{"gene":"TPI1","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2024,"finding":"Dopamine directly modifies TPI1 by dopaminylating the glutamine 65 (Q65) residue in endothelial cells. This post-translational modification directionally enhances TPI1's catalytic activity to convert DHAP to GAP, shifting ether phospholipid synthesis toward glucose metabolism, thereby attenuating lipid peroxidation and blocking ferroptosis in regenerating lung endothelial cells.","method":"Chemoproteomic approach identifying dopaminylation site; in vitro TPI1 activity assays; metabolic flux measurements; mutagenesis of Q65; loss-of-function and rescue experiments in endothelial cells and mouse lung injury models","journal":"Cell metabolism","confidence":"High","confidence_rationale":"Tier 1 / Strong — site-specific mutagenesis, in vitro enzymatic assays, chemoproteomic identification of modification site, and in vivo rescue experiments all in one rigorous study","pmids":["39111287"],"is_preprint":false},{"year":2023,"finding":"In human lung adenocarcinoma (hLUAD), TPI1 activity is regulated by phosphorylation at Ser21 by salt-inducible kinases (SIKs) in an LKB1-dependent manner. This phosphorylation modulates metabolic flux between the completion of glycolysis and production of glycerol lipids. Mouse TPI1 has a Cys at the equivalent position that can be oxidized to alter activity, representing an evolutionary divergence that explains why LKB1 loss creates a metabolic liability specifically in human tumors with KRAS/TP53 mutations.","method":"Phosphoproteomics; metabolomics; genetically engineered human cell lines with LKB1/SIK manipulation; genetically engineered mouse models (GEMM) comparison","journal":"Cancer discovery","confidence":"High","confidence_rationale":"Tier 1 / Strong — phosphoproteomics identifying specific phosphorylation site, metabolomics, and orthogonal genetic models (human cell lines + GEMMs) in a single rigorous study","pmids":["36715544"],"is_preprint":false},{"year":2022,"finding":"TPI1 undergoes nuclear translocation in lung adenocarcinoma tumor tissues (compared to cytoplasmic localization in adjacent normal tissues), and this nuclear localization—rather than its glycolytic catalytic activity—is required for its oncogenic function and for promoting chemoresistance. Nuclear translocation is induced by extracellular stresses including chemotherapy agents and peroxide.","method":"Subcellular fractionation; immunofluorescence; knockdown of TPI1; catalytic mutant experiments; xenograft tumor growth assays; TCGA data analysis","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiments with functional consequence (catalytic mutant vs. localization mutant), single lab with multiple orthogonal methods","pmids":["35246510"],"is_preprint":false},{"year":2022,"finding":"TPI1 interacts with SQSTM1/P62, which promotes ubiquitin-dependent proteasomal degradation of TPI1, decreasing TPI1 protein levels in breast cancer cells. TPI1 also interacts with and stabilizes CDCA5, activating the PI3K/AKT/mTOR pathway to regulate EMT and aerobic glycolysis.","method":"Co-immunoprecipitation; mass spectrometric analysis; ubiquitination assay; immunofluorescence; overexpression and knockdown functional experiments; Western blotting; in vivo mouse models","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and ubiquitination assay with multiple orthogonal methods, single lab","pmids":["35509067"],"is_preprint":false},{"year":2019,"finding":"The Arg189 residue in TPI1 participates in two salt bridges on the backside of the TPI enzyme dimer; mutation of this residue (Arg189Gln) alters the coordination of the substrate-binding site and important catalytic residues, causing reduced protein stability and loss of function. This was demonstrated by homologous mutagenesis in Drosophila using genomic engineering, showing motor behavioral deficits and markedly reduced protein levels.","method":"Genomic engineering in Drosophila (homologous mutagenesis); compound heterozygote animal generation; patient fibroblast analysis; structural analysis of salt bridge coordination","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vivo mutagenesis in model organism, patient fibroblast validation, and structural rationale for catalytic site disruption, multiple orthogonal methods","pmids":["31075491"],"is_preprint":false},{"year":2025,"finding":"TPI1 interacts with AKT and MDM2 to form a protein complex that enhances AKT-driven phosphorylation of MDM2 at serine 166, thereby promoting p53 ubiquitination and degradation in bladder cancer cells. The MDM2-F2 truncation mutant (spanning residues 181–360) with amino acid 317 was identified as critical for TPI1 binding. Reducing AKT expression counteracted p53 ubiquitination triggered by elevated TPI1.","method":"Co-immunoprecipitation; truncation mutagenesis of MDM2; AKT knockdown rescue experiments; functional cell assays; in vivo xenograft models","journal":"Pharmacological research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mapping mutagenesis and genetic rescue experiments, single lab","pmids":["40097123"],"is_preprint":false},{"year":2021,"finding":"TPI1 is incorporated into extracellular vesicles (EVs) in a manner positively regulated by Rab20 expression in the releasing hepatocellular carcinoma cells. EVs with reduced TPI1 (from Rab20-knockdown cells) enhance aerobic glycolysis in recipient cells, promoting HCC cell growth and motility; this promoting effect is blocked by a glycolytic inhibitor.","method":"Rab20 restoration and knockdown in HCC cells; proteomic profiling of EVs; TPI1 targeted expression in EVs; glycolytic inhibitor rescue experiments; cell proliferation and motility assays","journal":"Journal of extracellular vesicles","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomic profiling of EVs combined with genetic manipulation and pharmacological rescue, single lab with multiple orthogonal methods","pmids":["34401050"],"is_preprint":false},{"year":2025,"finding":"TPI1 directly binds to the BH3 domain of Beclin-1, competitively disrupting the Bcl-2/Beclin-1 interaction and relieving Bcl-2-mediated inhibition of Beclin-1. This interaction promotes PIK3C3-C1 complex formation and enhances its interaction with the ULK1 complex, increasing Beclin-1 phosphorylation at Ser15 and promoting autophagy, which contributes to gemcitabine resistance in bladder cancer cells. c-Myc was identified as a transcription factor that binds the TPI1 promoter to regulate its expression.","method":"Mass spectrometry; co-immunoprecipitation; transcriptome sequencing; transmission electron microscopy; dual luciferase assay; ChIP-qPCR; in vivo xenograft models","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified interaction confirmed by Co-IP with domain specificity, multiple orthogonal methods in single lab","pmids":["41429797"],"is_preprint":false},{"year":2024,"finding":"In LPS-induced OA chondrocytes, LDHA mediates H3K18 lactylation (H3K18la) at the TPI1 promoter, enhancing TPI1 transcriptional activity and glycolysis. Mutation of the K69 site ameliorated LPS-induced glycolysis, and LDHA knockout recovered cartilage injury in OA mice.","method":"LDHA knockdown and knockout (loss-of-function); H3K18la ChIP at TPI1 promoter; K69 mutation in TPI1; glycolysis functional assays (glucose consumption, lactate production); in vivo OA mouse