{"gene":"TUBA1B","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2022,"finding":"Forward-genetics screening in Ewing sarcoma (EWS) cell lines identified recurrent mutations in TUBA1B (encoding α-tubulin) that are sufficient to drive resistance to TK216. Using reconstituted microtubule (MT) polymerization in vitro and cell-based chemical probe competition assays, TK216 was demonstrated to act as an MT destabilizing agent that directly targets TUBA1B-containing microtubules.","method":"Forward-genetics hypermutation screen, in vitro reconstituted microtubule polymerization assay, cell-based chemical probe competition assay, active-site mutagenesis via spontaneous resistance mutations","journal":"Cell chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro MT polymerization combined with genetic resistance mutations and cell-based competition assays in a single rigorous study","pmids":["35803262"],"is_preprint":false},{"year":2001,"finding":"Mass spectrometry analysis of human breast (MDA-MB-231) and lung (A549) carcinoma cell lines identified k-alpha 1 (TUBA1B) as the major α-tubulin isotype present. Only low-level mono-glutamylation of k-alpha 1 was detected; extensive polyglutamylation and detyrosination (Glu-tubulin) were absent in these non-neuronal lines.","method":"SDS-PAGE, CNBr digestion, MALDI-TOF mass spectrometry of C-terminal tubulin peptides","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — direct biochemical characterization by mass spectrometry in two independent human cell lines, single lab","pmids":["11329278"],"is_preprint":false},{"year":2018,"finding":"Co-immunoprecipitation and LC-MS/MS proteomics in NPC cells identified TUBA1B as a binding partner of the tight junction protein CLDN11. The interaction was mediated through the intracellular loop and C-terminus of CLDN11, and CLDN11 was shown to block tubulin polymerization and inhibit cell migration through this interaction.","method":"Co-immunoprecipitation, LC-MS/MS proteomics, domain-deletion functional assays, tubulin polymerization inhibitor (nocodazole) rescue experiment","journal":"Journal of experimental & clinical cancer research : CR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with MS identification, domain mapping, and functional rescue with polymerization inhibitor; single lab","pmids":["29747653"],"is_preprint":false},{"year":2009,"finding":"In MeCP2-deficient mouse embryonic fibroblasts (MeCP2−/y MEFs), TUBA1B expression and tyrosinated α-tubulin protein levels were markedly reduced, causing deteriorated cell morphology. Reintroduction of human MeCP2 completely reversed these deficits, establishing MeCP2 as a positive regulator of TUBA1B expression in neuronal cells.","method":"Expression analysis of brain tissue from RTT/AS patients, Western blotting for tyrosinated α-tubulin in MeCP2−/y MEFs, genetic rescue by MeCP2 re-expression","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function with defined molecular phenotype and genetic rescue; single lab, two orthogonal readouts (mRNA and protein)","pmids":["19174478"],"is_preprint":false},{"year":2024,"finding":"Chemical proteomics (AfBPP photoaffinity probe with diazirine and alkyne), surface plasmon resonance, pull-down, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assay collectively identified TUBA1B (α-tubulin alpha-1B chain) as a direct binding target of asiatic acid (AA) in HepG2 hepatoma cells. Molecular docking further characterized the interaction interface.","method":"Photoaffinity-based chemical proteomics (AfBPP), mass spectrometry, surface plasmon resonance, pull-down assay, CETSA, DARTS, molecular docking","journal":"Organic & biomolecular chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal biochemical methods (SPR, pull-down, CETSA, DARTS, photoaffinity labeling) all confirming direct TUBA1B-AA interaction in a single rigorous study","pmids":["39479883"],"is_preprint":false},{"year":2023,"finding":"Expression of EGFP-tagged Δ3-tubulin driven by the endogenous TUBA1B promoter in PANC-1 pancreatic cancer cells impaired spindle body morphology and orientation during cell division (increased spindle bending, condensation defects) and increased nuclear size in a dose-dependent manner, demonstrating that the C-terminal processing state of TUBA1B-encoded α-tubulin directly controls mitotic spindle integrity.","method":"Endogenous promoter-driven EGFP-Δ3-tubulin expression (TUBA1B locus), live-cell/fixed imaging of spindle morphology, nuclear size quantification, cell proliferation assay","journal":"Medical molecular morphology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined genetic construct driven from TUBA1B endogenous promoter