{"gene":"TTLL12","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2016,"finding":"TTLL12 acts as a negative regulator of RIG-I-mediated antiviral signaling by directly interacting with VISA/MAVS, TBK1, and IKKε, and inhibiting the interactions of VISA with other signaling molecules. TTLL12 is localized in the cytosol. Mutagenesis studies indicated that TTLL12's repressive function is probably not dependent on its predicted enzymatic (methyltransferase or tubulin tyrosine ligase) activities.","method":"Co-immunoprecipitation, cell fractionation, fluorescent staining, CRISPR/RNA interference knockdown, mutagenesis, global gene expression profiling, Sendai virus infection assay","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, CRISPR and RNAi KO with defined IFN-β phenotype, localization, and mutagenesis to test enzymatic dependency; multiple orthogonal methods in a single focused study","pmids":["28011935"],"is_preprint":false},{"year":2023,"finding":"TTLL12 localizes to the base of primary cilia and is required for primary cilia formation in polarized renal epithelial cells. TTLL12 directly binds to the α/β-tubulin heterodimer in vitro and regulates microtubule dynamics, stability, and post-translational modifications. Unlike other TTLL family members, TTLL12 promotes both microtubule lysine acetylation and arginine methylation rather than adding glutamate or glycine to microtubule C-terminal tails.","method":"In vitro tubulin-binding assay, immunofluorescence/live imaging localization, loss-of-function in polarized renal epithelial cells, microtubule dynamics and PTM assays (acetylation, arginine methylation readouts)","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro reconstitution of tubulin binding, KO with defined ciliogenesis phenotype, multiple PTM readouts, published in peer-reviewed journal with preprint corroboration","pmids":["38177908","37546873"],"is_preprint":false},{"year":2024,"finding":"TTLL12 suppresses the ligation of nitrotyrosine to the C-terminus of detyrosinated α-tubulin (tubulin nitrotyrosination), acting as a pseudo-enzyme without predicted enzymatic activities. TTLL12 overexpression reduces tubulin nitrotyrosination in cells, and this activity was used to develop a cell-based ELISA assay for high-throughput screening.","method":"TTLL12 overexpression in cells, cell-based ELISA for nitrotyrosinated α-tubulin, high-throughput compound screen","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — cell-based overexpression assay with ELISA readout for tubulin PTM, single lab, no direct in vitro reconstitution of suppression mechanism","pmids":["38394155"],"is_preprint":false},{"year":2020,"finding":"TTLL12 overexpression enhances SCC-25 cell survival in the presence of nitrotyrosine by disrupting/reducing nitration of the tyrosine residues of tubulin. TTLL12 silencing significantly inhibits cell proliferation.","method":"TTLL12-overexpressing and TTLL12-silenced cell lines, western blot, immunofluorescence, MTT proliferation assay","journal":"Oncology letters","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — gain- and loss-of-function in cell lines with proliferation readout and tubulin nitration detection, but limited mechanistic depth; single lab","pmids":["33123251"],"is_preprint":false},{"year":2025,"finding":"Tumor-intrinsic TTLL12 drives immunosuppression by promoting secretion of chemokine CCL9, likely through binding to the promoter region of CCL9 (shown by chromatin immunoprecipitation and mass spectrometry), leading to recruitment and expansion of myeloid-derived suppressor cells (MDSCs) and resulting in a suppressive tumor immune microenvironment.","method":"Gain- and loss-of-function in syngeneic mouse models, flow cytometry, MDSC migration/proliferation assays in vitro, mass spectrometry, chromatin immunoprecipitation","journal":"Journal for immunotherapy of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and MS to identify CCL9 promoter binding, in vivo syngeneic models with immune readouts, single lab with multiple orthogonal methods","pmids":["40461158"],"is_preprint":false},{"year":2026,"finding":"TTLL12 counteracts BPOZ-2-mediated degradation of eEF1A1 by competing with BPOZ-2 for eEF1A1 binding, thereby preventing BPOZ-2 from recruiting eEF1A1 to CULLIN3 (CUL3) for ubiquitin-proteasome degradation. This stabilization of eEF1A1 promotes hepatocellular carcinoma cell proliferation.","method":"Co-immunoprecipitation, ubiquitin-proteasome degradation assays, TTLL12 knockdown/overexpression with proliferation readout, in vivo tumor growth models","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP to define TTLL12/BPOZ-2/eEF1A1 interactions, proteasome inhibition assays, in vivo models; single lab, not yet independently