model","journal":"Autoimmunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter-level ChIP for H3K18la combined with site mutagenesis and in vivo validation, single lab","pmids":["39086231"],"is_preprint":false},{"year":2025,"finding":"TPI1 nuclear phosphorylation is increased by GRK4 R65L in high-salt conditions; this reduces nuclear DHAP levels, increases H3K27ac at the Hao2 promoter, and upregulates Hao2 expression, leading to increased renal oxidative stress and salt-sensitive hypertension. DHAP (downstream metabolite of TPI1) directly reduced H3K27ac and Hao2 levels in cells.","method":"Immunoprecipitation-mass spectrometry (identifying TPI1-GRK4 interaction and TPI1 phosphorylation); GRK4 depletion via AAV9; DHAP supplementation rescue; H3K27ac inhibitor C646 treatment; in vivo GRK4 R65L mouse model; nuclear fractionation","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — IP-MS identifying phosphorylation, in vivo genetic model, pharmacological rescue with DHAP and C646, single lab with multiple orthogonal methods","pmids":["41407053"],"is_preprint":false},{"year":2025,"finding":"USP5 deubiquitinates TPI1, stabilizing its protein levels. Propofol increases TPI1 ubiquitination and reduces TPI1 protein stability, and the inhibitory effects of propofol on lung cancer glycolysis and progression are mediated through this USP5/TPI1 axis.","method":"Ubiquitination analysis; Western blot for TPI1 protein levels upon USP5 manipulation; qRT-PCR; xenograft mouse models; IHC","journal":"Biochemical genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, ubiquitination assay and protein level measurements without in vitro reconstitution of deubiquitination","pmids":["40956511"],"is_preprint":false},{"year":2026,"finding":"NOP2 promotes m5C methylation of TPI1 mRNA, enhancing its stability. Knockdown of NOP2 reduced m5C modification on TPI1 mRNA and decreased TPI1 expression; overexpression of TPI1 rescued glycolysis impaired by NOP2 knockdown in larynx cancer cells.","method":"MeRIP (methylated RNA immunoprecipitation) for m5C on TPI1 mRNA; RIP; dual-luciferase reporter assay; NOP2 knockdown and TPI1 overexpression rescue; xenograft tumor models; IHC","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP directly demonstrating m5C modification on TPI1 mRNA with rescue experiments, single lab","pmids":["41498196"],"is_preprint":false},{"year":2026,"finding":"NSUN2 promotes m5C methylation of TPI1 mRNA in a manner recognized by YBX1, enhancing TPI1 mRNA stability. TPI1 overexpression reversed the inhibition of breast cancer glycolysis, immune evasion, and tumor growth caused by NSUN2 silencing.","method":"RNA immunoprecipitation; methylated RNA immunoprecipitation (MeRIP); dual-luciferase reporter assay; NSUN2 knockdown/TPI1 overexpression rescue; tumor-bearing mouse model","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP demonstrating m5C modification on TPI1 mRNA, YBX1 as reader identified by RIP, genetic rescue experiments, single lab","pmids":["42251361"],"is_preprint":false},{"year":2026,"finding":"EIF4A3 interacts with TPI1 mRNA to stabilize it. OTUB2 deubiquitinates and stabilizes EIF4A3 protein, which in turn promotes TPI1 expression and glycolysis in triple-negative breast cancer.","method":"Co-immunoprecipitation; RNA immunoprecipitation; Western blot for protein stability; OTUB2/EIF4A3/TPI1 knockdown and overexpression rescue; xenograft mouse model","journal":"Breast cancer research : BCR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP confirming EIF4A3-TPI1 mRNA interaction and OTUB2 deubiquitination of EIF4A3, with genetic rescue, single lab","pmids":["41857621"],"is_preprint":false},{"year":2025,"finding":"lncRNA HANR directly interacts with TPI1 protein to stabilize it, promoting aerobic glycolysis and tumor growth in prostate cancer. Silencing HANR or TPI1 reduced prostate tumor growth in vitro and in vivo.","method":"RNA immunoprecipitation/pulldown (HANR-TPI1 interaction); HANR and TPI1 knockdown functional assays; in vivo xenograft model","journal":"Experimental cell research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, RIP/pulldown showing lncRNA-protein interaction without detailed mechanistic dissection of stabilization mechanism","pmids":["40921293"],"is_preprint":false},{"year":2024,"finding":"In GBM cells, TPI1 interacts with PKM2 and the lncRNA Linc00942 (ChIRP-MS and ChIRP-WB assays). This interaction promotes phosphorylation, dimerization, and nuclear translocation of TPI1 and PKM2, leading to increased H3K4 acetylation and STAT3/P300 axis activation, which transcriptionally activates SOX9 to drive TMZ resistance and self-renewal.","method":"ChIRP-MS (chromatin isolation by RNA purification followed by mass spectrometry); ChIRP-WB; Co-immunoprecipitation; in vitro and in vivo functional assays","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — ChIRP-MS identifies interaction, mechanistic follow-up is partial; single lab study; the lncRNA is not TPI1's canonical product but TPI1 protein is the subject here","pmids":["39342418"],"is_preprint":false},{"year":2025,"finding":"Hypoxia upregulates HK2 and TPI1 protein levels in non-neuronal C6 glioma cells without increasing their mRNA levels, implicating post-transcriptional regulation. Using dicistronic and promoter-less dicistronic reporter assays, IRES (internal ribosome entry site) elements were identified in the 5'UTR of TPI1 mRNA that are more active in C6 glioma cells, with PTB (polypyrimidine tract binding) protein involved.","method":"Dicistronic reporter assays; promoter-less dicistronic assays; MTT assay; LDH leakage assay; Western blot; qRT-PCR","journal":"Artificial cells, nanomedicine, and biotechnology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, IRES activity demonstrated by reporter assays but PTB-TPI1 mRNA interaction not directly validated by RIP or pulldown","pmids":["40105374"],"is_preprint":false},{"year":2008,"finding":"Deletion of the tpi1 (triosephosphate isomerase) gene in Saccharomyces cerevisiae increases carbon flux to DHAP in glycolysis, resulting in increased glycerol production. Introduction of bacterial mgs and gldA genes (converting DHAP to 1,2-propanediol) into the tpi1-deleted strain confirmed that DHAP accumulation from TPI1 loss drives metabolic flux to methylglyoxal and then 1,2-propanediol.","method":"Genetic deletion of tpi1 in yeast; metabolic engineering with heterologous gene expression; metabolite measurement in flask culture","journal":"Journal of microbiology and biotechnology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic KO with defined metabolic phenotype and metabolite quantification; yeast ortholog establishes canonical TPI1 function in DHAP/GAP interconversion","pmids":["19047824"],"is_preprint":false},{"year":2025,"finding":"A compound heterozygous TPI1 allele carrying an R5G missense mutation results in markedly reduced steady-state TPI protein levels despite the purified TPIR5G protein retaining wild-type catalytic activity and modestly increased dimer stability, indicating protein instability (not catalytic defect) as the pathogenic mechanism. Three newly identified compounds significantly increased TPI protein levels in patient cells, also increasing TPI enzymatic activity.","method":"Recombinant protein expression and purification; in vitro TPI activity assay; dimer stability assay; patient cell (TPIpatient) Western blot; treatment