with quantitative phenotypic readout; single lab, single method set","pmids":["37930423"],"is_preprint":false},{"year":2019,"finding":"CRISPR-Cas9 knock-in at the TUBA1B locus was used to introduce fluorescent protein (mEos) tags for single-molecule localization microscopy. More monomeric and codon-optimized mEos variants showed improved expression at the TUBA1B locus, enabling accurate single-molecule quantification of endogenously tagged protein compared to overexpressed constructs.","method":"CRISPR-Cas9 knock-in at TUBA1B locus, PALM (single-molecule localization microscopy), quantitative single-molecule imaging","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct endogenous locus tagging with functional imaging validation; single lab, novel methodological finding about TUBA1B localization at single-molecule level","pmids":["31578415"],"is_preprint":false},{"year":2026,"finding":"In colorectal cancer cells, malate (produced via elevated DLD-driven TCA cycle metabolism under mechanical compression) was found to interact directly with TUBA1B to promote microtubule assembly, facilitating confined cell migration and metastasis. Disruption of the malate-TUBA1B interaction significantly suppressed tumor metastasis in vivo.","method":"CRISPR metabolic enzyme screen, biochemical interaction assays between malate and TUBA1B, in vivo xenograft/metastasis models with TUBA1B interaction mutants, ARE-deleted knock-in mice","journal":"Cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic screen plus in vivo functional validation of TUBA1B-malate interaction; single lab, multiple orthogonal approaches","pmids":["42151565"],"is_preprint":false},{"year":2025,"finding":"In glioma cells, siRNA-mediated knockdown of TUBA1B reduced tumor cell proliferation, migration, invasion, and autophagy in vitro, and suppressed tumor growth in mouse xenograft models, placing TUBA1B upstream of cell cycle pathway regulation and intercellular communication in glioma progression.","method":"siRNA knockdown, in vitro proliferation/migration/invasion assays, autophagy assays, mouse xenograft model, qRT-PCR","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with multiple cellular readouts and in vivo validation; single lab","pmids":["40094001"],"is_preprint":false},{"year":2024,"finding":"siRNA-mediated silencing of TUBA1B in colorectal cancer cell lines (Caco-2 and Colo-205) significantly reduced expression of pro-inflammatory cytokines IL-6, IL-7, CXCL1, and CXCL2, and inhibited tumor cell growth, identifying TUBA1B as a regulator of inflammatory signaling and tumor cell proliferation.","method":"siRNA knockdown, cytokine expression measurement, cell growth assay","journal":"Discover oncology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single method (siRNA + cytokine measurement), no pathway placement beyond association","pmids":["39259234"],"is_preprint":false},{"year":1996,"finding":"Semiquantitative RT-PCR analysis of paclitaxel-resistant MES-SA sarcoma mutants showed reduced transcript levels of specific β-tubulin isotypes (5β and β4) across all clones, while K-alpha-1 (TUBA1B) transcript levels were examined but not directly implicated in resistance; the study established that tubulin isotype expression changes (not involving TUBA1B) accompany selection for paclitaxel resistance.","method":"Semiquantitative RT-PCR, immunoblotting for total tubulin content, fluctuation analysis","journal":"Cancer research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — TUBA1B (K-alpha-1) was analyzed as part of a broader isotype survey; negative/neutral result for TUBA1B-specific involvement in resistance; single lab","pmids":["8640766"],"is_preprint":false},{"year":2025,"finding":"Proteomics of stress granules (SGs) immunoprecipitated from postmortem frontal cortex of Alzheimer's disease patients showed reduced abundance of TUBA1B within SGs in rapidly progressive AD (rpAD) compared to controls and slowly progressive AD. Dysregulation of TUBA1B was also observed in 3xTg mouse model cortical tissue and human cortical homogenates, indicating cytoskeletal vulnerability and altered SG composition during aggressive AD progression.","method":"Anti-TIAR immunoprecipitation of stress granules, LC-MS/MS proteomics, Western blotting in human postmortem tissue and 3xTg mouse model","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single lab, SG proteomics with Western blot validation; no direct mechanistic manipulation of TUBA1B","pmids":["41847015"],"is_preprint":true}],"current_model":"TUBA1B encodes the major ubiquitous α-tubulin isoform (K-alpha-1) that incorporates into microtubules and is subject to C-terminal post-translational modifications (including limited mono-glutamylation in non-neuronal cells); its expression is positively regulated by MeCP2, its polymerization is promoted by malate binding and inhibited by CLDN11 interaction, it is directly targeted by small molecules including asiatic acid and the MT-destabilizing agent TK216 (resistance to which is conferred by TUBA1B mutations), and its C-terminal processing state (Δ3 form expressed from the endogenous TUBA1B promoter) directly controls mitotic spindle integrity and cell division."