{"gene":"TTC36","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2019,"finding":"TTC36 acts as a molecular chaperone that directly associates with HPD (4-hydroxyphenylpyruvic acid dioxygenase) and reduces binding of protein kinase STK33 to HPD, thereby inhibiting STK33-mediated phosphorylation of HPD at T382. Loss of T382 phosphorylation impairs recruitment of the FHA domain-containing E3 ligase adaptor PELI1, preventing PELI1-mediated HPD polyubiquitylation and proteasomal degradation. TTC36 deficiency in mice (Ttc36-/-) leads to enhanced STK33-mediated HPD T382 phosphorylation, increased PELI1 binding, HPD downregulation in liver, and consequent tyrosinemia with hippocampal neuronal damage and memory deficits.","method":"Co-immunoprecipitation, in vitro kinase assay, phosphorylation-site mutagenesis (T382), ubiquitylation assay, Ttc36 knockout mouse model with phenotypic analysis","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal biochemical methods (Co-IP, kinase assay, mutagenesis, ubiquitylation assay) plus in vivo genetic knockout with defined molecular and physiological phenotype","pmids":["31537781"],"is_preprint":false},{"year":2008,"finding":"TTC36/HBP21 physically interacts with the C-terminal domain of HSP70 via its three tandem TPR motifs. Truncation and amino acid substitution mutants of HSP70 mapped the interaction to the EEVD/PTIEEVD C-terminal residues of HSP70, which are required for binding HBP21.","method":"Yeast two-hybrid assay, GST pull-down assay, Hsp70 truncation and substitution mutant analysis","journal":"Molecular Biotechnology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — reciprocal binding demonstrated by two orthogonal methods (yeast two-hybrid + GST pull-down) with mutagenesis mapping, but from a single lab","pmids":["18587674"],"is_preprint":false},{"year":2015,"finding":"HBP21/TTC36 inhibits the HSP70–Bax interaction, promoting Bax translocation from the cytoplasm to mitochondria, cytochrome c release into the cytoplasm, and subsequent apoptosis induction, particularly under heat or chemotherapeutic stress. This function was demonstrated by transfection of HBP21 into HCC cells followed by assessment of Bax localization and cytochrome c release.","method":"Transfection/overexpression in HCC cells, Co-IP (HSP70–Bax interaction), subcellular fractionation (Bax mitochondrial translocation), cytochrome c release assay, apoptosis assays","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple biochemical readouts (Co-IP, fractionation, cytochrome c) from a single lab establishing a mechanistic pathway","pmids":["26246424"],"is_preprint":false},{"year":2016,"finding":"Ttc36 protein is expressed in mouse liver and kidney from embryonic day E15.5 through adulthood and in testis. In kidney, Ttc36 co-localizes with the proximal tubule marker Lotus Tetragonolobus Lectin (LTL) but not with the distal tubule marker Calbindin-D28k, establishing its specific subcellular/tissue localization in proximal tubules.","method":"Immunofluorescence staining with validated polyclonal antibody, double immunofluorescence co-localization with proximal and distal tubule markers","journal":"Gene Expression Patterns","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization by immunofluorescence with validated antibody and orthogonal marker comparison; single lab","pmids":["27826126"],"is_preprint":false},{"year":2021,"finding":"TTC36 inactivation in gastric carcinoma cells activates the Wnt/β-catenin pathway. TTC36 overexpression reduced levels of β-catenin and its downstream effectors (TCF4, c-jun, pAKT), while TTC36 knockdown had the opposite effect. Treatment with the Wnt/β-catenin inhibitor XAV939 attenuated TTC36 loss-of-function effects, placing TTC36 upstream of β-catenin signaling.","method":"Lentiviral overexpression and knockdown in GC cell lines, Western blot for β-catenin/TCF4/c-jun/pAKT, GSEA pathway analysis, XAV939 pharmacological rescue","journal":"Journal of Cancer","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, primarily Western blot with pharmacological rescue; no direct biochemical interaction demonstrated between TTC36 and Wnt pathway components","pmids":["33854620"],"is_preprint":false},{"year":2025,"finding":"TTC36 promotes c-Myc protein accumulation in hepatocellular carcinoma cells by disrupting the interaction between SET and PPP2R1A, thereby suppressing PP2A phosphatase