with pharmacological compounds and TPI activity measurement","journal":"Genes","confidence":"High","confidence_rationale":"Tier 1 / Moderate — purified recombinant protein biochemistry (activity, stability) combined with patient cell validation; single lab but multiple orthogonal methods","pmids":["41153421"],"is_preprint":false}],"current_model":"TPI1 (triosephosphate isomerase 1) is a glycolytic enzyme that interconverts DHAP and GAP; its activity is regulated by site-specific post-translational modifications including phosphorylation at Ser21 by LKB1-dependent SIKs (in humans) and dopaminylation at Q65 (which directionally enhances DHAP-to-GAP conversion to suppress ferroptosis), while its protein stability is controlled by ubiquitin-mediated proteasomal degradation (via P62/SQSTM1 or USP5-dependent deubiquitination) and mRNA stability is regulated by m5C modification (through NSUN2/YBX1 or NOP2); beyond cytoplasmic glycolysis, TPI1 undergoes nuclear translocation under stress conditions where it promotes oncogenic transcriptional programs (including SOX9 via STAT3/P300) and interacts with Beclin-1 to drive autophagy-mediated chemoresistance, and its metabolic product DHAP functions as an epigenetic regulator by modulating H3K27 acetylation."},"narrative":{"mechanistic_narrative":"TPI1 is the glycolytic isomerase that interconverts DHAP and GAP, a reaction central to controlling carbon partitioning between glycolytic completion and glycerolipid synthesis [PMID:19047824, PMID:36715544]. Its catalytic output is tuned by site-specific post-translational modifications: dopaminylation at Gln65 directionally enhances DHAP-to-GAP conversion to redirect ether phospholipid synthesis, attenuate lipid peroxidation, and block ferroptosis [PMID:39111287], while phosphorylation at Ser21 by LKB1-dependent salt-inducible kinases (SIKs) modulates flux between glycolysis and glycerol lipids in human lung adenocarcinoma [PMID:36715544]. Enzyme integrity depends on structural elements whose disruption causes loss of function through protein destabilization rather than direct catalytic impairment, as shown for the dimer-interface Arg189 and the R5G allele, in which purified mutant protein retains wild-type activity yet is depleted at steady state [PMID:31075491, PMID:41153421]. TPI1 abundance is set by competing protein-stability inputs—SQSTM1/P62-mediated ubiquitin-dependent degradation versus USP5-dependent deubiquitination—and by mRNA-level control through m5C methylation (NSUN2/YBX1 and NOP2) and EIF4A3-mediated transcript stabilization [PMID:35509067, PMID:40956511, PMID:41498196, PMID:42251361, PMID:41857621]. Beyond cytoplasmic glycolysis, TPI1 translocates to the nucleus under chemotherapy and peroxide stress, where its nuclear localization, independent of catalytic activity, drives oncogenic transcriptional programs and chemoresistance, including STAT3/P300-dependent SOX9 activation in glioblastoma [PMID:35246510, PMID:39342418]. In bladder cancer it acts non-glycolytically by binding the Beclin-1 BH3 domain to disrupt the Bcl-2/Beclin-1 interaction and promote autophagy [PMID:41429797], and by forming an AKT–MDM2 complex that enhances MDM2 Ser166 phosphorylation to drive p53 ubiquitination and degradation [PMID:40097123]. The TPI1 product DHAP itself functions as an epigenetic signal, with nuclear DHAP levels modulating H3K27 acetylation at target promoters [PMID:41407053]. Compound heterozygous TPI1 mutations cause a human enzymopathy presenting with reduced enzyme protein and neuromuscular dysfunction [PMID:31075491, PMID:41153421].","teleology":[{"year":2008,"claim":"Establishing TPI1's canonical metabolic role: deleting the gene demonstrated it governs the DHAP/GAP equilibrium and thereby controls carbon flux toward glycerol and downstream metabolites.","evidence":"Genetic deletion of tpi1 in S. cerevisiae with heterologous metabolic engineering and metabolite quantification","pmids":["19047824"],"confidence":"Medium","gaps":["Yeast ortholog; does not address regulation of human TPI1","No structural or PTM-level mechanism for flux control"]},{"year":2019,"claim":"Defined how point mutations cause disease by showing that an interface salt-bridge residue (Arg189) is required for protein stability and substrate-site coordination, linking structural destabilization to loss of function and motor deficits.","evidence":"Homologous genomic mutagenesis in Drosophila, patient fibroblast analysis, and structural rationale","pmids":["31075491"],"confidence":"High","gaps":["Does not resolve whether instability or catalytic loss dominates for all pathogenic alleles","No human in vivo validation"]},{"year":2022,"claim":"Revealed a moonlighting function: stress-induced nuclear translocation, not glycolytic catalysis, drives TPI1's oncogenic and chemoresistance activity.","evidence":"Subcellular fractionation, immunofluorescence, catalytic mutant vs. localization experiments, and xenografts in lung adenocarcinoma","pmids":["35246510"],"confidence":"Medium","gaps":["Nuclear import mechanism and transcriptional targets not defined here","Single tumor type"]},{"year":2022,"claim":"Identified protein-turnover and signaling partners: SQSTM1/P62 drives ubiquitin-dependent degradation while CDCA5 stabilization links TPI1 to PI3K/AKT/mTOR-driven EMT and glycolysis.","evidence":"Reciprocal Co-IP, ubiquitination assays, and functional knockdown/overexpression in breast cancer cells","pmids":["35509067"],"confidence":"Medium","gaps":["E3 ligase mediating P62-dependent degradation not identified","Direct vs. indirect CDCA5 stabilization unresolved"]},{"year":2023,"claim":"Connected TPI1 to a kinase signaling axis: LKB1-dependent SIK phosphorylation at Ser21 modulates the glycolysis/glycerolipid branch point, explaining a human-specific metabolic liability not present in mouse (which has an oxidizable Cys).","evidence":"Phosphoproteomics, metabolomics, and orthogonal human cell line plus GEMM models","pmids":["36715544"],"confidence":"High","gaps":["Structural basis of how Ser21 phosphorylation alters activity not fully resolved","Cross-species divergence complicates mouse modeling"]},{"year":2024,"claim":"Demonstrated a direct PTM that tunes catalytic directionality: dopaminylation at Q65 enhances DHAP-to-GAP conversion to suppress ferroptosis through redirected ether phospholipid synthesis.","evidence":"Chemoproteomic site identification, in vitro activity assays, Q65 mutagenesis, and rescue in endothelial cells and mouse lung injury","pmids":["39111287"],"confidence":"High","gaps":["Enzyme(s) installing/removing dopaminylation not defined","Generality beyond endothelial regeneration unknown"]},{"year":2024,"claim":"Linked TPI1 transcription to metabolic-epigenetic feedback, showing LDHA-driven H3K18 lactylation at the TPI1 promoter upregulates its expression to amplify glycolysis.","evidence":"LDHA knockdown/knockout, H3K18la ChIP at the TPI1 promoter, K69 mutation, and OA mouse model","pmids":["39086231"],"confidence":"Medium","gaps":["Direct lactylation writer at TPI1 locus not isolated","Limited to OA chondrocyte context"]},{"year":2024,"claim":"Extended the nuclear-moonlighting model mechanistically, showing TPI1 partners with PKM2 and Linc00942 to undergo nuclear translocation and activate a STAT3/P300–SOX9 transcriptional program driving therapy resistance.","evidence":"ChIRP-MS/WB, Co-IP, and functional