{"gene":"TTI1","run_date":"2026-04-28T21:43:00","timeline":{"discoveries":[{"year":2010,"finding":"Mammalian TTI1 (KIAA0406) constitutively interacts with mTOR in both mTORC1 and mTORC2, binds Tel2, and is required for stability of all six PIKK family members (mTOR, ATM, ATR, DNA-PKcs, SMG-1, TRRAP). Knockdown of TTI1 causes disassembly of mTORC1 and mTORC2, suppresses phosphorylation of mTORC1 substrates (S6K1, 4E-BP1) and mTORC2 substrate (Akt), and induces autophagy.","method":"Co-immunoprecipitation, size-exclusion chromatography, siRNA knockdown, western blot for PIKK levels and substrate phosphorylation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, size-exclusion chromatography, multiple substrates tested, >200 citations and independently replicated","pmids":["20427287"],"is_preprint":false},{"year":2013,"finding":"CK2 phosphorylates TTI1 (and Tel2) within mTORC1 upon growth factor withdrawal, targeting them for ubiquitination and degradation by the SCFFbxo9 E3 ubiquitin ligase, leading to mTORC1-specific inactivation while relieving feedback to sustain PI3K/mTORC2/Akt signaling.","method":"Co-immunoprecipitation, ubiquitination assays, CK2 kinase assay, siRNA knockdown, reconstitution of SCFFbxo9 ubiquitin ligase activity","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1–2 — biochemical reconstitution of ubiquitin ligase activity, kinase assay, multiple orthogonal methods in a high-impact journal","pmids":["23263282"],"is_preprint":false},{"year":2014,"finding":"IP7 (generated by IP6K2) binds CK2 and enhances CK2-mediated phosphorylation of the TTT complex (Tel2/TTI1/TTI2), which stabilizes DNA-PKcs and ATM, thereby promoting p53 phosphorylation at serine 15 and apoptosis.","method":"In vitro CK2 kinase assay with IP7, co-immunoprecipitation, IP6K2 knockdown/overexpression, western blot for PIKK levels and p53 phosphorylation","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase assay with defined small molecule, multiple orthogonal methods, >100 citations","pmids":["24657168"],"is_preprint":false},{"year":2021,"finding":"Cryo-EM structure of the human R2TP-TTT complex reveals that the HEAT-repeat TTT complex binds the kinase domain of TOR (without blocking kinase activity) and delivers TOR to the R2TP chaperone; TTT also inhibits RUVBL1-RUVBL2 ATPase activity and modulates PIH1D1 and RPAP3 conformations within R2TP.","method":"Cryo-EM structure determination, biochemical ATPase assays, co-immunoprecipitation, crosslinking mass spectrometry","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure with biochemical validation of mechanism, multiple orthogonal assays","pmids":["34233195"],"is_preprint":false},{"year":2021,"finding":"Cryo-EM structure of the TTT complex at 4.2 Å resolution shows TTI1 as a central scaffold with TELO2 binding its central region and TTI2 binding its C-terminal end; TTI1 N- and C-terminal segments contact the FAT domain and N-terminal HEAT repeats of ATM respectively, and the TELO2 CTD is required for interaction with TTI1 and ATM recruitment. TTI1 N- and C-terminal segments are required for cell survival after ionizing radiation.","method":"Cryo-EM structure determination, deletion mutagenesis, co-immunoprecipitation, cell survival assays after ionizing radiation","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure with mutagenesis and functional validation","pmids":["34838521"],"is_preprint":false},{"year":2019,"finding":"In fission yeast, destabilization of the TTT complex (Tel2-Tti1-Tti2) by a tel2 mutation nearly abolishes Rad3 (ATR ortholog) checkpoint signaling in the DNA replication checkpoint and causes telomere shortening, demonstrating that TTT complex integrity is required for ATR-mediated checkpoint kinase signaling.","method":"Genetic screen, co-immunoprecipitation to assess TTT complex stability, western blot for Rad3-mediated phospho-signaling, telomere length assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with biochemical validation in fission yeast ortholog","pmids":["31332096"],"is_preprint":false},{"year":2021,"finding":"In S. cerevisiae, single-residue substitutions in Tti1 suppress lethality caused by Sis1 (Hsp70 cochaperone) depletion; Sis1 depletion reduces levels of essential PIKKs (Mec1/ATR, Tra1/TRRAP, Tor2/mTOR), and overexpression of Tti1 alone (without increasing Tel2 or Tti2) restores growth, indicating Tti1 can function