activity. When TTC36 is depleted, activated PP2A dephosphorylates p-c-MycS62 and activates GSK3β (via AKT), leading to increased p-c-MycT58 phosphorylation, FBXW7-mediated polyubiquitination, and c-Myc degradation. GSK3β and PP2A inhibitors reverse sorafenib resistance conferred by TTC36.","method":"Co-IP (SET–PPP2R1A interaction), PP2A activity assay, phosphorylation analysis of c-Myc (S62/T58), ubiquitination assay, TTC36 depletion in HCC cell lines, pharmacological inhibitor rescue","journal":"Cell Death & Disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biochemical assays (Co-IP, phosphorylation, ubiquitination, PP2A activity) in a single lab establishing a mechanistic cascade","pmids":["40274799"],"is_preprint":false},{"year":2025,"finding":"TTC36 directly binds YBX3 and masks its ubiquitination sites (K311/K350), inhibiting proteasomal degradation of YBX3. Stabilized YBX3 enhances SPRED1 mRNA stability by binding the CACAUC motif in SPRED1's 3'UTR, thereby suppressing Ras/MAPK signaling. This TTC36/YBX3/SPRED1 axis inhibits HCC tumor growth but paradoxically induces sorafenib resistance via compensatory PI3K/Akt activation.","method":"Mass spectrometry, RNA pulldown, dual-luciferase reporter assay, molecular docking, ubiquitination site mutagenesis (K311/K350), RNA-seq, in vivo tumor models, Akt inhibitor (MK-2206) rescue","journal":"International Journal of Biological Sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (MS, RNA pulldown, luciferase, mutagenesis, in vivo) from a single lab; not yet independently replicated","pmids":["41208883"],"is_preprint":false},{"year":2024,"finding":"TTC36 overexpression in HK2 renal tubular epithelial cells increases IκBα protein stability (prolonged half-life after cycloheximide chase, reversed by MG132), thereby inhibiting NF-κB p65 phosphorylation and activation, reducing inflammatory cytokine expression (TNF-α, iNOS, IL-6, CCL2, IL-1β), suppressing apoptosis, and promoting proliferation.","method":"Lentiviral overexpression in HK2 cells, Western blot, qRT-PCR, cycloheximide chase assay, MG132 proteasome inhibitor experiment, flow cytometry, CCK-8 proliferation assay","journal":"Chinese Journal of Cellular and Molecular Immunology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, cell-line overexpression only, no direct biochemical interaction between TTC36 and IκBα demonstrated","pmids":["39215665"],"is_preprint":false},{"year":2025,"finding":"HBP21/TTC36 overexpression in LPS-stimulated macrophages promotes M2 polarization and activates PI3K/AKT signaling, while HBP21 knockdown promotes M1 polarization and suppresses PI3K/AKT. The PI3K inhibitor LY294002 and activator 740Y-P reverse HBP21-mediated effects, confirming PI3K/AKT pathway involvement. In a rat CLP sepsis model, HBP21 overexpression reduced renal injury markers and shifted renal macrophages toward M2 phenotype.","method":"Lentiviral overexpression/knockdown in RAW264.7 macrophages, flow cytometry (M1/M2 markers), Western blot (PI3K/AKT), pharmacological inhibitor/activator (LY294002/740Y-P), CLP rat model, co-culture with renal tubular cells (NRK-52E)","journal":"Mediators of Inflammation","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, pharmacological pathway manipulation without direct TTC36–PI3K biochemical interaction; primarily phenotypic readouts","pmids":["40757051"],"is_preprint":false}],"current_model":"TTC36 is a TPR-motif chaperone protein that protects multiple client proteins from phosphorylation-dependent ubiquitination and degradation: in hepatocytes it shields HPD from STK33-mediated T382 phosphorylation and subsequent PELI1-mediated degradation (maintaining tyrosine catabolism), stabilizes YBX3 by masking ubiquitination sites to suppress Ras/MAPK signaling, and modulates c-Myc stability by maintaining the SET–PP2A interaction; it also binds HSP70 via its TPR motifs to regulate Bax-mediated apoptosis, and in renal cells stabilizes IκBα protein to suppress NF-κB-driven inflammation."