assays in glioblastoma","pmids":["39342418"],"confidence":"Low","gaps":["ChIRP-MS interaction with partial mechanistic follow-up; single lab","Direct vs. scaffold role of TPI1 in the complex unclear"]},{"year":2025,"claim":"Established a non-glycolytic autophagy mechanism: TPI1 binds the Beclin-1 BH3 domain to displace Bcl-2 and promote PIK3C3-C1/ULK1 assembly, driving autophagy and gemcitabine resistance.","evidence":"MS, Co-IP with domain specificity, ChIP-qPCR for c-Myc regulation, and xenografts in bladder cancer","pmids":["41429797"],"confidence":"Medium","gaps":["Whether catalytic activity is dispensable for Beclin-1 binding not stated","Single cancer context"]},{"year":2025,"claim":"Defined a tumor-suppressor-antagonizing complex: TPI1 scaffolds AKT and MDM2 to enhance MDM2 Ser166 phosphorylation and p53 degradation.","evidence":"Co-IP, MDM2 truncation mapping (residues 181–360), AKT knockdown rescue, and xenografts in bladder cancer","pmids":["40097123"],"confidence":"Medium","gaps":["Direct vs. AKT-bridged TPI1–MDM2 contact not fully separated","Single lab"]},{"year":2025,"claim":"Showed TPI1 product DHAP acts as an epigenetic signal: GRK4-driven nuclear TPI1 phosphorylation lowers nuclear DHAP, raising H3K27ac at the Hao2 promoter to promote oxidative stress and salt-sensitive hypertension.","evidence":"IP-MS, GRK4 R65L mouse model, DHAP supplementation and C646 rescue, nuclear fractionation","pmids":["41407053"],"confidence":"Medium","gaps":["Mechanism by which DHAP modulates acetyltransferase activity not defined","Phosphosite on nuclear TPI1 not mapped here"]},{"year":2025,"claim":"Identified deubiquitination as a stabilization input: USP5 stabilizes TPI1 protein, and propofol inhibits lung cancer glycolysis by increasing TPI1 ubiquitination through this axis.","evidence":"Ubiquitination analysis, USP5 manipulation, and xenografts","pmids":["40956511"],"confidence":"Low","gaps":["No in vitro reconstitution of USP5 deubiquitination of TPI1","Direct vs. indirect USP5–TPI1 action not shown"]},{"year":2025,"claim":"Added a lncRNA stabilization input, showing HANR directly binds and stabilizes TPI1 protein to support prostate tumor glycolysis.","evidence":"RIP/pulldown and knockdown functional and xenograft assays","pmids":["40921293"],"confidence":"Low","gaps":["Stabilization mechanism not dissected","RIP/pulldown without orthogonal validation"]},{"year":2025,"claim":"Confirmed the destabilization-disease paradigm and offered a therapeutic strategy: the R5G allele retains catalytic activity but is depleted, and small molecules restore TPI protein and activity in patient cells.","evidence":"Recombinant protein activity/stability assays, patient cell Western blot, and pharmacological rescue","pmids":["41153421"],"confidence":"High","gaps":["Mechanism of compound-mediated stabilization not defined","In vivo efficacy untested"]},{"year":2026,"claim":"Defined mRNA-stability control of TPI1 through m5C methylation and translation-associated factors, linking RNA modification machinery to glycolytic output and immune evasion.","evidence":"MeRIP, RIP, dual-luciferase reporters and rescue across larynx, breast, and TNBC models (NOP2; NSUN2/YBX1; OTUB2/EIF4A3)","pmids":["41498196","42251361","41857621"],"confidence":"Medium","gaps":["Whether these RNA inputs converge on the same regulatory elements unknown","Relative contribution versus protein-level control unquantified"]},{"year":2026,"claim":"Identified post-transcriptional translational control under hypoxia via an IRES element in the TPI1 5'UTR.","evidence":"Dicistronic reporter assays with PTB involvement in C6 glioma cells","pmids":["40105374"],"confidence":"Low","gaps":["PTB–TPI1 mRNA interaction not directly validated by RIP/pulldown","Single context"]},{"year":null,"claim":"How the diverse regulatory layers—catalytic-tuning PTMs, nuclear moonlighting, and the multiple protein- and mRNA-stability inputs—are integrated to set TPI1 output in a given cell type, and which are dispensable for its enzymatic versus non-enzymatic functions, remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unified model coordinating PTMs, localization, and stability","Catalysis-independent functions not systematically separated from glycolytic role","Nuclear import mechanism undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016853","term_label":"isomerase activity","supporting_discovery_ids":[0,1,17,18]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[5,7]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,17]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,15,9]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,1,17]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[7]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[15,9]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[11,12,13]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[0,5]}],"complexes":[],"partners":["SQSTM1","CDCA5","AKT1","MDM2","BECN1","PKM","GRK4","EIF4A3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P60174","full_name":"Triosephosphate isomerase","aliases":["Methylglyoxal synthase","Triose-phosphate isomerase"],"length_aa":249,"mass_kda":26.7,"function":"Triosephosphate isomerase is an extremely efficient metabolic enzyme that catalyzes the interconversion between dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (G3P) in glycolysis and gluconeogenesis It is also responsible for the non-negligible production of methylglyoxal a reactive cytotoxic side-product that modifies and can alter proteins, DNA and lipids","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/P60174/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/TPI1","classification":"Common Essential","n_dependent_lines":1041,"n_total_lines":1208,"dependency_fraction":0.8617549668874173},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TPI1","total_profiled":1310},"omim":[{"mim_id":"615512","title":"TRIOSEPHOSPHATE ISOMERASE DEFICIENCY; TPID","url":"https://www.omim.org/entry/615512"},{"mim_id":"600414","title":"PEROXISOME BIOGENESIS FACTOR 5; PEX5","url":"https://www.omim.org/entry/600414"},{"mim_id":"190450","title":"TRIOSEPHOSPHATE ISOMERASE 1; TPI1","url":"https://www.omim.org/entry/190450"},{"mim_id":"176267","title":"POTASSIUM CHANNEL, VOLTAGE-GATED, SHAKER-RELATED SUBFAMILY, MEMBER 5; KCNA5","url":"https://www.omim.org/entry/176267"},{"mim_id":"176260","title":"POTASSIUM CHANNEL, VOLTAGE-GATED, SHAKER-RELATED SUBFAMILY, MEMBER 1; KCNA1","url":"https://www.omim.org/entry/176260"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"skeletal muscle","ntpm":2315.7},{"tissue":"tongue","ntpm":1873.5}],"url":"https://www.proteinatlas.org/search/TPI1"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P60174","domains":[{"cath_id":"3.20.20.70","chopping":"6-245","consensus_level":"high","plddt":97.4416,"start":6,"end":245}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P60174","model_url":"https://alphafold.ebi.ac.uk/files/AF-P60174-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P60174-F1-predicted_aligned_error_v6.png","plddt_mean":96.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TPI1","jax_strain_url":"https://www.jax.org/strain/search?query=TPI1"},"sequence":{"accession":"P60174","fasta_url":"https://rest.uniprot.org/uniprotkb/P60174.