},"narrative":{"mechanistic_narrative":"TUBA1B encodes the major ubiquitous α-tubulin isoform (K-alpha-1) that constitutes the principal α-tubulin isotype incorporated into microtubules in non-neuronal carcinoma cells and is subject to only low-level C-terminal mono-glutamylation, with extensive polyglutamylation and detyrosination absent in these contexts [PMID:11329278]. Its incorporation into microtubules and the polymerization state of those microtubules are directly controlled by the C-terminal processing state of the encoded α-tubulin: expression of a Δ3-tubulin form from the endogenous TUBA1B promoter impairs mitotic spindle morphology and orientation and increases nuclear size, demonstrating direct control of spindle integrity during cell division [PMID:37930423]. TUBA1B-containing microtubule polymerization is positively modulated by direct binding of the TCA-cycle metabolite malate, which promotes microtubule assembly to facilitate confined cell migration and metastasis [PMID:42151565], and is inhibited by direct interaction with the tight junction protein CLDN11 through its intracellular loop and C-terminus, which blocks tubulin polymerization and suppresses cell migration [PMID:29747653]. TUBA1B is a direct pharmacological target: the microtubule-destabilizing agent TK216 binds TUBA1B-containing microtubules and resistance arises through TUBA1B mutations [PMID:35803262], and the small molecule asiatic acid binds TUBA1B directly [PMID:39479883]. Loss-of-function studies place TUBA1B as a driver of tumor cell proliferation, migration, and invasion across glioma and colorectal cancer models [PMID:40094001, PMID:39259234], and its expression is positively regulated by MeCP2 [PMID:19174478].","teleology":[{"year":2001,"claim":"Establishing which α-tubulin isotype and post-translational modification state predominates in non-neuronal human cells defined the molecular identity of the TUBA1B gene product in cancer contexts.","evidence":"MALDI-TOF mass spectrometry of C-terminal tubulin peptides from breast and lung carcinoma cell lines","pmids":["11329278"],"confidence":"Medium","gaps":["Does not address modification state in neuronal or other tissues","Functional consequence of mono-glutamylation not tested"]},{"year":2009,"claim":"Identifying an upstream transcriptional regulator addressed how TUBA1B expression is controlled, linking it to MeCP2 function and cell morphology.","evidence":"Western blotting and genetic rescue in MeCP2-deficient mouse embryonic fibroblasts","pmids":["19174478"],"confidence":"Medium","gaps":["Direct vs indirect transcriptional regulation not resolved","Whether MeCP2 binds the TUBA1B locus not shown"]},{"year":2018,"claim":"Discovery of a direct protein partner that inhibits polymerization explained one mechanism restraining TUBA1B-dependent microtubule assembly and migration.","evidence":"Reciprocal Co-IP/LC-MS/MS, domain mapping, and nocodazole rescue in NPC cells","pmids":["29747653"],"confidence":"Medium","gaps":["Single lab","Structural basis of CLDN11-tubulin interaction not defined"]},{"year":2019,"claim":"Endogenous-locus fluorescent tagging established methodology for accurate single-molecule quantification of TUBA1B without overexpression artifacts.","evidence":"CRISPR-Cas9 knock-in of mEos variants at TUBA1B locus with PALM imaging","pmids":["31578415"],"confidence":"Medium","gaps":["Primarily methodological","No new functional mechanism established"]},{"year":2022,"claim":"Genetic resistance mutations and reconstituted polymerization defined TUBA1B as the direct target of the microtubule-destabilizing agent TK216.","evidence":"Forward-genetics resistance screen, in vitro reconstituted MT polymerization, cell-based chemical probe competition in Ewing sarcoma cells","pmids":["35803262"],"confidence":"High","gaps":["Precise binding-site residues not fully mapped","Isoform selectivity vs other α-tubulins not addressed"]},{"year":2023,"claim":"Driving a C-terminally processed Δ3-tubulin from the endogenous promoter showed that TUBA1B processing state directly governs mitotic spindle integrity.","evidence":"Endogenous TUBA1B-promoter-driven EGFP-Δ3-tubulin, spindle