independently of the full TTT complex as a PIKK-specific chaperone.","method":"Genetic suppressor analysis, protein level measurement by western blot, rapamycin sensitivity assay, overexpression experiments","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with biochemical readouts, yeast ortholog","pmids":["34935410"],"is_preprint":false},{"year":2023,"finding":"Bi-allelic loss-of-function variants in TTI1 impair TTT complex integrity and reduce mTOR pathway activity (mTORC1 signaling) in patient-derived cells; rapamycin treatment partially restores mTOR pathway activity, placing TTI1 upstream of mTOR in the signaling cascade in a human disease context.","method":"Functional studies in HEK293T cells and patient-derived fibroblasts/lymphoblastoid cells, western blot for mTOR pathway substrates, rapamycin rescue experiment","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 — patient-derived cell functional studies with pharmacological rescue, multiple cell types","pmids":["36724785"],"is_preprint":false},{"year":2025,"finding":"TTI1 promotes ATM signaling pathway activation in rectal cancer cells; TTI1 knockdown reduces ATM-dependent DNA damage repair after irradiation, increasing radiosensitivity, while TTI1 overexpression enhances repair and radioresistance.","method":"siRNA knockdown, overexpression, western blot for ATM pathway activation, comet assay, colony formation assay, in vivo xenograft, organoid and PDX models","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 — loss- and gain-of-function with multiple functional readouts including in vivo models","pmids":["40514657"],"is_preprint":false}],"current_model":"TTI1 is a central scaffold subunit of the heterotrimeric TTT (TELO2-TTI1-TTI2) co-chaperone complex that binds newly synthesized PIKKs (mTOR, ATM, ATR, DNA-PKcs, SMG-1, TRRAP) via defined HEAT-repeat interfaces and delivers them to the R2TP-HSP90 chaperone system for folding, assembly, and stabilization; TTI1 is required for mTORC1 and mTORC2 complex assembly and activity, is targeted for CK2-primed SCFFbxo9-mediated degradation to attenuate mTORC1 signaling upon growth factor withdrawal, and is phosphorylated by CK2 in a manner enhanced by the inositol pyrophosphate IP7 to stabilize ATM/DNA-PKcs and activate p53-dependent apoptosis."},"narrative":{"teleology":[{"year":2010,"claim":"Identifying TTI1 as a constitutive PIKK-binding partner resolved how all six PIKKs are co-stabilized and how mTORC1/mTORC2 assembly depends on a shared chaperone adaptor.","evidence":"Co-IP, gel filtration, and siRNA knockdown in mammalian cells showing loss of all PIKK proteins and mTOR complex activity upon TTI1 depletion","pmids":["20427287"],"confidence":"High","gaps":["No structural information on how TTI1 contacts PIKKs","Mechanism of selectivity for PIKK family over other kinases unknown","Role beyond protein stability (e.g., kinase activation) not addressed"]},{"year":2013,"claim":"Demonstrating that CK2-primed SCF^Fbxo9 ubiquitination degrades TTI1 upon growth-factor withdrawal established the first regulated proteolytic switch that selectively inactivates mTORC1 while sustaining mTORC2/Akt signaling.","evidence":"Reconstituted SCF^Fbxo9 ubiquitin ligase activity, CK2 kinase assays, and siRNA in mammalian cells","pmids":["23263282"],"confidence":"High","gaps":["Specific CK2 phosphosites on TTI1 not mapped at residue resolution","Whether other E3 ligases target TTI1 under different stress conditions unknown"]},{"year":2014,"claim":"Showing that IP7 enhances CK2-mediated TTT complex phosphorylation to stabilize ATM/DNA-PKcs and activate p53-dependent apoptosis linked metabolite signaling to PIKK homeostasis through TTI1.","evidence":"In vitro CK2 kinase assays with IP7, IP6K2 manipulation, and western blot for PIKK levels and p53 Ser15 phosphorylation","pmids":["24657168"],"confidence":"High","gaps":["Direct IP7 binding site on TTI1/TTT versus CK2 not resolved","Context-dependent outcome (mTORC1 degradation vs. ATM stabilization) of TTI1 phosphorylation not mechanistically reconciled"]},{"year":2019,"claim":"Genetic destabilization of the TTT complex in fission yeast confirmed that TTT integrity is required for ATR checkpoint signaling and telomere maintenance, extending the mammalian findings to a conserved function.","evidence":"Genetic screen with tel2 mutant, co-IP for TTT stability, Rad3 phospho-signaling, and