replicated","pmids":["42014684"],"is_preprint":false}],"current_model":"TTLL12 is an atypical, pseudo-enzyme member of the TTLL family that lacks conventional enzymatic activities but mechanistically acts as: (1) a cytosolic negative regulator of RIG-I/VISA antiviral innate immune signaling via direct protein–protein interactions with VISA, TBK1, and IKKε; (2) a regulator of primary ciliogenesis and microtubule dynamics in polarized epithelial cells through direct α/β-tubulin binding and promotion of lysine acetylation and arginine methylation; (3) a suppressor of tubulin nitrotyrosination; (4) a driver of tumor immune evasion by promoting CCL9 transcription and MDSC recruitment; and (5) an oncogenic stabilizer of eEF1A1 by competing with BPOZ-2 to prevent CUL3-mediated ubiquitin-proteasome degradation."},"narrative":{"mechanistic_narrative":"TTLL12 is an atypical member of the tubulin tyrosine ligase-like family that functions as a pseudo-enzyme, exerting its cellular roles through direct protein–protein interactions rather than the methyltransferase or tubulin tyrosine ligase activities predicted from its sequence [PMID:28011935, PMID:38394155]. In innate immunity, cytosolic TTLL12 acts as a negative regulator of RIG-I-mediated antiviral signaling by binding directly to VISA/MAVS, TBK1, and IKKε and disrupting VISA's association with downstream signaling partners, dampening interferon-β induction independently of any catalytic activity [PMID:28011935]. At the cytoskeleton, TTLL12 binds the α/β-tubulin heterodimer directly and localizes to the base of primary cilia, where it is required for ciliogenesis in polarized renal epithelial cells; rather than adding glutamate or glycine to tubulin tails like canonical TTLL enzymes, it promotes microtubule lysine acetylation and arginine methylation and modulates microtubule dynamics and stability [PMID:38177908, PMID:37546873]. TTLL12 additionally suppresses ligation of nitrotyrosine onto detyrosinated α-tubulin, reducing tubulin nitrotyrosination in cells [PMID:38394155]. These tubulin-related and signaling activities converge on pro-tumorigenic functions: TTLL12 enhances cancer cell survival and proliferation [PMID:33123251], drives an immunosuppressive tumor microenvironment by promoting CCL9 transcription and recruitment of myeloid-derived suppressor cells [PMID:40461158], and stabilizes eEF1A1 by competing with BPOZ-2 to block CUL3-mediated ubiquitin-proteasome degradation, thereby promoting hepatocellular carcinoma cell proliferation [PMID:42014684].","teleology":[{"year":2016,"claim":"Established the first defined molecular function for TTLL12 as a cytosolic brake on antiviral innate immune signaling, answering whether this predicted enzyme has a non-cytoskeletal role.","evidence":"Reciprocal Co-IP, cell fractionation, CRISPR/RNAi knockdown with IFN-β phenotype, and Sendai virus infection assays, plus mutagenesis testing enzymatic dependency","pmids":["28011935"],"confidence":"High","gaps":["Structural basis of how TTLL12 disrupts VISA's interactions is undefined","Whether this immune role is shared across cell types is not addressed","No direct demonstration of catalytic inactivity, only mutagenesis inference"]},{"year":2023,"claim":"Resolved whether TTLL12 acts on microtubules like other TTLL family members, showing it binds tubulin directly but promotes atypical PTMs and is required for ciliogenesis.","evidence":"In vitro tubulin-binding assay, localization imaging, loss-of-function in polarized renal epithelial cells, and acetylation/arginine-methylation PTM readouts","pmids":["38177908","37546873"],"confidence":"High","gaps":["Whether TTLL12 directly catalyzes acetylation/methylation or recruits other enzymes is unresolved","Mechanism linking microtubule PTM changes to cilium assembly not detailed","Connection to the immune-signaling role is not established"]},{"year":2020,"claim":"Linked TTLL12 to cancer cell survival, showing it protects cells from nitrotyrosine stress and supports proliferation.","evidence":"Gain- and loss-of-function SCC-25 cell lines with western blot, immunofluorescence, and MTT proliferation assay","pmids":["33123251"],"confidence":"Medium","gaps":["Single lab, limited mechanistic depth on how nitration protection drives survival","No in vivo confirmation in this study","Tubulin nitration detection not reconstituted biochemically"]},{"year":2024,"claim":"Defined a specific biochemical output of TTLL12's pseudo-enzymatic action—suppression of tubulin nitrotyrosination—and converted it into a screening readout.","evidence":"TTLL12 overexpression with cell-based ELISA for nitrotyrosinated α-tubulin and high-throughput compound screening","pmids":["38394155"],"confidence":"Medium","gaps":["No direct in