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P60174/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P60174"}},"corpus_meta":[{"pmid":"35509067","id":"PMC_35509067","title":"TPI1 activates the PI3K/AKT/mTOR signaling pathway to induce breast cancer progression by stabilizing CDCA5.","date":"2022","source":"Journal of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35509067","citation_count":54,"is_preprint":false},{"pmid":"35246510","id":"PMC_35246510","title":"Elevated nuclear localization of glycolytic enzyme TPI1 promotes lung adenocarcinoma and enhances chemoresistance.","date":"2022","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/35246510","citation_count":45,"is_preprint":false},{"pmid":"34401050","id":"PMC_34401050","title":"TPI1-reduced extracellular vesicles mediated by Rab20 downregulation promotes aerobic glycolysis to drive hepatocarcinogenesis.","date":"2021","source":"Journal of extracellular vesicles","url":"https://pubmed.ncbi.nlm.nih.gov/34401050","citation_count":44,"is_preprint":false},{"pmid":"39086231","id":"PMC_39086231","title":"LDHA-induced histone lactylation mediates the development of osteoarthritis 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This post-translational modification directionally enhances TPI1's catalytic activity to convert DHAP to GAP, shifting ether phospholipid synthesis toward glucose metabolism, thereby attenuating lipid peroxidation and blocking ferroptosis in regenerating lung endothelial cells.\",\n      \"method\": \"Chemoproteomic approach identifying dopaminylation site; in vitro TPI1 activity assays; metabolic flux measurements; mutagenesis of Q65; loss-of-function and rescue experiments in endothelial cells and mouse lung injury models\",\n      \"journal\": \"Cell metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — site-specific mutagenesis, in vitro enzymatic assays, chemoproteomic identification of modification site, and in vivo rescue experiments all in one rigorous study\",\n      \"pmids\": [\"39111287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In human lung adenocarcinoma (hLUAD), TPI1 activity is regulated by phosphorylation at Ser21 by salt-inducible kinases (SIKs) in an LKB1-dependent manner. This phosphorylation modulates metabolic flux between the completion of glycolysis and production of glycerol lipids. Mouse TPI1 has a Cys at the equivalent position that can be oxidized to alter activity, representing an evolutionary divergence that explains why LKB1 loss creates a metabolic liability specifically in human tumors with KRAS/TP53 mutations.\",\n      \"method\": \"Phosphoproteomics; metabolomics; genetically engineered human cell lines with LKB1/SIK manipulation; genetically engineered mouse models (GEMM) comparison\",\n      \"journal\": \"Cancer discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — phosphoproteomics identifying specific phosphorylation site, metabolomics, and orthogonal genetic models (human cell lines + GEMMs) in a single rigorous study\",\n      \"pmids\": [\"36715544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TPI1 undergoes nuclear translocation in lung adenocarcinoma tumor tissues (compared to cytoplasmic localization in adjacent normal tissues), and this nuclear localization—rather than its glycolytic catalytic activity—is required for its oncogenic function and for promoting chemoresistance. Nuclear translocation is induced by extracellular stresses including chemotherapy agents and peroxide.\",\n      \"method\": \"Subcellular fractionation; immunofluorescence; knockdown of TPI1; catalytic mutant experiments; xenograft tumor growth assays; TCGA data analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiments with functional consequence (catalytic mutant vs. localization mutant), single lab with multiple orthogonal methods\",\n      \"pmids\": [\"35246510\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TPI1 interacts with SQSTM1/P62, which promotes ubiquitin-dependent proteasomal degradation of TPI1, decreasing TPI1 protein levels in breast cancer cells. TPI1 also interacts with and stabilizes CDCA5, activating the PI3K/AKT/mTOR pathway to regulate EMT and aerobic glycolysis.\",\n      \"method\": \"Co-immunoprecipitation; mass spectrometric analysis; ubiquitination assay; immunofluorescence; overexpression and knockdown functional experiments; Western blotting; in vivo mouse models\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and ubiquitination assay with multiple orthogonal methods, single lab\",\n      \"pmids\": [\"35509067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The Arg189 residue in TPI1 participates in two salt bridges on the backside of the TPI enzyme dimer; mutation of this residue (Arg189Gln) alters the coordination of the substrate-binding site and important catalytic residues, causing reduced protein stability and loss of function. This was demonstrated by homologous mutagenesis in Drosophila using genomic engineering, showing motor behavioral deficits and markedly reduced protein levels.\",\n      \"method\": \"Genomic engineering in Drosophila (homologous mutagenesis); compound heterozygote animal generation; patient fibroblast analysis; structural analysis of salt bridge coordination\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vivo mutagenesis in model organism, patient fibroblast validation, and structural rationale for catalytic site disruption, multiple orthogonal methods\",\n      \"pmids\": [\"31075491\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TPI1 interacts with AKT and MDM2 to form a protein complex that enhances AKT-driven phosphorylation of MDM2 at serine 166, thereby promoting p53 ubiquitination and degradation in bladder cancer cells. The MDM2-F2 truncation mutant (spanning residues 181–360) with amino acid 317 was identified as critical for TPI1 binding. Reducing AKT expression counteracted p53 ubiquitination triggered by elevated TPI1.\",\n      \"method\": \"Co-immunoprecipitation; truncation mutagenesis of MDM2; AKT knockdown rescue experiments; functional cell assays; in vivo xenograft models\",\n      \"journal\": \"Pharmacological research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mapping mutagenesis and genetic rescue experiments, single lab\",\n      \"pmids\": [\"40097123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TPI1 is incorporated into extracellular vesicles (EVs) in a manner positively regulated by Rab20 expression in the releasing hepatocellular carcinoma cells. EVs with reduced TPI1 (from Rab20-knockdown cells) enhance aerobic glycolysis in recipient cells, promoting HCC cell growth and motility; this promoting effect is blocked by a glycolytic inhibitor.\",\n      \"method\": \"Rab20 restoration and knockdown in HCC cells; proteomic profiling of EVs; TPI1 targeted expression in EVs; glycolytic inhibitor rescue experiments; cell proliferation and motility assays\",\n      \"journal\": \"Journal of extracellular vesicles\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomic profiling of EVs combined with genetic manipulation and pharmacological rescue, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"34401050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TPI1 directly binds to the BH3 domain of Beclin-1, competitively disrupting the Bcl-2/Beclin-1 interaction and relieving Bcl-2-mediated inhibition of Beclin-1. This interaction promotes PIK3C3-C1 complex formation and enhances its interaction with the ULK1 complex, increasing Beclin-1 phosphorylation at Ser15 and promoting autophagy, which contributes to gemcitabine resistance in bladder cancer cells. c-Myc was identified as a transcription factor that binds the TPI1 promoter to regulate its expression.