telomere length assay in S. pombe","pmids":["31332096"],"confidence":"Medium","gaps":["Tti1-specific contributions versus Tel2/Tti2 not separated in this system","Telomere maintenance mechanism (via ATR vs. other PIKKs) not distinguished"]},{"year":2021,"claim":"Cryo-EM structures of the TTT complex alone and bound to R2TP revealed that TTI1 HEAT repeats serve as the central scaffold bridging TELO2 and TTI2, directly contacting PIKK FAT-domain and kinase-domain surfaces, and delivering clients to the R2TP–HSP90 system while inhibiting RUVBL1/2 ATPase activity.","evidence":"Cryo-EM at 4.2 Å (TTT–ATM) and of R2TP–TTT, deletion mutagenesis, crosslinking mass spectrometry, ATPase assays","pmids":["34233195","34838521"],"confidence":"High","gaps":["High-resolution map of TTI1–PIKK interface residues needed for drug targeting","How ATPase inhibition of RUVBL1/2 is relieved to complete client handoff is unknown","Structure of TTT bound to PIKKs other than mTOR/ATM not determined"]},{"year":2021,"claim":"Yeast suppressor analysis showed Tti1 can function independently of the full TTT complex, with Tti1 overexpression alone rescuing PIKK levels after Hsp70-cochaperone depletion, implying an intrinsic PIKK-specific chaperone capacity.","evidence":"Genetic suppressor screen, protein level measurements, and rapamycin sensitivity in S. cerevisiae with Sis1 depletion","pmids":["34935410"],"confidence":"Medium","gaps":["Whether mammalian TTI1 retains TTT-independent chaperone activity not tested","Physical interaction between Tti1 and PIKKs in the absence of Tel2/Tti2 not demonstrated biochemically"]},{"year":2023,"claim":"Patient studies established that bi-allelic TTI1 loss-of-function causes a neurodevelopmental disorder with reduced mTOR signaling, directly linking TTI1 to human Mendelian disease.","evidence":"Functional studies in patient-derived fibroblasts and lymphoblastoid cells, HEK293T reconstitution, rapamycin rescue","pmids":["36724785"],"confidence":"Medium","gaps":["Impact on non-mTOR PIKKs (ATM, ATR) in patient cells not characterized","Genotype-phenotype correlation across different TTI1 variants not established"]},{"year":2025,"claim":"Demonstrating that TTI1 promotes ATM-dependent DNA damage repair in rectal cancer cells linked TTI1 expression to radioresistance and identified it as a potential therapeutic target.","evidence":"siRNA knockdown and overexpression with comet assay, colony formation, xenograft, organoid, and PDX models","pmids":["40514657"],"confidence":"Medium","gaps":["Whether TTI1 contribution to radioresistance is solely through ATM or involves other PIKKs not resolved","No pharmacological inhibitor of TTI1 tested"]},{"year":null,"claim":"The precise mechanism by which TTI1 coordinates opposing phospho-dependent outcomes — mTORC1 degradation versus ATM/DNA-PKcs stabilization — and whether TTI1 has PIKK-independent functions remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["Site-resolved phospho-regulation of TTI1 determining client-specific fate is unknown","No high-resolution structure of TTI1 with a full-length non-mTOR/ATM PIKK client","Whether TTI1 functions outside of PIKK biology is unexplored"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[0,3,4,6]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,3,4]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,3]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,7]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[4,5,8]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,3,4,6]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[0]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[2]}],"complexes":["TTT complex (TELO2–TTI1–TTI2)","R2TP–TTT complex"],"partners":["TELO2","TTI2","MTOR","ATM","ATR","PRKDC","FBXO9","CSNK2A1"],"other_free_text":[]},"mechanistic_narrative":"TTI1 is the central scaffold subunit of the TTT (TELO2–TTI1–TTI2) co-chaperone complex that is essential for the folding, stabilization, and assembly of all six PIKK family kinases (mTOR, ATM, ATR, DNA-PKcs, SMG-1, TRRAP) [PMID:20427287]. Cryo-EM structures show that TTI1 HEAT repeats organize the complex, with TELO2 binding its central region and TTI2 its C-terminus, while TTI1 N- and C-terminal segments directly contact the FAT domain and N-terminal HEAT repeats of client PIKKs; the TTT complex delivers clients to the R2TP–HSP90 chaperone machinery by