vitro reconstitution of the suppression mechanism","Whether suppression is via competition for tubulin C-terminus or another route is unclear","Single-lab overexpression assay only"]},{"year":2025,"claim":"Connected tumor-intrinsic TTLL12 to immune evasion via a transcriptional/chemokine axis recruiting suppressive myeloid cells.","evidence":"Gain/loss-of-function in syngeneic mouse models, flow cytometry, MDSC migration/proliferation assays, ChIP and mass spectrometry implicating CCL9 promoter binding","pmids":["40461158"],"confidence":"Medium","gaps":["Direct DNA-binding versus indirect promoter recruitment not firmly distinguished","How a cytosolic/microtubule-associated protein engages chromatin is mechanistically unexplained","Single lab, not independently replicated"]},{"year":2026,"claim":"Identified a protein-stabilization mechanism whereby TTLL12 promotes oncogenesis by shielding eEF1A1 from ubiquitin-proteasome degradation.","evidence":"Co-IP mapping TTLL12/BPOZ-2/eEF1A1 interactions, ubiquitin-proteasome degradation assays, knockdown/overexpression proliferation readouts, and in vivo tumor models","pmids":["42014684"],"confidence":"Medium","gaps":["Single lab, not yet independently replicated","Competition stoichiometry and binding interface with BPOZ-2 undefined","Relationship to TTLL12's tubulin and immune roles unknown"]},{"year":null,"claim":"Whether TTLL12's diverse activities (immune signaling, microtubule PTMs, transcription, protein stabilization) reflect one unifying biochemical mechanism or independent moonlighting functions remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model explaining how a single pseudo-enzyme engages tubulin, signaling adaptors, chromatin, and eEF1A1","No reconciliation of cytosolic versus ciliary-base versus nuclear localizations","No direct evidence of any catalytic activity for TTLL12"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,5]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[1]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1]}],"complexes":[],"partners":["MAVS","TBK1","IKBKE","TUBA","TUBB","EEF1A1","BPOZ-2","CUL3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q14166","full_name":"Tubulin--tyrosine ligase-like protein 12","aliases":["Inactive tubulin--tyrosine ligase-like protein 12"],"length_aa":644,"mass_kda":74.4,"function":"Negatively regulates post-translational modifications of tubulin, including detyrosination of the C-terminus and polyglutamylation of glutamate residues (PubMed:20162578, PubMed:23251473). Also, indirectly promotes histone H4 trimethylation at 'Lys-20' (H4K20me3) (PubMed:23251473). Probably by controlling tubulin and/or histone H4 post-translational modifications, plays a role in mitosis and in maintaining chromosome number stability (PubMed:20162578, PubMed:23251473). During RNA virus-mediated infection, acts as a negative regulator of the RIG-I pathway by preventing MAVS binding to TBK1 and IKBKE (PubMed:28011935)","subcellular_location":"Cytoplasm; Midbody; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Cytoplasm, cytoskeleton, spindle; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q14166/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TTLL12","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TTLL12","total_profiled":1310},"omim":[{"mim_id":"619410","title":"TUBULIN TYROSINE LIGASE-LIKE 12; TTLL12","url":"https://www.omim.org/entry/619410"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Plasma membrane","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"esophagus","ntpm":98.6}],"url":"https://www.proteinatlas.org/search/TTLL12"},"hgnc":{"alias_symbol":["KIAA0153"],"prev_symbol":[]},"alphafold":{"accession":"Q14166","domains":[{"cath_id":"2.170.270.10","chopping":"25-72_95-247","consensus_level":"medium","plddt":93.1853,"start":25,"end":247},{"cath_id":"-","chopping":"289-355","consensus_level":"medium","plddt":94.9728,"start":289,"end":355}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14166","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q14166-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q14166-F1-predicted_aligned_error_v6.png","plddt_mean":91.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TTLL12","jax_strain_url":"https://www.jax.org/strain/search?query=TTLL12"},"sequence":{"accession":"Q14166","fasta_url":"https://rest.uniprot.org/uniprotkb/Q14166.