},"narrative":{"mechanistic_narrative":"TTC36 (HBP21) is a TPR-motif chaperone protein that stabilizes client proteins by preventing their phosphorylation-dependent ubiquitination, with documented roles in hepatic amino-acid catabolism and tumor signaling [PMID:31537781, PMID:41208883]. Its three tandem TPR motifs bind the C-terminal EEVD/PTIEEVD residues of HSP70, defining a canonical co-chaperone binding mode [PMID:18587674]. In hepatocytes, TTC36 associates with HPD and excludes the kinase STK33, blocking STK33-mediated phosphorylation of HPD at T382; loss of this phosphorylation prevents recruitment of the FHA-domain E3 adaptor PELI1 and the resulting polyubiquitylation and proteasomal degradation of HPD, so that Ttc36 deficiency in mice causes HPD loss, tyrosinemia, hippocampal neuronal damage, and memory deficits [PMID:31537781]. TTC36 similarly stabilizes YBX3 by masking its K311/K350 ubiquitination sites, and the stabilized YBX3 binds the CACAUC motif in the SPRED1 3'UTR to enhance SPRED1 mRNA stability and suppress Ras/MAPK signaling [PMID:41208883]. It also promotes c-Myc accumulation by disrupting the SET–PPP2R1A interaction to suppress PP2A activity, thereby preserving stabilizing c-Myc phosphorylation and blocking FBXW7-mediated degradation [PMID:40274799]. Through its HSP70 interaction TTC36 inhibits the HSP70–Bax association, promoting Bax mitochondrial translocation, cytochrome c release, and apoptosis under stress [PMID:26246424]. TTC36 is expressed in liver and kidney and localizes specifically to renal proximal tubules [PMID:27826126].","teleology":[{"year":2008,"claim":"Established the physical basis for TTC36 acting as a co-chaperone by defining how it engages HSP70, the first molecular interaction assigned to the protein.","evidence":"Yeast two-hybrid and GST pull-down with HSP70 truncation/substitution mutants mapping the interaction to HSP70's C-terminal EEVD/PTIEEVD residues via TTC36's three TPR motifs","pmids":["18587674"],"confidence":"Medium","gaps":["Functional consequence of the HSP70 interaction not addressed","Single-lab in vitro mapping without cellular validation"]},{"year":2015,"claim":"Connected the HSP70 interaction to a cellular outcome, showing TTC36 modulates apoptosis by interfering with HSP70's anti-apoptotic sequestration of Bax.","evidence":"HBP21 transfection in HCC cells with Co-IP of HSP70–Bax, subcellular fractionation for Bax translocation, and cytochrome c release/apoptosis assays","pmids":["26246424"],"confidence":"Medium","gaps":["Whether the HSP70 TPR interaction is mechanistically required for Bax displacement not directly tested","Single-cell-line, overexpression-based"]},{"year":2016,"claim":"Defined the tissue and subcellular context of TTC36 expression, anchoring its function to renal proximal tubules and liver.","evidence":"Immunofluorescence in mouse tissues with co-localization against proximal (LTL) and distal (Calbindin-D28k) tubule markers across developmental stages","pmids":["27826126"],"confidence":"Medium","gaps":["Localization is descriptive and not linked to a molecular function","No subcellular compartment resolved within tubule cells"]},{"year":2019,"claim":"Provided the definitive in vivo mechanism: TTC36 protects HPD from a kinase/E3-ligase degradation axis, establishing a physiological role in tyrosine catabolism.","evidence":"Co-IP, in vitro kinase assay, T382 phospho-site mutagenesis, ubiquitylation assay, and Ttc36-knockout mouse with tyrosinemia and neurological phenotype","pmids":["31537781"],"confidence":"High","gaps":["Whether the same STK33/PELI1 axis operates on other clients unknown","Structural detail of HPD–TTC36 binding not resolved"]},{"year":2021,"claim":"Implicated TTC36 as a negative regulator of Wnt/β-catenin signaling in gastric carcinoma, suggesting a tumor-suppressive role.","evidence":"Lentiviral overexpression/knockdown in GC cells with Western blot for β-catenin/TCF4/c-jun/pAKT and XAV939 pharmacological rescue","pmids":["33854620"],"confidence":"Low","gaps":["No direct biochemical interaction between TTC36 and Wnt components demonstrated","Mechanism inferred pharmacologically","Single lab"]},{"year":2024,"claim":"Extended TTC36's protein-stabilizing role to inflammation, indicating it suppresses NF-κB signaling in renal tubular cells.","evidence":"Lentiviral overexpression in HK2 cells with cycloheximide chase, MG132 reversal of IκBα stabilization, and cytokine/apoptosis/proliferation readouts","pmids":["39215665"],"confidence":"Low","gaps":["No direct TTC36–IκBα interaction shown","Overexpression-only in a single cell line"]},{"year":2025,"claim":"Resolved two parallel client-stabilization mechanisms in hepatocellular carcinoma — masking YBX3 ubiquitination sites and disrupting the SET–PP2A interaction to control c-Myc — linking TTC36 to Ras/MAPK suppression