imaging, and nuclear size quantification in PANC-1 cells","pmids":["37930423"],"confidence":"Medium","gaps":["Enzymes generating Δ3 form in vivo not identified","Single cell line"]},{"year":2024,"claim":"Multi-method target identification established asiatic acid as a direct TUBA1B binder, extending the gene's role as a druggable target.","evidence":"Photoaffinity chemical proteomics, SPR, pull-down, CETSA, DARTS, and docking in HepG2 cells","pmids":["39479883"],"confidence":"High","gaps":["Downstream microtubule/functional consequence of asiatic acid binding not detailed","Binding site only modeled by docking"]},{"year":2024,"claim":"Loss-of-function linked TUBA1B to inflammatory cytokine expression and proliferation in colorectal cancer cells.","evidence":"siRNA knockdown with cytokine expression and growth assays in Caco-2 and Colo-205","pmids":["39259234"],"confidence":"Low","gaps":["Single method beyond knockdown, no pathway placement beyond association","Mechanism connecting tubulin to cytokine expression unknown"]},{"year":2025,"claim":"Knockdown across glioma models placed TUBA1B upstream of proliferation, invasion, and autophagy, supporting a pro-tumorigenic role.","evidence":"siRNA knockdown with proliferation/migration/invasion/autophagy assays and mouse xenografts","pmids":["40094001"],"confidence":"Medium","gaps":["Mechanistic link to autophagy not resolved","Direct effectors downstream of TUBA1B not identified"]},{"year":2026,"claim":"Identifying malate as a direct metabolite ligand revealed a metabolism-cytoskeleton axis whereby TUBA1B couples TCA-cycle output to microtubule assembly and metastasis.","evidence":"CRISPR metabolic screen, malate-TUBA1B interaction assays, and in vivo metastasis models with interaction mutants and ARE-deleted knock-in mice","pmids":["42151565"],"confidence":"Medium","gaps":["Structural detail of malate binding site limited","Generality beyond colorectal cancer under mechanical compression not established"]},{"year":null,"claim":"How the enzymes and signals that set TUBA1B's C-terminal processing and modification state are regulated, and how these integrate with its ligand-dependent assembly control, remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No identification of the enzymes generating the Δ3 form in vivo","No structural model unifying malate, CLDN11, TK216, and asiatic acid binding sites","Tissue-specific modification regulation uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,1,5]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,5]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[5,8]}],"complexes":[],"partners":["CLDN11","MECP2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P68363","full_name":"Tubulin alpha-1B chain","aliases":["Alpha-tubulin ubiquitous","Tubulin K-alpha-1","Tubulin alpha-ubiquitous chain"],"length_aa":451,"mass_kda":50.2,"function":"Tubulin is the major constituent of microtubules, protein filaments consisting of alpha- and beta-tubulin heterodimers (PubMed:38305685, PubMed:34996871, PubMed:38609661). Microtubules grow by the addition of GTP-tubulin dimers to the microtubule end, where a stabilizing cap forms (PubMed:38305685, PubMed:34996871, PubMed:38609661). Below the cap, tubulin dimers are in GDP-bound state, owing to GTPase activity of alpha-tubulin (PubMed:34996871, PubMed:38609661)","subcellular_location":"Cytoplasm, cytoskeleton","url":"https://www.uniprot.org/uniprotkb/P68363/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/TUBA1B","classification":"Common Essential","n_dependent_lines":1128,"n_total_lines":1208,"dependency_fraction":0.9337748344370861},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000123416","cell_line_id":"CID000825","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"cytoskeleton","grade":2}],"interactors":[{"gene":"DNAJA1","stoichiometry":10.0},{"gene":"TUBB4B","stoichiometry":10.0},{"gene":"DNAJA2","stoichiometry":0.2},{"gene":"DNAJB6","stoichiometry":0.2},{"gene":"MAP4","stoichiometry":0.2},{"gene":"TUBB","stoichiometry":0.2},{"gene":"GCC2","stoichiometry":0.2},{"gene":"CEP97","stoichiometry":0.2},{"gene":"TCP11L2","stoichiometry":0.2},{"gene":"AFTPH","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000825","total_profiled":1310},"omim":[{"mim_id":"619014","title":"TETRATRICOPEPTIDE REPEAT DOMAIN-CONTAINING PROTEIN 5; TTC5","url":"https://www.omim.org/entry/619014"},{"mim_id":"616144","title":"WD REPEAT-CONTAINING PROTEIN 73; WDR73","url":"https://www.omim.org/entry/616144"},{"mim_id":"602530","title":"TUBULIN, ALPHA-1B; TUBA1B","url":"https://www.omim.org/entry/602530"},{"mim_id":"300005","title":"METHYL-CpG-BINDING PROTEIN 