engaging the PIKK kinase domain and modulating RUVBL1/2 ATPase activity [PMID:34838521, PMID:34233195]. TTI1 is required for mTORC1 and mTORC2 assembly and signaling, and upon growth-factor withdrawal CK2 phosphorylates TTI1, targeting it for SCF^Fbxo9-mediated ubiquitination and degradation to selectively inactivate mTORC1 [PMID:23263282]; CK2-mediated phosphorylation of the TTT complex is enhanced by the inositol pyrophosphate IP7, which stabilizes DNA-PKcs and ATM to activate p53-dependent apoptosis [PMID:24657168]. Bi-allelic loss-of-function variants in TTI1 cause a neurodevelopmental disorder with impaired mTOR signaling in patient-derived cells [PMID:36724785]."},"prefetch_data":{"uniprot":{"accession":"O43156","full_name":"TELO2-interacting protein 1 homolog","aliases":["Protein SMG10"],"length_aa":1089,"mass_kda":122.1,"function":"Regulator of the DNA damage response (DDR). Part of the TTT complex that is required to stabilize protein levels of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family proteins. The TTT complex is involved in the cellular resistance to DNA damage stresses, like ionizing radiation (IR), ultraviolet (UV) and mitomycin C (MMC). Together with the TTT complex and HSP90 may participate in the proper folding of newly synthesized PIKKs. Promotes assembly, stabilizes and maintains the activity of mTORC1 and mTORC2 complexes, which regulate cell growth and survival in response to nutrient and hormonal signals","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/O43156/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/TTI1","classification":"Common Essential","n_dependent_lines":1145,"n_total_lines":1208,"dependency_fraction":0.9478476821192053},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"FKBP5","stoichiometry":0.2},{"gene":"POLR2E","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TTI1","total_profiled":1310},"omim":[{"mim_id":"620445","title":"NEURODEVELOPMENTAL DISORDER WITH MICROCEPHALY AND MOVEMENT ABNORMALITIES; NEDMIM","url":"https://www.omim.org/entry/620445"},{"mim_id":"616954","title":"YOU-HOOVER-FONG SYNDROME; YHFS","url":"https://www.omim.org/entry/616954"},{"mim_id":"615541","title":"INTELLECTUAL DEVELOPMENTAL DISORDER, AUTOSOMAL RECESSIVE 39; MRT39","url":"https://www.omim.org/entry/615541"},{"mim_id":"614426","title":"TELO2-INTERACTING PROTEIN 2; TTI2","url":"https://www.omim.org/entry/614426"},{"mim_id":"614425","title":"TELO2-INTERACTING PROTEIN 1; TTI1","url":"https://www.omim.org/entry/614425"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TTI1"},"hgnc":{"alias_symbol":["smg-10"],"prev_symbol":["KIAA0406"]},"alphafold":{"accession":"O43156","domains":[{"cath_id":"-","chopping":"194-367","consensus_level":"medium","plddt":84.4153,"start":194,"end":367},{"cath_id":"-","chopping":"400-451_475-580_617-684","consensus_level":"medium","plddt":85.8945,"start":400,"end":684},{"cath_id":"1.25.40","chopping":"692-772_858-899","consensus_level":"medium","plddt":88.7598,"start":692,"end":899}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O43156","model_url":"https://alphafold.ebi.ac.uk/files/AF-O43156-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O43156-F1-predicted_aligned_error_v6.png","plddt_mean":81.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TTI1","jax_strain_url":"https://www.jax.org/strain/search?query=TTI1"},"sequence":{"accession":"O43156","fasta_url":"https://rest.uniprot.org/uniprotkb/O43156.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O43156/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O43156"}},"corpus_meta":[{"pmid":"20427287","id":"PMC_20427287","title":"Tti1 and Tel2 are critical factors in mammalian target of rapamycin complex assembly.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20427287","citation_count":204,"is_preprint":false},{"pmid":"24657168","id":"PMC_24657168","title":"Inositol pyrophosphates mediate the DNA-PK/ATM-p53 cell death pathway by regulating CK2 phosphorylation of Tti1/Tel2.","date":"2014","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/24657168","citation_count":105,"is_preprint":false},{"pmid":"23263282","id":"PMC_23263282","title":"SCFFbxo9 and CK2 direct the cellular response to growth factor withdrawal via Tel2/Tti1 degradation and promote survival in multiple myeloma.","date":"2013","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/23263282","citation_count":82,"is_preprint":false},{"pmid":"34233195","id":"PMC_34233195","title":"Structure