and to sorafenib resistance.","evidence":"MS, RNA pulldown, luciferase, K311/K350 mutagenesis, in vivo tumor models (YBX3 axis); Co-IP, PP2A activity, c-Myc S62/T58 phospho and ubiquitination assays with inhibitor rescue (c-Myc axis)","pmids":["41208883","40274799"],"confidence":"Medium","gaps":["The two axes are characterized separately; their integration in the same cells unclear","Single-lab, not independently replicated","Direct binding interfaces not structurally resolved"]},{"year":2025,"claim":"Linked TTC36 to macrophage polarization and PI3K/AKT signaling in a sepsis-associated renal injury context.","evidence":"Lentiviral overexpression/knockdown in RAW264.7 macrophages with flow cytometry, PI3K inhibitor/activator rescue, and a CLP rat model","pmids":["40757051"],"confidence":"Low","gaps":["No direct TTC36–PI3K biochemical interaction","Predominantly phenotypic readouts","Single lab"]},{"year":null,"claim":"It remains unknown whether TTC36's diverse client interactions (HPD, YBX3, SET/PP2A, IκBα) share a common TPR-mediated recognition mechanism or reflect distinct binding modes.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of TTC36 bound to any client","Whether HSP70 co-chaperone activity underlies all client-stabilization events untested","Generality of the kinase/E3-ligase shielding mechanism across clients unestablished"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0044183","term_label":"protein folding chaperone","supporting_discovery_ids":[0,1]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,5,6]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[0,6]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,5,6]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[2]}],"complexes":[],"partners":["HPD","HSP70","YBX3","SET","PPP2R1A"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"A6NLP5","full_name":"Tetratricopeptide repeat protein 36","aliases":["HSP70-binding protein 21"],"length_aa":189,"mass_kda":20.9,"function":"","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/A6NLP5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TTC36","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TTC36","total_profiled":1310},"omim":[{"mim_id":"620701","title":"TETRATRICOPEPTIDE REPEAT DOMAIN 36; TTC36","url":"https://www.omim.org/entry/620701"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Nucleoplasm","reliability":"Uncertain"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"liver","ntpm":235.3}],"url":"https://www.proteinatlas.org/search/TTC36"},"hgnc":{"alias_symbol":["HBP21"],"prev_symbol":[]},"alphafold":{"accession":"A6NLP5","domains":[{"cath_id":"1.25.40.10","chopping":"44-107","consensus_level":"medium","plddt":96.7589,"start":44,"end":107},{"cath_id":"-","chopping":"117-182","consensus_level":"medium","plddt":89.5717,"start":117,"end":182}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/A6NLP5","model_url":"https://alphafold.ebi.ac.uk/files/AF-A6NLP5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-A6NLP5-F1-predicted_aligned_error_v6.png","plddt_mean":85.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TTC36","jax_strain_url":"https://www.jax.org/strain/search?query=TTC36"},"sequence":{"accession":"A6NLP5","fasta_url":"https://rest.uniprot.org/uniprotkb/A6NLP5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/A6NLP5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/A6NLP5"}},"corpus_meta":[{"pmid":"31537781","id":"PMC_31537781","title":"HPD degradation regulated by the TTC36-STK33-PELI1 signaling axis induces tyrosinemia and neurological damage.","date":"2019","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/31537781","citation_count":29,"is_preprint":false},{"pmid":"33854620","id":"PMC_33854620","title":"TTC36 inactivation induce malignant properties via Wnt-β-catenin pathway in gastric carcinoma.","date":"2021","source":"Journal of Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/33854620","citation_count":16,"is_preprint":false},{"pmid":"18587674","id":"PMC_18587674","title":"HBP21: a novel member of TPR motif family, as a potential chaperone of heat shock protein 70 in proliferative vitreoretinopathy (PVR) and breast cancer.","date":"2008","source":"Molecular biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/18587674","citation_count":15,"is_preprint":false},{"pmid":"26246424","id":"PMC_26246424","title":"HBP21, a chaperone of heat shock protein 