2; MECP2","url":"https://www.omim.org/entry/300005"},{"mim_id":"191130","title":"TUBULIN, BETA; TUBB","url":"https://www.omim.org/entry/191130"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Microtubules","reliability":"Enhanced"},{"location":"Primary cilium","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TUBA1B"},"hgnc":{"alias_symbol":["K-ALPHA-1"],"prev_symbol":[]},"alphafold":{"accession":"P68363","domains":[{"cath_id":"3.40.50.1440","chopping":"2-35_54-385","consensus_level":"medium","plddt":94.2909,"start":2,"end":385}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P68363","model_url":"https://alphafold.ebi.ac.uk/files/AF-P68363-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P68363-F1-predicted_aligned_error_v6.png","plddt_mean":91.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TUBA1B","jax_strain_url":"https://www.jax.org/strain/search?query=TUBA1B"},"sequence":{"accession":"P68363","fasta_url":"https://rest.uniprot.org/uniprotkb/P68363.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P68363/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P68363"}},"corpus_meta":[{"pmid":"20868794","id":"PMC_20868794","title":"Antibodies to 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Using reconstituted microtubule (MT) polymerization in vitro and cell-based chemical probe competition assays, TK216 was demonstrated to act as an MT destabilizing agent that directly targets TUBA1B-containing microtubules.\",\n      \"method\": \"Forward-genetics hypermutation screen, in vitro reconstituted microtubule polymerization assay, cell-based chemical probe competition assay, active-site mutagenesis via spontaneous resistance mutations\",\n      \"journal\": \"Cell chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro MT polymerization combined with genetic resistance mutations and cell-based competition assays in a single rigorous study\",\n      \"pmids\": [\"35803262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Mass spectrometry analysis of human breast (MDA-MB-231) and lung (A549) carcinoma cell lines identified k-alpha 1 (TUBA1B) as the major α-tubulin isotype present. Only low-level mono-glutamylation of k-alpha 1 was detected; extensive polyglutamylation and detyrosination (Glu-tubulin) were absent in these non-neuronal lines.\",\n      \"method\": \"SDS-PAGE, CNBr digestion, MALDI-TOF mass spectrometry of C-terminal tubulin peptides\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct biochemical characterization by mass spectrometry in two independent human cell lines, single lab\",\n      \"pmids\": [\"11329278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Co-immunoprecipitation and LC-MS/MS proteomics in NPC cells identified TUBA1B as a binding partner of the tight junction protein CLDN11. The interaction was mediated through the intracellular loop and C-terminus of CLDN11, and CLDN11 was shown to block tubulin polymerization and inhibit cell migration through this interaction.\",\n      \"method\": \"Co-immunoprecipitation, LC-MS/MS proteomics, domain-deletion functional assays, tubulin polymerization inhibitor (nocodazole) rescue experiment\",\n      \"journal\": \"Journal of experimental & clinical cancer research : CR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with MS identification, domain mapping, and functional rescue with polymerization inhibitor; single lab\",\n      \"pmids\": [\"29747653\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In MeCP2-deficient mouse embryonic fibroblasts (MeCP2−/y MEFs), TUBA1B expression and tyrosinated α-tubulin protein levels were markedly reduced, causing deteriorated cell morphology. Reintroduction of human MeCP2 completely reversed these deficits, establishing MeCP2 as a positive regulator of TUBA1B expression in neuronal cells.\",\n      \"method\": \"Expression analysis of brain tissue from RTT/AS patients, Western blotting for tyrosinated α-tubulin in MeCP2−/y MEFs, genetic rescue by MeCP2 re-expression\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function with defined molecular phenotype and genetic rescue; single lab, two orthogonal readouts (mRNA and protein)\",\n      \"pmids\": [\"19174478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Chemical proteomics (AfBPP photoaffinity probe with diazirine and alkyne), surface plasmon resonance, pull-down, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assay collectively identified TUBA1B (α-tubulin alpha-1B chain) as a direct binding target of asiatic acid (AA) in HepG2 hepatoma cells. Molecular docking further characterized the interaction interface.