\",\n      \"method\": \"Mass spectrometry; co-immunoprecipitation; transcriptome sequencing; transmission electron microscopy; dual luciferase assay; ChIP-qPCR; in vivo xenograft models\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified interaction confirmed by Co-IP with domain specificity, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"41429797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In LPS-induced OA chondrocytes, LDHA mediates H3K18 lactylation (H3K18la) at the TPI1 promoter, enhancing TPI1 transcriptional activity and glycolysis. Mutation of the K69 site ameliorated LPS-induced glycolysis, and LDHA knockout recovered cartilage injury in OA mice.\",\n      \"method\": \"LDHA knockdown and knockout (loss-of-function); H3K18la ChIP at TPI1 promoter; K69 mutation in TPI1; glycolysis functional assays (glucose consumption, lactate production); in vivo OA mouse model\",\n      \"journal\": \"Autoimmunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter-level ChIP for H3K18la combined with site mutagenesis and in vivo validation, single lab\",\n      \"pmids\": [\"39086231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TPI1 nuclear phosphorylation is increased by GRK4 R65L in high-salt conditions; this reduces nuclear DHAP levels, increases H3K27ac at the Hao2 promoter, and upregulates Hao2 expression, leading to increased renal oxidative stress and salt-sensitive hypertension. DHAP (downstream metabolite of TPI1) directly reduced H3K27ac and Hao2 levels in cells.\",\n      \"method\": \"Immunoprecipitation-mass spectrometry (identifying TPI1-GRK4 interaction and TPI1 phosphorylation); GRK4 depletion via AAV9; DHAP supplementation rescue; H3K27ac inhibitor C646 treatment; in vivo GRK4 R65L mouse model; nuclear fractionation\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — IP-MS identifying phosphorylation, in vivo genetic model, pharmacological rescue with DHAP and C646, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"41407053\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP5 deubiquitinates TPI1, stabilizing its protein levels. Propofol increases TPI1 ubiquitination and reduces TPI1 protein stability, and the inhibitory effects of propofol on lung cancer glycolysis and progression are mediated through this USP5/TPI1 axis.\",\n      \"method\": \"Ubiquitination analysis; Western blot for TPI1 protein levels upon USP5 manipulation; qRT-PCR; xenograft mouse models; IHC\",\n      \"journal\": \"Biochemical genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, ubiquitination assay and protein level measurements without in vitro reconstitution of deubiquitination\",\n      \"pmids\": [\"40956511\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"NOP2 promotes m5C methylation of TPI1 mRNA, enhancing its stability. Knockdown of NOP2 reduced m5C modification on TPI1 mRNA and decreased TPI1 expression; overexpression of TPI1 rescued glycolysis impaired by NOP2 knockdown in larynx cancer cells.\",\n      \"method\": \"MeRIP (methylated RNA immunoprecipitation) for m5C on TPI1 mRNA; RIP; dual-luciferase reporter assay; NOP2 knockdown and TPI1 overexpression rescue; xenograft tumor models; IHC\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP directly demonstrating m5C modification on TPI1 mRNA with rescue experiments, single lab\",\n      \"pmids\": [\"41498196\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"NSUN2 promotes m5C methylation of TPI1 mRNA in a manner recognized by YBX1, enhancing TPI1 mRNA stability. TPI1 overexpression reversed the inhibition of breast cancer glycolysis, immune evasion, and tumor growth caused by NSUN2 silencing.\",\n      \"method\": \"RNA immunoprecipitation; methylated RNA immunoprecipitation (MeRIP); dual-luciferase reporter assay; NSUN2 knockdown/TPI1 overexpression rescue; tumor-bearing mouse model\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP demonstrating m5C modification on TPI1 mRNA, YBX1 as reader identified by RIP, genetic rescue experiments, single lab\",\n      \"pmids\": [\"42251361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"EIF4A3 interacts with TPI1 mRNA to stabilize it. OTUB2 deubiquitinates and stabilizes EIF4A3 protein, which in turn promotes TPI1 expression and glycolysis in triple-negative breast cancer.\",\n      \"method\": \"Co-immunoprecipitation; RNA immunoprecipitation; Western blot for protein stability; OTUB2/EIF4A3/TPI1 knockdown and overexpression rescue; xenograft mouse model\",\n      \"journal\": \"Breast cancer research : BCR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP confirming EIF4A3-TPI1 mRNA interaction and OTUB2 deubiquitination of EIF4A3, with genetic rescue, single lab\",\n      \"pmids\": [\"41857621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"lncRNA HANR directly interacts with TPI1 protein to stabilize it, promoting aerobic glycolysis and tumor growth in prostate cancer. Silencing HANR or TPI1 reduced prostate tumor growth in vitro and in vivo.\",\n      \"method\": \"RNA immunoprecipitation/pulldown (HANR-TPI1 interaction); HANR and TPI1 knockdown functional assays; in vivo xenograft model\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, RIP/pulldown showing lncRNA-protein interaction without detailed mechanistic dissection of stabilization mechanism\",\n      \"pmids\": [\"40921293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In GBM cells, TPI1 interacts with PKM2 and the lncRNA Linc00942 (ChIRP-MS and ChIRP-WB assays). This interaction promotes phosphorylation, dimerization, and nuclear translocation of TPI1 and PKM2, leading to increased H3K4 acetylation and STAT3/P300 axis activation, which transcriptionally activates SOX9 to drive TMZ resistance and self-renewal.\",\n      \"method\": \"ChIRP-MS (chromatin isolation by RNA purification followed by mass spectrometry); ChIRP-WB; Co-immunoprecipitation; in vitro and in vivo functional assays\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — ChIRP-MS identifies interaction, mechanistic follow-up is partial; single lab study; the lncRNA is not TPI1's canonical product but TPI1 protein is the subject here\",\n      \"pmids\": [\"39342418\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Hypoxia upregulates HK2 and TPI1 protein levels in non-neuronal C6 glioma cells without increasing their mRNA levels, implicating post-transcriptional regulation. Using dicistronic and promoter-less dicistronic reporter assays, IRES (internal ribosome entry site) elements were identified in the 5'UTR of TPI1 mRNA that are more active in C6 glioma cells, with PTB (polypyrimidine tract binding) protein involved.\",\n      \"method\": \"Dicistronic reporter assays; promoter-less dicistronic assays; MTT assay; LDH leakage assay; Western blot; qRT-PCR\",\n      \"journal\": \"Artificial cells, nanomedicine, and biotechnology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, IRES activity demonstrated by reporter assays but PTB-TPI1 mRNA interaction not directly validated by RIP or pulldown\",\n      \"pmids\": [\"40105374\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Deletion of the tpi1 (triosephosphate isomerase) gene in Saccharomyces cerevisiae increases carbon flux to DHAP in glycolysis, resulting in increased glycerol production. Introduction of bacterial mgs and gldA genes (converting DHAP to 1,2-propanediol) into the tpi1-deleted strain confirmed that DHAP accumulation from TPI1 loss drives metabolic flux to methylglyoxal and then 1,2-propanediol.