70, functions as a tumor suppressor in hepatocellular carcinoma.","date":"2015","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/26246424","citation_count":14,"is_preprint":false},{"pmid":"27826126","id":"PMC_27826126","title":"The expression patterns of Tetratricopeptide repeat domain 36 (Ttc36).","date":"2016","source":"Gene expression patterns : GEP","url":"https://pubmed.ncbi.nlm.nih.gov/27826126","citation_count":11,"is_preprint":false},{"pmid":"40274799","id":"PMC_40274799","title":"TTC36 promotes proliferation and drug resistance in hepatocellular carcinoma cells by inhibiting c-Myc degradation.","date":"2025","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/40274799","citation_count":7,"is_preprint":false},{"pmid":"41208883","id":"PMC_41208883","title":"TTC36-Mediated Tumor Suppression via YBX3/SPRED1 Axis Paradoxically Reduces Sorafenib Sensitivity in Hepatocellular Carcinoma.","date":"2025","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/41208883","citation_count":0,"is_preprint":false},{"pmid":"40757051","id":"PMC_40757051","title":"HBP21 Alleviates Sepsis-Induced Acute Kidney Injury by Targeting PI3K/AKT-Mediated M1 Macrophage Polarization.","date":"2025","source":"Mediators of inflammation","url":"https://pubmed.ncbi.nlm.nih.gov/40757051","citation_count":0,"is_preprint":false},{"pmid":"39215665","id":"PMC_39215665","title":"[Tetrapeptide repeat domain 36 (TTC36) inhibits the inflammatory response of HK2 cells by enhancing the expression of IκBα to inhibit the activation of NF-κB signaling pathway].","date":"2024","source":"Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/39215665","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7172,"output_tokens":2907,"usd":0.03256,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10273,"output_tokens":3106,"usd":0.064507,"stage2_stop_reason":"end_turn"},"total_usd":0.097067,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2019,\n      \"finding\": \"TTC36 acts as a molecular chaperone that directly associates with HPD (4-hydroxyphenylpyruvic acid dioxygenase) and reduces binding of protein kinase STK33 to HPD, thereby inhibiting STK33-mediated phosphorylation of HPD at T382. Loss of T382 phosphorylation impairs recruitment of the FHA domain-containing E3 ligase adaptor PELI1, preventing PELI1-mediated HPD polyubiquitylation and proteasomal degradation. TTC36 deficiency in mice (Ttc36-/-) leads to enhanced STK33-mediated HPD T382 phosphorylation, increased PELI1 binding, HPD downregulation in liver, and consequent tyrosinemia with hippocampal neuronal damage and memory deficits.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, phosphorylation-site mutagenesis (T382), ubiquitylation assay, Ttc36 knockout mouse model with phenotypic analysis\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal biochemical methods (Co-IP, kinase assay, mutagenesis, ubiquitylation assay) plus in vivo genetic knockout with defined molecular and physiological phenotype\",\n      \"pmids\": [\"31537781\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TTC36/HBP21 physically interacts with the C-terminal domain of HSP70 via its three tandem TPR motifs. Truncation and amino acid substitution mutants of HSP70 mapped the interaction to the EEVD/PTIEEVD C-terminal residues of HSP70, which are required for binding HBP21.\",\n      \"method\": \"Yeast two-hybrid assay, GST pull-down assay, Hsp70 truncation and substitution mutant analysis\",\n      \"journal\": \"Molecular Biotechnology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — reciprocal binding demonstrated by two orthogonal methods (yeast two-hybrid + GST pull-down) with mutagenesis mapping, but from a single lab\",\n      \"pmids\": [\"18587674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"HBP21/TTC36 inhibits the HSP70–Bax interaction, promoting Bax translocation from the cytoplasm to mitochondria, cytochrome c release into the cytoplasm, and subsequent apoptosis induction, particularly under heat or chemotherapeutic stress. This function was demonstrated by transfection of HBP21 into HCC cells followed by assessment of Bax localization and cytochrome c release.