of the TELO2-TTI1-TTI2 complex and its function in TOR recruitment to the R2TP chaperone.","date":"2021","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/34233195","citation_count":26,"is_preprint":false},{"pmid":"34838521","id":"PMC_34838521","title":"Structure of the Human TELO2-TTI1-TTI2 Complex.","date":"2021","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/34838521","citation_count":10,"is_preprint":false},{"pmid":"31332096","id":"PMC_31332096","title":"A tel2 Mutation That Destabilizes the Tel2-Tti1-Tti2 Complex Eliminates Rad3ATR Kinase Signaling in the DNA Replication Checkpoint and Leads to Telomere Shortening in Fission Yeast.","date":"2019","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/31332096","citation_count":10,"is_preprint":false},{"pmid":"36403197","id":"PMC_36403197","title":"TTI1 promotes non-small-cell lung cancer progression by regulating the mTOR signaling pathway.","date":"2022","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/36403197","citation_count":9,"is_preprint":false},{"pmid":"34935410","id":"PMC_34935410","title":"Essentiality of Sis1, a J-domain protein Hsp70 cochaperone, can be overcome by Tti1, a specialized PIKK chaperone.","date":"2021","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/34935410","citation_count":8,"is_preprint":false},{"pmid":"37175973","id":"PMC_37175973","title":"TTT (Tel2-Tti1-Tti2) Complex, the Co-Chaperone of PIKKs and a Potential Target for Cancer Chemotherapy.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/37175973","citation_count":6,"is_preprint":false},{"pmid":"35116463","id":"PMC_35116463","title":"A role of TTI1 in the colorectal cancer by promoting proliferation.","date":"2021","source":"Translational cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/35116463","citation_count":6,"is_preprint":false},{"pmid":"36724785","id":"PMC_36724785","title":"Bi-allelic TTI1 variants cause an autosomal-recessive neurodevelopmental disorder with microcephaly.","date":"2023","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/36724785","citation_count":5,"is_preprint":false},{"pmid":"38501918","id":"PMC_38501918","title":"ALKBH5 promotes hepatocellular carcinoma cell proliferation, migration and invasion by regulating TTI1 expression.","date":"2024","source":"Biomolecules & biomedicine","url":"https://pubmed.ncbi.nlm.nih.gov/38501918","citation_count":3,"is_preprint":false},{"pmid":"40514657","id":"PMC_40514657","title":"TTI1 contributes to radioresistance by activating ATM pathway in rectal cancer.","date":"2025","source":"Journal of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/40514657","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8040,"output_tokens":2433,"usd":0.030308},"stage2":{"model":"claude-opus-4-6","input_tokens":5729,"output_tokens":2624,"usd":0.141368},"total_usd":0.171676,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"Mammalian TTI1 (KIAA0406) constitutively interacts with mTOR in both mTORC1 and mTORC2, binds Tel2, and is required for stability of all six PIKK family members (mTOR, ATM, ATR, DNA-PKcs, SMG-1, TRRAP). Knockdown of TTI1 causes disassembly of mTORC1 and mTORC2, suppresses phosphorylation of mTORC1 substrates (S6K1, 4E-BP1) and mTORC2 substrate (Akt), and induces autophagy.\",\n      \"method\": \"Co-immunoprecipitation, size-exclusion chromatography, siRNA knockdown, western blot for PIKK levels and substrate phosphorylation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, size-exclusion chromatography, multiple substrates tested, >200 citations and independently replicated\",\n      \"pmids\": [\"20427287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CK2 phosphorylates TTI1 (and Tel2) within mTORC1 upon growth factor withdrawal, targeting them for ubiquitination and degradation by the SCFFbxo9 E3 ubiquitin ligase, leading to mTORC1-specific inactivation while relieving feedback to sustain PI3K/mTORC2/Akt signaling.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, CK2 kinase assay, siRNA knockdown, reconstitution of SCFFbxo9 ubiquitin ligase activity\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — biochemical reconstitution of ubiquitin ligase activity, kinase assay, multiple orthogonal methods in a high-impact journal\",\n      \"pmids\": [\"23263282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"IP7 (generated by IP6K2) binds CK2 and enhances CK2-mediated phosphorylation of the TTT complex (Tel2/TTI1/TTI2), which stabilizes DNA-PKcs and ATM, thereby promoting p53 phosphorylation at serine 15 and apoptosis.