\",\n      \"method\": \"Photoaffinity-based chemical proteomics (AfBPP), mass spectrometry, surface plasmon resonance, pull-down assay, CETSA, DARTS, molecular docking\",\n      \"journal\": \"Organic & biomolecular chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal biochemical methods (SPR, pull-down, CETSA, DARTS, photoaffinity labeling) all confirming direct TUBA1B-AA interaction in a single rigorous study\",\n      \"pmids\": [\"39479883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Expression of EGFP-tagged Δ3-tubulin driven by the endogenous TUBA1B promoter in PANC-1 pancreatic cancer cells impaired spindle body morphology and orientation during cell division (increased spindle bending, condensation defects) and increased nuclear size in a dose-dependent manner, demonstrating that the C-terminal processing state of TUBA1B-encoded α-tubulin directly controls mitotic spindle integrity.\",\n      \"method\": \"Endogenous promoter-driven EGFP-Δ3-tubulin expression (TUBA1B locus), live-cell/fixed imaging of spindle morphology, nuclear size quantification, cell proliferation assay\",\n      \"journal\": \"Medical molecular morphology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined genetic construct driven from TUBA1B endogenous promoter with quantitative phenotypic readout; single lab, single method set\",\n      \"pmids\": [\"37930423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CRISPR-Cas9 knock-in at the TUBA1B locus was used to introduce fluorescent protein (mEos) tags for single-molecule localization microscopy. More monomeric and codon-optimized mEos variants showed improved expression at the TUBA1B locus, enabling accurate single-molecule quantification of endogenously tagged protein compared to overexpressed constructs.\",\n      \"method\": \"CRISPR-Cas9 knock-in at TUBA1B locus, PALM (single-molecule localization microscopy), quantitative single-molecule imaging\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct endogenous locus tagging with functional imaging validation; single lab, novel methodological finding about TUBA1B localization at single-molecule level\",\n      \"pmids\": [\"31578415\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"In colorectal cancer cells, malate (produced via elevated DLD-driven TCA cycle metabolism under mechanical compression) was found to interact directly with TUBA1B to promote microtubule assembly, facilitating confined cell migration and metastasis. Disruption of the malate-TUBA1B interaction significantly suppressed tumor metastasis in vivo.\",\n      \"method\": \"CRISPR metabolic enzyme screen, biochemical interaction assays between malate and TUBA1B, in vivo xenograft/metastasis models with TUBA1B interaction mutants, ARE-deleted knock-in mice\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic screen plus in vivo functional validation of TUBA1B-malate interaction; single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"42151565\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In glioma cells, siRNA-mediated knockdown of TUBA1B reduced tumor cell proliferation, migration, invasion, and autophagy in vitro, and suppressed tumor growth in mouse xenograft models, placing TUBA1B upstream of cell cycle pathway regulation and intercellular communication in glioma progression.\",\n      \"method\": \"siRNA knockdown, in vitro proliferation/migration/invasion assays, autophagy assays, mouse xenograft model, qRT-PCR\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with multiple cellular readouts and in vivo validation; single lab\",\n      \"pmids\": [\"40094001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"siRNA-mediated silencing of TUBA1B in colorectal cancer cell lines (Caco-2 and Colo-205) significantly reduced expression of pro-inflammatory cytokines IL-6, IL-7, CXCL1, and CXCL2, and inhibited tumor cell growth, identifying TUBA1B as a regulator of inflammatory signaling and tumor cell proliferation.\",\n      \"method\": \"siRNA knockdown, cytokine expression measurement, cell growth assay\",\n      \"journal\": \"Discover oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single method (siRNA + cytokine measurement), no pathway placement beyond association\",\n      \"pmids\": [\"39259234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Semiquantitative RT-PCR analysis of paclitaxel-resistant MES-SA sarcoma mutants showed reduced transcript levels of specific β-tubulin isotypes (5β and β4) across all clones, while K-alpha-1 (TUBA1B) transcript levels were examined but not directly implicated in resistance; the study established that tubulin isotype expression changes (not involving TUBA1B) accompany selection for paclitaxel resistance.