\",\n      \"method\": \"Transfection/overexpression in HCC cells, Co-IP (HSP70–Bax interaction), subcellular fractionation (Bax mitochondrial translocation), cytochrome c release assay, apoptosis assays\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple biochemical readouts (Co-IP, fractionation, cytochrome c) from a single lab establishing a mechanistic pathway\",\n      \"pmids\": [\"26246424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Ttc36 protein is expressed in mouse liver and kidney from embryonic day E15.5 through adulthood and in testis. In kidney, Ttc36 co-localizes with the proximal tubule marker Lotus Tetragonolobus Lectin (LTL) but not with the distal tubule marker Calbindin-D28k, establishing its specific subcellular/tissue localization in proximal tubules.\",\n      \"method\": \"Immunofluorescence staining with validated polyclonal antibody, double immunofluorescence co-localization with proximal and distal tubule markers\",\n      \"journal\": \"Gene Expression Patterns\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization by immunofluorescence with validated antibody and orthogonal marker comparison; single lab\",\n      \"pmids\": [\"27826126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TTC36 inactivation in gastric carcinoma cells activates the Wnt/β-catenin pathway. TTC36 overexpression reduced levels of β-catenin and its downstream effectors (TCF4, c-jun, pAKT), while TTC36 knockdown had the opposite effect. Treatment with the Wnt/β-catenin inhibitor XAV939 attenuated TTC36 loss-of-function effects, placing TTC36 upstream of β-catenin signaling.\",\n      \"method\": \"Lentiviral overexpression and knockdown in GC cell lines, Western blot for β-catenin/TCF4/c-jun/pAKT, GSEA pathway analysis, XAV939 pharmacological rescue\",\n      \"journal\": \"Journal of Cancer\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, primarily Western blot with pharmacological rescue; no direct biochemical interaction demonstrated between TTC36 and Wnt pathway components\",\n      \"pmids\": [\"33854620\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TTC36 promotes c-Myc protein accumulation in hepatocellular carcinoma cells by disrupting the interaction between SET and PPP2R1A, thereby suppressing PP2A phosphatase activity. When TTC36 is depleted, activated PP2A dephosphorylates p-c-MycS62 and activates GSK3β (via AKT), leading to increased p-c-MycT58 phosphorylation, FBXW7-mediated polyubiquitination, and c-Myc degradation. GSK3β and PP2A inhibitors reverse sorafenib resistance conferred by TTC36.\",\n      \"method\": \"Co-IP (SET–PPP2R1A interaction), PP2A activity assay, phosphorylation analysis of c-Myc (S62/T58), ubiquitination assay, TTC36 depletion in HCC cell lines, pharmacological inhibitor rescue\",\n      \"journal\": \"Cell Death & Disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical assays (Co-IP, phosphorylation, ubiquitination, PP2A activity) in a single lab establishing a mechanistic cascade\",\n      \"pmids\": [\"40274799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TTC36 directly binds YBX3 and masks its ubiquitination sites (K311/K350), inhibiting proteasomal degradation of YBX3. Stabilized YBX3 enhances SPRED1 mRNA stability by binding the CACAUC motif in SPRED1's 3'UTR, thereby suppressing Ras/MAPK signaling. This TTC36/YBX3/SPRED1 axis inhibits HCC tumor growth but paradoxically induces sorafenib resistance via compensatory PI3K/Akt activation.\",\n      \"method\": \"Mass spectrometry, RNA pulldown, dual-luciferase reporter assay, molecular docking, ubiquitination site mutagenesis (K311/K350), RNA-seq, in vivo tumor models, Akt inhibitor (MK-2206) rescue\",\n      \"journal\": \"International Journal of Biological Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (MS, RNA pulldown, luciferase, mutagenesis, in vivo) from a single lab; not yet independently replicated\",\n      \"pmids\": [\"41208883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TTC36 overexpression in HK2 renal tubular epithelial cells increases IκBα protein stability (prolonged half-life after cycloheximide chase, reversed by MG132), thereby inhibiting NF-κB p65 phosphorylation and activation, reducing inflammatory cytokine expression (TNF-α, iNOS, IL-6, CCL2, IL-1β), suppressing apoptosis, and promoting proliferation.