\",\n      \"method\": \"In vitro CK2 kinase assay with IP7, co-immunoprecipitation, IP6K2 knockdown/overexpression, western blot for PIKK levels and p53 phosphorylation\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase assay with defined small molecule, multiple orthogonal methods, >100 citations\",\n      \"pmids\": [\"24657168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structure of the human R2TP-TTT complex reveals that the HEAT-repeat TTT complex binds the kinase domain of TOR (without blocking kinase activity) and delivers TOR to the R2TP chaperone; TTT also inhibits RUVBL1-RUVBL2 ATPase activity and modulates PIH1D1 and RPAP3 conformations within R2TP.\",\n      \"method\": \"Cryo-EM structure determination, biochemical ATPase assays, co-immunoprecipitation, crosslinking mass spectrometry\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure with biochemical validation of mechanism, multiple orthogonal assays\",\n      \"pmids\": [\"34233195\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structure of the TTT complex at 4.2 Å resolution shows TTI1 as a central scaffold with TELO2 binding its central region and TTI2 binding its C-terminal end; TTI1 N- and C-terminal segments contact the FAT domain and N-terminal HEAT repeats of ATM respectively, and the TELO2 CTD is required for interaction with TTI1 and ATM recruitment. TTI1 N- and C-terminal segments are required for cell survival after ionizing radiation.\",\n      \"method\": \"Cryo-EM structure determination, deletion mutagenesis, co-immunoprecipitation, cell survival assays after ionizing radiation\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure with mutagenesis and functional validation\",\n      \"pmids\": [\"34838521\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In fission yeast, destabilization of the TTT complex (Tel2-Tti1-Tti2) by a tel2 mutation nearly abolishes Rad3 (ATR ortholog) checkpoint signaling in the DNA replication checkpoint and causes telomere shortening, demonstrating that TTT complex integrity is required for ATR-mediated checkpoint kinase signaling.\",\n      \"method\": \"Genetic screen, co-immunoprecipitation to assess TTT complex stability, western blot for Rad3-mediated phospho-signaling, telomere length assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with biochemical validation in fission yeast ortholog\",\n      \"pmids\": [\"31332096\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In S. cerevisiae, single-residue substitutions in Tti1 suppress lethality caused by Sis1 (Hsp70 cochaperone) depletion; Sis1 depletion reduces levels of essential PIKKs (Mec1/ATR, Tra1/TRRAP, Tor2/mTOR), and overexpression of Tti1 alone (without increasing Tel2 or Tti2) restores growth, indicating Tti1 can function independently of the full TTT complex as a PIKK-specific chaperone.\",\n      \"method\": \"Genetic suppressor analysis, protein level measurement by western blot, rapamycin sensitivity assay, overexpression experiments\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with biochemical readouts, yeast ortholog\",\n      \"pmids\": [\"34935410\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Bi-allelic loss-of-function variants in TTI1 impair TTT complex integrity and reduce mTOR pathway activity (mTORC1 signaling) in patient-derived cells; rapamycin treatment partially restores mTOR pathway activity, placing TTI1 upstream of mTOR in the signaling cascade in a human disease context.\",\n      \"method\": \"Functional studies in HEK293T cells and patient-derived fibroblasts/lymphoblastoid cells, western blot for mTOR pathway substrates, rapamycin rescue experiment\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — patient-derived cell functional studies with pharmacological rescue, multiple cell types\",\n      \"pmids\": [\"36724785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TTI1 promotes ATM signaling pathway activation in rectal cancer cells; TTI1 knockdown reduces ATM-dependent DNA damage repair after irradiation, increasing radiosensitivity, while TTI1 overexpression enhances repair and radioresistance.