\",\n      \"method\": \"Semiquantitative RT-PCR, immunoblotting for total tubulin content, fluctuation analysis\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — TUBA1B (K-alpha-1) was analyzed as part of a broader isotype survey; negative/neutral result for TUBA1B-specific involvement in resistance; single lab\",\n      \"pmids\": [\"8640766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Proteomics of stress granules (SGs) immunoprecipitated from postmortem frontal cortex of Alzheimer's disease patients showed reduced abundance of TUBA1B within SGs in rapidly progressive AD (rpAD) compared to controls and slowly progressive AD. Dysregulation of TUBA1B was also observed in 3xTg mouse model cortical tissue and human cortical homogenates, indicating cytoskeletal vulnerability and altered SG composition during aggressive AD progression.\",\n      \"method\": \"Anti-TIAR immunoprecipitation of stress granules, LC-MS/MS proteomics, Western blotting in human postmortem tissue and 3xTg mouse model\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single lab, SG proteomics with Western blot validation; no direct mechanistic manipulation of TUBA1B\",\n      \"pmids\": [\"41847015\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"TUBA1B encodes the major ubiquitous α-tubulin isoform (K-alpha-1) that incorporates into microtubules and is subject to C-terminal post-translational modifications (including limited mono-glutamylation in non-neuronal cells); its expression is positively regulated by MeCP2, its polymerization is promoted by malate binding and inhibited by CLDN11 interaction, it is directly targeted by small molecules including asiatic acid and the MT-destabilizing agent TK216 (resistance to which is conferred by TUBA1B mutations), and its C-terminal processing state (Δ3 form expressed from the endogenous TUBA1B promoter) directly controls mitotic spindle integrity and cell division.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TUBA1B encodes the major ubiquitous \\u03b1-tubulin isoform (K-alpha-1) that constitutes the principal \\u03b1-tubulin isotype incorporated into microtubules in non-neuronal carcinoma cells and is subject to only low-level C-terminal mono-glutamylation, with extensive polyglutamylation and detyrosination absent in these contexts [#1]. Its incorporation into microtubules and the polymerization state of those microtubules are directly controlled by the C-terminal processing state of the encoded \\u03b1-tubulin: expression of a \\u03943-tubulin form from the endogenous TUBA1B promoter impairs mitotic spindle morphology and orientation and increases nuclear size, demonstrating direct control of spindle integrity during cell division [#5]. TUBA1B-containing microtubule polymerization is positively modulated by direct binding of the TCA-cycle metabolite malate, which promotes microtubule assembly to facilitate confined cell migration and metastasis [#7], and is inhibited by direct interaction with the tight junction protein CLDN11 through its intracellular loop and C-terminus, which blocks tubulin polymerization and suppresses cell migration [#2]. TUBA1B is a direct pharmacological target: the microtubule-destabilizing agent TK216 binds TUBA1B-containing microtubules and resistance arises through TUBA1B mutations [#0], and the small molecule asiatic acid binds TUBA1B directly [#4]. Loss-of-function studies place TUBA1B as a driver of tumor cell proliferation, migration, and invasion across glioma and colorectal cancer models [#8, #9], and its expression is positively regulated by MeCP2 [#3].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Establishing which \\u03b1-tubulin isotype and post-translational modification state predominates in non-neuronal human cells defined the molecular identity of the TUBA1B gene product in cancer contexts.\",\n      \"evidence\": \"MALDI-TOF mass spectrometry of C-terminal tubulin peptides from breast and lung carcinoma cell lines\",\n      \"pmids\": [\"11329278\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not address modification state in neuronal or other tissues\", \"Functional consequence of mono-glutamylation not tested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identifying an upstream transcriptional regulator addressed how TUBA1B expression is controlled, linking it to MeCP2 function and cell morphology.\",\n      \"evidence\": \"Western blotting and genetic rescue in MeCP2-deficient mouse embryonic fibroblasts\",\n      \"pmids\": [\"19174478\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect transcriptional regulation not resolved\", \"Whether MeCP2 binds the TUBA1B locus not shown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Discovery of a direct protein partner that inhibits polymerization explained one mechanism restraining TUBA1B-dependent microtubule assembly and migration.