\",\n      \"method\": \"Lentiviral overexpression in HK2 cells, Western blot, qRT-PCR, cycloheximide chase assay, MG132 proteasome inhibitor experiment, flow cytometry, CCK-8 proliferation assay\",\n      \"journal\": \"Chinese Journal of Cellular and Molecular Immunology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, cell-line overexpression only, no direct biochemical interaction between TTC36 and IκBα demonstrated\",\n      \"pmids\": [\"39215665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HBP21/TTC36 overexpression in LPS-stimulated macrophages promotes M2 polarization and activates PI3K/AKT signaling, while HBP21 knockdown promotes M1 polarization and suppresses PI3K/AKT. The PI3K inhibitor LY294002 and activator 740Y-P reverse HBP21-mediated effects, confirming PI3K/AKT pathway involvement. In a rat CLP sepsis model, HBP21 overexpression reduced renal injury markers and shifted renal macrophages toward M2 phenotype.\",\n      \"method\": \"Lentiviral overexpression/knockdown in RAW264.7 macrophages, flow cytometry (M1/M2 markers), Western blot (PI3K/AKT), pharmacological inhibitor/activator (LY294002/740Y-P), CLP rat model, co-culture with renal tubular cells (NRK-52E)\",\n      \"journal\": \"Mediators of Inflammation\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, pharmacological pathway manipulation without direct TTC36–PI3K biochemical interaction; primarily phenotypic readouts\",\n      \"pmids\": [\"40757051\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TTC36 is a TPR-motif chaperone protein that protects multiple client proteins from phosphorylation-dependent ubiquitination and degradation: in hepatocytes it shields HPD from STK33-mediated T382 phosphorylation and subsequent PELI1-mediated degradation (maintaining tyrosine catabolism), stabilizes YBX3 by masking ubiquitination sites to suppress Ras/MAPK signaling, and modulates c-Myc stability by maintaining the SET–PP2A interaction; it also binds HSP70 via its TPR motifs to regulate Bax-mediated apoptosis, and in renal cells stabilizes IκBα protein to suppress NF-κB-driven inflammation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TTC36 (HBP21) is a TPR-motif chaperone protein that stabilizes client proteins by preventing their phosphorylation-dependent ubiquitination, with documented roles in hepatic amino-acid catabolism and tumor signaling [#0, #6]. Its three tandem TPR motifs bind the C-terminal EEVD/PTIEEVD residues of HSP70, defining a canonical co-chaperone binding mode [#1]. In hepatocytes, TTC36 associates with HPD and excludes the kinase STK33, blocking STK33-mediated phosphorylation of HPD at T382; loss of this phosphorylation prevents recruitment of the FHA-domain E3 adaptor PELI1 and the resulting polyubiquitylation and proteasomal degradation of HPD, so that Ttc36 deficiency in mice causes HPD loss, tyrosinemia, hippocampal neuronal damage, and memory deficits [#0]. TTC36 similarly stabilizes YBX3 by masking its K311/K350 ubiquitination sites, and the stabilized YBX3 binds the CACAUC motif in the SPRED1 3'UTR to enhance SPRED1 mRNA stability and suppress Ras/MAPK signaling [#6]. It also promotes c-Myc accumulation by disrupting the SET–PPP2R1A interaction to suppress PP2A activity, thereby preserving stabilizing c-Myc phosphorylation and blocking FBXW7-mediated degradation [#5]. Through its HSP70 interaction TTC36 inhibits the HSP70–Bax association, promoting Bax mitochondrial translocation, cytochrome c release, and apoptosis under stress [#2]. TTC36 is expressed in liver and kidney and localizes specifically to renal proximal tubules [#3].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Established the physical basis for TTC36 acting as a co-chaperone by defining how it engages HSP70, the first molecular interaction assigned to the protein.\",\n      \"evidence\": \"Yeast two-hybrid and GST pull-down with HSP70 truncation/substitution mutants mapping the interaction to HSP70's C-terminal EEVD/PTIEEVD residues via TTC36's three TPR motifs\",\n      \"pmids\": [\"18587674\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of the HSP70 interaction not addressed\", \"Single-lab in vitro mapping without cellular validation\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected the HSP70 interaction to a cellular outcome, showing TTC36 modulates apoptosis by interfering with HSP70's anti-apoptotic sequestration of Bax.\",\n      \"evidence\": \"HBP21 transfection in HCC cells with Co-IP of HSP70–Bax, subcellular fractionation for Bax translocation, and cytochrome c release/apoptosis assays\",\n      \"pmids\": [\"26246424\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the HSP70 TPR interaction is mechanistically required for Bax displacement not directly tested\", \"Single-cell-line, overexpression-based\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined the tissue and subcellular context of TTC36 expression, anchoring its function to renal proximal tubules and liver.