\",\n      \"method\": \"siRNA knockdown, overexpression, western blot for ATM pathway activation, comet assay, colony formation assay, in vivo xenograft, organoid and PDX models\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — loss- and gain-of-function with multiple functional readouts including in vivo models\",\n      \"pmids\": [\"40514657\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TTI1 is a central scaffold subunit of the heterotrimeric TTT (TELO2-TTI1-TTI2) co-chaperone complex that binds newly synthesized PIKKs (mTOR, ATM, ATR, DNA-PKcs, SMG-1, TRRAP) via defined HEAT-repeat interfaces and delivers them to the R2TP-HSP90 chaperone system for folding, assembly, and stabilization; TTI1 is required for mTORC1 and mTORC2 complex assembly and activity, is targeted for CK2-primed SCFFbxo9-mediated degradation to attenuate mTORC1 signaling upon growth factor withdrawal, and is phosphorylated by CK2 in a manner enhanced by the inositol pyrophosphate IP7 to stabilize ATM/DNA-PKcs and activate p53-dependent apoptosis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TTI1 is the central scaffold subunit of the TTT (TELO2–TTI1–TTI2) co-chaperone complex that is essential for the folding, stabilization, and assembly of all six PIKK family kinases (mTOR, ATM, ATR, DNA-PKcs, SMG-1, TRRAP) [PMID:20427287]. Cryo-EM structures show that TTI1 HEAT repeats organize the complex, with TELO2 binding its central region and TTI2 its C-terminus, while TTI1 N- and C-terminal segments directly contact the FAT domain and N-terminal HEAT repeats of client PIKKs; the TTT complex delivers clients to the R2TP–HSP90 chaperone machinery by engaging the PIKK kinase domain and modulating RUVBL1/2 ATPase activity [PMID:34838521, PMID:34233195]. TTI1 is required for mTORC1 and mTORC2 assembly and signaling, and upon growth-factor withdrawal CK2 phosphorylates TTI1, targeting it for SCF^Fbxo9-mediated ubiquitination and degradation to selectively inactivate mTORC1 [PMID:23263282]; CK2-mediated phosphorylation of the TTT complex is enhanced by the inositol pyrophosphate IP7, which stabilizes DNA-PKcs and ATM to activate p53-dependent apoptosis [PMID:24657168]. Bi-allelic loss-of-function variants in TTI1 cause a neurodevelopmental disorder with impaired mTOR signaling in patient-derived cells [PMID:36724785].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Identifying TTI1 as a constitutive PIKK-binding partner resolved how all six PIKKs are co-stabilized and how mTORC1/mTORC2 assembly depends on a shared chaperone adaptor.\",\n      \"evidence\": \"Co-IP, gel filtration, and siRNA knockdown in mammalian cells showing loss of all PIKK proteins and mTOR complex activity upon TTI1 depletion\",\n      \"pmids\": [\"20427287\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural information on how TTI1 contacts PIKKs\", \"Mechanism of selectivity for PIKK family over other kinases unknown\", \"Role beyond protein stability (e.g., kinase activation) not addressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrating that CK2-primed SCF^Fbxo9 ubiquitination degrades TTI1 upon growth-factor withdrawal established the first regulated proteolytic switch that selectively inactivates mTORC1 while sustaining mTORC2/Akt signaling.\",\n      \"evidence\": \"Reconstituted SCF^Fbxo9 ubiquitin ligase activity, CK2 kinase assays, and siRNA in mammalian cells\",\n      \"pmids\": [\"23263282\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific CK2 phosphosites on TTI1 not mapped at residue resolution\", \"Whether other E3 ligases target TTI1 under different stress conditions unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showing that IP7 enhances CK2-mediated TTT complex phosphorylation to stabilize ATM/DNA-PKcs and activate p53-dependent apoptosis linked metabolite signaling to PIKK homeostasis through TTI1.\",\n      \"evidence\": \"In vitro CK2 kinase assays with IP7, IP6K2 manipulation, and western blot for PIKK levels and p53 Ser15 phosphorylation\",\n      \"pmids\": [\"24657168\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct IP7 binding site on TTI1/TTT versus CK2 not resolved\", \"Context-dependent outcome (mTORC1 degradation vs. ATM stabilization) of TTI1 phosphorylation not mechanistically reconciled\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Genetic destabilization of the TTT complex in fission yeast confirmed that TTT integrity is required for ATR checkpoint signaling and telomere maintenance, extending the mammalian findings to a conserved function.