\",\n      \"method\": \"Genetic deletion of tpi1 in yeast; metabolic engineering with heterologous gene expression; metabolite measurement in flask culture\",\n      \"journal\": \"Journal of microbiology and biotechnology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic KO with defined metabolic phenotype and metabolite quantification; yeast ortholog establishes canonical TPI1 function in DHAP/GAP interconversion\",\n      \"pmids\": [\"19047824\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A compound heterozygous TPI1 allele carrying an R5G missense mutation results in markedly reduced steady-state TPI protein levels despite the purified TPIR5G protein retaining wild-type catalytic activity and modestly increased dimer stability, indicating protein instability (not catalytic defect) as the pathogenic mechanism. Three newly identified compounds significantly increased TPI protein levels in patient cells, also increasing TPI enzymatic activity.\",\n      \"method\": \"Recombinant protein expression and purification; in vitro TPI activity assay; dimer stability assay; patient cell (TPIpatient) Western blot; treatment with pharmacological compounds and TPI activity measurement\",\n      \"journal\": \"Genes\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — purified recombinant protein biochemistry (activity, stability) combined with patient cell validation; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"41153421\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TPI1 (triosephosphate isomerase 1) is a glycolytic enzyme that interconverts DHAP and GAP; its activity is regulated by site-specific post-translational modifications including phosphorylation at Ser21 by LKB1-dependent SIKs (in humans) and dopaminylation at Q65 (which directionally enhances DHAP-to-GAP conversion to suppress ferroptosis), while its protein stability is controlled by ubiquitin-mediated proteasomal degradation (via P62/SQSTM1 or USP5-dependent deubiquitination) and mRNA stability is regulated by m5C modification (through NSUN2/YBX1 or NOP2); beyond cytoplasmic glycolysis, TPI1 undergoes nuclear translocation under stress conditions where it promotes oncogenic transcriptional programs (including SOX9 via STAT3/P300) and interacts with Beclin-1 to drive autophagy-mediated chemoresistance, and its metabolic product DHAP functions as an epigenetic regulator by modulating H3K27 acetylation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TPI1 is the glycolytic isomerase that interconverts DHAP and GAP, a reaction central to controlling carbon partitioning between glycolytic completion and glycerolipid synthesis [#17, #1]. Its catalytic output is tuned by site-specific post-translational modifications: dopaminylation at Gln65 directionally enhances DHAP-to-GAP conversion to redirect ether phospholipid synthesis, attenuate lipid peroxidation, and block ferroptosis [#0], while phosphorylation at Ser21 by LKB1-dependent salt-inducible kinases (SIKs) modulates flux between glycolysis and glycerol lipids in human lung adenocarcinoma [#1]. Enzyme integrity depends on structural elements whose disruption causes loss of function through protein destabilization rather than direct catalytic impairment, as shown for the dimer-interface Arg189 and the R5G allele, in which purified mutant protein retains wild-type activity yet is depleted at steady state [#4, #18]. TPI1 abundance is set by competing protein-stability inputs—SQSTM1/P62-mediated ubiquitin-dependent degradation versus USP5-dependent deubiquitination—and by mRNA-level control through m5C methylation (NSUN2/YBX1 and NOP2) and EIF4A3-mediated transcript stabilization [#3, #10, #11, #12, #13]. Beyond cytoplasmic glycolysis, TPI1 translocates to the nucleus under chemotherapy and peroxide stress, where its nuclear localization, independent of catalytic activity, drives oncogenic transcriptional programs and chemoresistance, including STAT3/P300-dependent SOX9 activation in glioblastoma [#2, #15]. In bladder cancer it acts non-glycolytically by binding the Beclin-1 BH3 domain to disrupt the Bcl-2/Beclin-1 interaction and promote autophagy [#7], and by forming an AKT–MDM2 complex that enhances MDM2 Ser166 phosphorylation to drive p53 ubiquitination and degradation [#5]. The TPI1 product DHAP itself functions as an epigenetic signal, with nuclear DHAP levels modulating H3K27 acetylation at target promoters [#9]. Compound heterozygous TPI1 mutations cause a human enzymopathy presenting with reduced enzyme protein and neuromuscular dysfunction [#4, #18].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Establishing TPI1's canonical metabolic role: deleting the gene demonstrated it governs the DHAP/GAP equilibrium and thereby controls carbon flux toward glycerol and downstream metabolites.\",\n      \"evidence\": \"Genetic deletion of tpi1 in S. cerevisiae with heterologous metabolic engineering and metabolite quantification\",\n      \"pmids\": [\"19047824\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Yeast ortholog; does not address regulation of human TPI1\", \"No structural or PTM-level mechanism for flux control\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined how point mutations cause disease by showing that an interface salt-bridge residue (Arg189) is required for protein stability and substrate-site coordination, linking structural destabilization to loss of function and motor deficits.\",\n      \"evidence\": \"Homologous genomic mutagenesis in Drosophila, patient fibroblast analysis, and structural rationale\",\n      \"pmids\": [\"31075491\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not resolve whether instability or catalytic loss dominates for all pathogenic alleles\", \"No human in vivo validation\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed a moonlighting function: stress-induced nuclear translocation, not glycolytic catalysis, drives TPI1's oncogenic and chemoresistance activity.\",\n      \"evidence\": \"Subcellular fractionation, immunofluorescence, catalytic mutant vs. localization experiments, and xenografts in lung adenocarcinoma\",\n      \"pmids\": [\"35246510\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Nuclear import mechanism and transcriptional targets not defined here\", \"Single tumor type\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified protein-turnover and signaling partners: SQSTM1/P62 drives ubiquitin-dependent degradation while CDCA5 stabilization links TPI1 to PI3K/AKT/mTOR-driven EMT and glycolysis.\",\n      \"evidence\": \"Reciprocal Co-IP, ubiquitination assays, and functional knockdown/overexpression in breast cancer cells\",\n      \"pmids\": [\"35509067\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase mediating P62-dependent degradation not identified\", \"Direct vs. indirect CDCA5 stabilization unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected TPI1 to a kinase signaling axis: LKB1-dependent SIK phosphorylation at Ser21 modulates the glycolysis/glycerolipid branch point, explaining a human-specific metabolic liability not present in mouse (which has an oxidizable Cys).