\",\n      \"evidence\": \"Reciprocal Co-IP/LC-MS/MS, domain mapping, and nocodazole rescue in NPC cells\",\n      \"pmids\": [\"29747653\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Structural basis of CLDN11-tubulin interaction not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Endogenous-locus fluorescent tagging established methodology for accurate single-molecule quantification of TUBA1B without overexpression artifacts.\",\n      \"evidence\": \"CRISPR-Cas9 knock-in of mEos variants at TUBA1B locus with PALM imaging\",\n      \"pmids\": [\"31578415\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Primarily methodological\", \"No new functional mechanism established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Genetic resistance mutations and reconstituted polymerization defined TUBA1B as the direct target of the microtubule-destabilizing agent TK216.\",\n      \"evidence\": \"Forward-genetics resistance screen, in vitro reconstituted MT polymerization, cell-based chemical probe competition in Ewing sarcoma cells\",\n      \"pmids\": [\"35803262\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise binding-site residues not fully mapped\", \"Isoform selectivity vs other \\u03b1-tubulins not addressed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Driving a C-terminally processed \\u03943-tubulin from the endogenous promoter showed that TUBA1B processing state directly governs mitotic spindle integrity.\",\n      \"evidence\": \"Endogenous TUBA1B-promoter-driven EGFP-\\u03943-tubulin, spindle imaging, and nuclear size quantification in PANC-1 cells\",\n      \"pmids\": [\"37930423\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Enzymes generating \\u03943 form in vivo not identified\", \"Single cell line\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Multi-method target identification established asiatic acid as a direct TUBA1B binder, extending the gene's role as a druggable target.\",\n      \"evidence\": \"Photoaffinity chemical proteomics, SPR, pull-down, CETSA, DARTS, and docking in HepG2 cells\",\n      \"pmids\": [\"39479883\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream microtubule/functional consequence of asiatic acid binding not detailed\", \"Binding site only modeled by docking\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Loss-of-function linked TUBA1B to inflammatory cytokine expression and proliferation in colorectal cancer cells.\",\n      \"evidence\": \"siRNA knockdown with cytokine expression and growth assays in Caco-2 and Colo-205\",\n      \"pmids\": [\"39259234\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single method beyond knockdown, no pathway placement beyond association\", \"Mechanism connecting tubulin to cytokine expression unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Knockdown across glioma models placed TUBA1B upstream of proliferation, invasion, and autophagy, supporting a pro-tumorigenic role.\",\n      \"evidence\": \"siRNA knockdown with proliferation/migration/invasion/autophagy assays and mouse xenografts\",\n      \"pmids\": [\"40094001\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic link to autophagy not resolved\", \"Direct effectors downstream of TUBA1B not identified\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identifying malate as a direct metabolite ligand revealed a metabolism-cytoskeleton axis whereby TUBA1B couples TCA-cycle output to microtubule assembly and metastasis.\",\n      \"evidence\": \"CRISPR metabolic screen, malate-TUBA1B interaction assays, and in vivo metastasis models with interaction mutants and ARE-deleted knock-in mice\",\n      \"pmids\": [\"42151565\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural detail of malate binding site limited\", \"Generality beyond colorectal cancer under mechanical compression not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the enzymes and signals that set TUBA1B's C-terminal processing and modification state are regulated, and how these integrate with its ligand-dependent assembly control, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No identification of the enzymes generating the \\u03943 form in vivo\", \"No structural model unifying malate, CLDN11, TK216, and asiatic acid binding sites\", \"Tissue-specific modification regulation uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 1, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0005819\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [5, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CLDN11\", \"MeCP2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}