\",\n      \"pmids\": [\"40296701\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct competition with individual F-box proteins not measured\", \"In vivo contribution of dimerization to F-box selection not established\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"AUC and interface mutagenesis proposed a non-enzymatic role for GAT1 as a glycosylation-sensitive competitor at the F-box interface, refining how SKP1's binding preference is set.\",\n      \"evidence\": \"Sedimentation velocity AUC, computational modeling, hydrophobic-interface mutants, and gat1-knockout interactome (preprint)\",\n      \"pmids\": [\"42327091\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, single lab, not yet peer-reviewed\", \"In vivo relevance of GAT1-driven monomerization to specific F-box selection not demonstrated\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct ubiquitination substrates of the SKP1-based SCF complexes and how O2-tuned F-box selection reshapes the parasite proteome during replication and stage conversion remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No SCF substrate targets identified for any F-box receptor in the corpus\", \"Molecular basis for differential FBXO13/14 vs FBXO1 sensitivity to glycosylation state incomplete\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0019005\", \"supporting_discovery_ids\": [4, 7, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0043130\", \"supporting_discovery_ids\": [2, 4, 7, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0042803\", \"supporting_discovery_ids\": [10, 11], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005737\", \"supporting_discovery_ids\": [1, 3, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005938\", \"supporting_discovery_ids\": [4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0016567\", \"supporting_discovery_ids\": [4, 7, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0070482\", \"supporting_discovery_ids\": [0, 6, 7], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0051301\", \"supporting_discovery_ids\": [4, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"SCF (SKP1-Cullin-F-box) E3 ubiquitin ligase\",\n      \"SCF-FBXO1 complex (daughter-cell scaffold)\",\n      \"SCF-FBXL2 complex associated with the COP9 signalosome\",\n      \"SKP1 homodimer\",\n      \"SKP1-GAT1 complex\"\n    ],\n    \"partners\": [\n      \"TgPhyA/PHYa (prolyl 4-hydroxylase)\",\n      \"Gnt1\",\n      \"PgtA\",\n      \"Glt1\",\n      \"Gat1/GAT1\",\n      \"TgFBXO1\",\n      \"TgFBXO13\",\n      \"TgFBXO14\",\n      \"TgFBXL2\",\n      \"COP9 signalosome\"\n    ],\n    \"other_free_text\": [\n      \"SKP1 is modified at a specific hydroxyproline (Pro-143/Pro-154) by an O2-dependent prolyl hydroxylation followed by sequential assembly of a pentasaccharide (GlcNAc-Gal-Fuc-Glc-Gal).\",\n      \"Glycosylation opens the F-box-combining site (helix separation) and weakens electrostatically driven homodimerization of the disordered C-terminal region, promoting F-box protein binding.\",\n      \"PHYa-dependent glycosylation selectively stabilizes FBXO13 and FBXO14 (proteasomally degraded when SKP1 is hypoglycosylated) but not FBXO1.\",\n      \"The O2-sensing/SKP1 pathway is required for virulence, brain cyst formation, and survival of IFN\\u03b3-induced nutritional immunity via tryptophan scavenging under IDO induction (ME49).\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_207680","name":"SKP1","product":"suppressor of kinetochore protein 1","uniprot_acc":"A0A125YIV5","chromosome":"TGME49_chrIb","start_pos":207748},{"strain":"GT1","gene_id":"TGGT1_207680","name":null,"product":"putative suppressor of kinetochore protein 1","uniprot_acc":null,"chromosome":"TGGT1_chrIb","start_pos":223489},{"strain":"RH88","gene_id":"TGRH88_022970","name":null,"product":"putative suppressor of kinetochore protein 1","uniprot_acc":null,"chromosome":"CM023082","start_pos":1802837},{"strain":"VEG","gene_id":"TGVEG_207680","name":null,"product":"putative suppressor of kinetochore protein 1","uniprot_acc":"A0A0F7UNE6","chromosome":"TGVEG_chrIb","start_pos":205005}]}