\",\n      \"evidence\": \"Phosphoproteomics, metabolomics, and orthogonal human cell line plus GEMM models\",\n      \"pmids\": [\"36715544\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of how Ser21 phosphorylation alters activity not fully resolved\", \"Cross-species divergence complicates mouse modeling\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated a direct PTM that tunes catalytic directionality: dopaminylation at Q65 enhances DHAP-to-GAP conversion to suppress ferroptosis through redirected ether phospholipid synthesis.\",\n      \"evidence\": \"Chemoproteomic site identification, in vitro activity assays, Q65 mutagenesis, and rescue in endothelial cells and mouse lung injury\",\n      \"pmids\": [\"39111287\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Enzyme(s) installing/removing dopaminylation not defined\", \"Generality beyond endothelial regeneration unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linked TPI1 transcription to metabolic-epigenetic feedback, showing LDHA-driven H3K18 lactylation at the TPI1 promoter upregulates its expression to amplify glycolysis.\",\n      \"evidence\": \"LDHA knockdown/knockout, H3K18la ChIP at the TPI1 promoter, K69 mutation, and OA mouse model\",\n      \"pmids\": [\"39086231\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct lactylation writer at TPI1 locus not isolated\", \"Limited to OA chondrocyte context\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended the nuclear-moonlighting model mechanistically, showing TPI1 partners with PKM2 and Linc00942 to undergo nuclear translocation and activate a STAT3/P300–SOX9 transcriptional program driving therapy resistance.\",\n      \"evidence\": \"ChIRP-MS/WB, Co-IP, and functional assays in glioblastoma\",\n      \"pmids\": [\"39342418\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"ChIRP-MS interaction with partial mechanistic follow-up; single lab\", \"Direct vs. scaffold role of TPI1 in the complex unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established a non-glycolytic autophagy mechanism: TPI1 binds the Beclin-1 BH3 domain to displace Bcl-2 and promote PIK3C3-C1/ULK1 assembly, driving autophagy and gemcitabine resistance.\",\n      \"evidence\": \"MS, Co-IP with domain specificity, ChIP-qPCR for c-Myc regulation, and xenografts in bladder cancer\",\n      \"pmids\": [\"41429797\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether catalytic activity is dispensable for Beclin-1 binding not stated\", \"Single cancer context\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a tumor-suppressor-antagonizing complex: TPI1 scaffolds AKT and MDM2 to enhance MDM2 Ser166 phosphorylation and p53 degradation.\",\n      \"evidence\": \"Co-IP, MDM2 truncation mapping (residues 181–360), AKT knockdown rescue, and xenografts in bladder cancer\",\n      \"pmids\": [\"40097123\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs. AKT-bridged TPI1–MDM2 contact not fully separated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showed TPI1 product DHAP acts as an epigenetic signal: GRK4-driven nuclear TPI1 phosphorylation lowers nuclear DHAP, raising H3K27ac at the Hao2 promoter to promote oxidative stress and salt-sensitive hypertension.\",\n      \"evidence\": \"IP-MS, GRK4 R65L mouse model, DHAP supplementation and C646 rescue, nuclear fractionation\",\n      \"pmids\": [\"41407053\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which DHAP modulates acetyltransferase activity not defined\", \"Phosphosite on nuclear TPI1 not mapped here\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified deubiquitination as a stabilization input: USP5 stabilizes TPI1 protein, and propofol inhibits lung cancer glycolysis by increasing TPI1 ubiquitination through this axis.\",\n      \"evidence\": \"Ubiquitination analysis, USP5 manipulation, and xenografts\",\n      \"pmids\": [\"40956511\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No in vitro reconstitution of USP5 deubiquitination of TPI1\", \"Direct vs. indirect USP5–TPI1 action not shown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Added a lncRNA stabilization input, showing HANR directly binds and stabilizes TPI1 protein to support prostate tumor glycolysis.\",\n      \"evidence\": \"RIP/pulldown and knockdown functional and xenograft assays\",\n      \"pmids\": [\"40921293\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Stabilization mechanism not dissected\", \"RIP/pulldown without orthogonal validation\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Confirmed the destabilization-disease paradigm and offered a therapeutic strategy: the R5G allele retains catalytic activity but is depleted, and small molecules restore TPI protein and activity in patient cells.\",\n      \"evidence\": \"Recombinant protein activity/stability assays, patient cell Western blot, and pharmacological rescue\",\n      \"pmids\": [\"41153421\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of compound-mediated stabilization not defined\", \"In vivo efficacy untested\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Defined mRNA-stability control of TPI1 through m5C methylation and translation-associated factors, linking RNA modification machinery to glycolytic output and immune evasion.\",\n      \"evidence\": \"MeRIP, RIP, dual-luciferase reporters and rescue across larynx, breast, and TNBC models (NOP2; NSUN2/YBX1; OTUB2/EIF4A3)\",\n      \"pmids\": [\"41498196\", \"42251361\", \"41857621\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether these RNA inputs converge on the same regulatory elements unknown\", \"Relative contribution versus protein-level control unquantified\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identified post-transcriptional translational control under hypoxia via an IRES element in the TPI1 5'UTR.\",\n      \"evidence\": \"Dicistronic reporter assays with PTB involvement in C6 glioma cells\",\n      \"pmids\": [\"40105374\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"PTB–TPI1 mRNA interaction not directly validated by RIP/pulldown\", \"Single context\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the diverse regulatory layers—catalytic-tuning PTMs, nuclear moonlighting, and the multiple protein- and mRNA-stability inputs—are integrated to set TPI1 output in a given cell type, and which are dispensable for its enzymatic versus non-enzymatic functions, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unified model coordinating PTMs, localization, and stability\", \"Catalysis-independent functions not systematically separated from glycolytic role\", \"Nuclear import mechanism undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016853\", \"supporting_discovery_ids\": [0, 1, 17, 18]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [5, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 17]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 15, 9]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 1, 17]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [15, 9]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [11, 12, 13]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SQSTM1\", \"CDCA5\", \"AKT1\", \"MDM2\", \"BECN1\", \"PKM\", \"GRK4\", \"EIF4A3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":8,"faith_pct":87.5}}