\",\n      \"evidence\": \"Immunofluorescence in mouse tissues with co-localization against proximal (LTL) and distal (Calbindin-D28k) tubule markers across developmental stages\",\n      \"pmids\": [\"27826126\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Localization is descriptive and not linked to a molecular function\", \"No subcellular compartment resolved within tubule cells\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Provided the definitive in vivo mechanism: TTC36 protects HPD from a kinase/E3-ligase degradation axis, establishing a physiological role in tyrosine catabolism.\",\n      \"evidence\": \"Co-IP, in vitro kinase assay, T382 phospho-site mutagenesis, ubiquitylation assay, and Ttc36-knockout mouse with tyrosinemia and neurological phenotype\",\n      \"pmids\": [\"31537781\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the same STK33/PELI1 axis operates on other clients unknown\", \"Structural detail of HPD–TTC36 binding not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Implicated TTC36 as a negative regulator of Wnt/β-catenin signaling in gastric carcinoma, suggesting a tumor-suppressive role.\",\n      \"evidence\": \"Lentiviral overexpression/knockdown in GC cells with Western blot for β-catenin/TCF4/c-jun/pAKT and XAV939 pharmacological rescue\",\n      \"pmids\": [\"33854620\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct biochemical interaction between TTC36 and Wnt components demonstrated\", \"Mechanism inferred pharmacologically\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended TTC36's protein-stabilizing role to inflammation, indicating it suppresses NF-κB signaling in renal tubular cells.\",\n      \"evidence\": \"Lentiviral overexpression in HK2 cells with cycloheximide chase, MG132 reversal of IκBα stabilization, and cytokine/apoptosis/proliferation readouts\",\n      \"pmids\": [\"39215665\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct TTC36–IκBα interaction shown\", \"Overexpression-only in a single cell line\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved two parallel client-stabilization mechanisms in hepatocellular carcinoma — masking YBX3 ubiquitination sites and disrupting the SET–PP2A interaction to control c-Myc — linking TTC36 to Ras/MAPK suppression and to sorafenib resistance.\",\n      \"evidence\": \"MS, RNA pulldown, luciferase, K311/K350 mutagenesis, in vivo tumor models (YBX3 axis); Co-IP, PP2A activity, c-Myc S62/T58 phospho and ubiquitination assays with inhibitor rescue (c-Myc axis)\",\n      \"pmids\": [\"41208883\", \"40274799\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The two axes are characterized separately; their integration in the same cells unclear\", \"Single-lab, not independently replicated\", \"Direct binding interfaces not structurally resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked TTC36 to macrophage polarization and PI3K/AKT signaling in a sepsis-associated renal injury context.\",\n      \"evidence\": \"Lentiviral overexpression/knockdown in RAW264.7 macrophages with flow cytometry, PI3K inhibitor/activator rescue, and a CLP rat model\",\n      \"pmids\": [\"40757051\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct TTC36–PI3K biochemical interaction\", \"Predominantly phenotypic readouts\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown whether TTC36's diverse client interactions (HPD, YBX3, SET/PP2A, IκBα) share a common TPR-mediated recognition mechanism or reflect distinct binding modes.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of TTC36 bound to any client\", \"Whether HSP70 co-chaperone activity underlies all client-stabilization events untested\", \"Generality of the kinase/E3-ligase shielding mechanism across clients unestablished\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0044183\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 5, 6]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [0, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 5, 6]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"HPD\", \"HSP70\", \"YBX3\", \"SET\", \"PPP2R1A\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}