\",\n      \"evidence\": \"Genetic screen with tel2 mutant, co-IP for TTT stability, Rad3 phospho-signaling, and telomere length assay in S. pombe\",\n      \"pmids\": [\"31332096\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tti1-specific contributions versus Tel2/Tti2 not separated in this system\", \"Telomere maintenance mechanism (via ATR vs. other PIKKs) not distinguished\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Cryo-EM structures of the TTT complex alone and bound to R2TP revealed that TTI1 HEAT repeats serve as the central scaffold bridging TELO2 and TTI2, directly contacting PIKK FAT-domain and kinase-domain surfaces, and delivering clients to the R2TP–HSP90 system while inhibiting RUVBL1/2 ATPase activity.\",\n      \"evidence\": \"Cryo-EM at 4.2 Å (TTT–ATM) and of R2TP–TTT, deletion mutagenesis, crosslinking mass spectrometry, ATPase assays\",\n      \"pmids\": [\"34233195\", \"34838521\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution map of TTI1–PIKK interface residues needed for drug targeting\", \"How ATPase inhibition of RUVBL1/2 is relieved to complete client handoff is unknown\", \"Structure of TTT bound to PIKKs other than mTOR/ATM not determined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Yeast suppressor analysis showed Tti1 can function independently of the full TTT complex, with Tti1 overexpression alone rescuing PIKK levels after Hsp70-cochaperone depletion, implying an intrinsic PIKK-specific chaperone capacity.\",\n      \"evidence\": \"Genetic suppressor screen, protein level measurements, and rapamycin sensitivity in S. cerevisiae with Sis1 depletion\",\n      \"pmids\": [\"34935410\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether mammalian TTI1 retains TTT-independent chaperone activity not tested\", \"Physical interaction between Tti1 and PIKKs in the absence of Tel2/Tti2 not demonstrated biochemically\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Patient studies established that bi-allelic TTI1 loss-of-function causes a neurodevelopmental disorder with reduced mTOR signaling, directly linking TTI1 to human Mendelian disease.\",\n      \"evidence\": \"Functional studies in patient-derived fibroblasts and lymphoblastoid cells, HEK293T reconstitution, rapamycin rescue\",\n      \"pmids\": [\"36724785\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Impact on non-mTOR PIKKs (ATM, ATR) in patient cells not characterized\", \"Genotype-phenotype correlation across different TTI1 variants not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrating that TTI1 promotes ATM-dependent DNA damage repair in rectal cancer cells linked TTI1 expression to radioresistance and identified it as a potential therapeutic target.\",\n      \"evidence\": \"siRNA knockdown and overexpression with comet assay, colony formation, xenograft, organoid, and PDX models\",\n      \"pmids\": [\"40514657\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether TTI1 contribution to radioresistance is solely through ATM or involves other PIKKs not resolved\", \"No pharmacological inhibitor of TTI1 tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The precise mechanism by which TTI1 coordinates opposing phospho-dependent outcomes — mTORC1 degradation versus ATM/DNA-PKcs stabilization — and whether TTI1 has PIKK-independent functions remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Site-resolved phospho-regulation of TTI1 determining client-specific fate is unknown\", \"No high-resolution structure of TTI1 with a full-length non-mTOR/ATM PIKK client\", \"Whether TTI1 functions outside of PIKK biology is unexplored\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [0, 3, 4, 6]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 3, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 7]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [4, 5, 8]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 3, 4, 6]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [\n      \"TTT complex (TELO2–TTI1–TTI2)\",\n      \"R2TP–TTT complex\"\n    ],\n    \"partners\": [\n      \"TELO2\",\n      \"TTI2\",\n      \"MTOR\",\n      \"ATM\",\n      \"ATR\",\n      \"PRKDC\",\n      \"FBXO9\",\n      \"CSNK2A1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}