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q14166/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14166"}},"corpus_meta":[{"pmid":"28011935","id":"PMC_28011935","title":"TTLL12 Inhibits the Activation of Cellular Antiviral Signaling through Interaction with VISA/MAVS.","date":"2016","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/28011935","citation_count":24,"is_preprint":false},{"pmid":"32211104","id":"PMC_32211104","title":"TTLL12 expression in ovarian cancer correlates with a poor outcome.","date":"2020","source":"International journal of clinical and experimental pathology","url":"https://pubmed.ncbi.nlm.nih.gov/32211104","citation_count":10,"is_preprint":false},{"pmid":"27748896","id":"PMC_27748896","title":"Identification of a novel transcript isoform of the TTLL12 gene in human cancers.","date":"2016","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/27748896","citation_count":8,"is_preprint":false},{"pmid":"33123251","id":"PMC_33123251","title":"Effect of TTLL12 on tubulin tyrosine nitration as a novel target for screening anticancer drugs in vitro.","date":"2020","source":"Oncology letters","url":"https://pubmed.ncbi.nlm.nih.gov/33123251","citation_count":7,"is_preprint":false},{"pmid":"38394155","id":"PMC_38394155","title":"TTLL12 has a potential oncogenic activity, suppression of ligation of nitrotyrosine to the C-terminus of detyrosinated α-tubulin, that can be overcome by molecules identified by screening a compound library.","date":"2024","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/38394155","citation_count":4,"is_preprint":false},{"pmid":"38177908","id":"PMC_38177908","title":"TTLL12 is required for primary ciliary axoneme formation in polarized epithelial cells.","date":"2023","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/38177908","citation_count":3,"is_preprint":false},{"pmid":"40461158","id":"PMC_40461158","title":"Tumor-intrinsic TTLL12 drives resistance to cancer immunotherapy via modulating myeloid-derived suppressor cells.","date":"2025","source":"Journal for immunotherapy of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/40461158","citation_count":2,"is_preprint":false},{"pmid":"37546873","id":"PMC_37546873","title":"TTLL12 is required for primary ciliary axoneme formation in polarized epithelial cells.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/37546873","citation_count":2,"is_preprint":false},{"pmid":"42014684","id":"PMC_42014684","title":"TTLL12 counteracts BPOZ-2 to stabilize eEF1A1 and promote hepatocarcinogenesis.","date":"2026","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/42014684","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6798,"output_tokens":1881,"usd":0.024304,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8780,"output_tokens":2820,"usd":0.0572,"stage2_stop_reason":"end_turn"},"total_usd":0.081504,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2016,\n      \"finding\": \"TTLL12 acts as a negative regulator of RIG-I-mediated antiviral signaling by directly interacting with VISA/MAVS, TBK1, and IKKε, and inhibiting the interactions of VISA with other signaling molecules. TTLL12 is localized in the cytosol. Mutagenesis studies indicated that TTLL12's repressive function is probably not dependent on its predicted enzymatic (methyltransferase or tubulin tyrosine ligase) activities.\",\n      \"method\": \"Co-immunoprecipitation, cell fractionation, fluorescent staining, CRISPR/RNA interference knockdown, mutagenesis, global gene expression profiling, Sendai virus infection assay\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, CRISPR and RNAi KO with defined IFN-β phenotype, localization, and mutagenesis to test enzymatic dependency; multiple orthogonal methods in a single focused study\",\n      \"pmids\": [\"28011935\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TTLL12 localizes to the base of primary cilia and is required for primary cilia formation in polarized renal epithelial cells. TTLL12 directly binds to the α/β-tubulin heterodimer in vitro and regulates microtubule dynamics, stability, and post-translational modifications. Unlike other TTLL family members, TTLL12 promotes both microtubule lysine acetylation and arginine methylation rather than adding glutamate or glycine to microtubule C-terminal tails.\",\n      \"method\": \"In vitro tubulin-binding assay, immunofluorescence/live imaging localization, loss-of-function in polarized renal epithelial cells, microtubule dynamics and PTM assays (acetylation, arginine methylation readouts)\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro reconstitution of tubulin binding, KO with defined ciliogenesis phenotype, multiple PTM readouts, published in peer-reviewed journal with preprint corroboration\",\n      \"pmids\": [\"38177908\", \"37546873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TTLL12 suppresses the ligation of nitrotyrosine to the C-terminus of detyrosinated α-tubulin (tubulin nitrotyrosination), acting as a pseudo-enzyme without predicted enzymatic activities. TTLL12 overexpression reduces tubulin nitrotyrosination in cells, and this activity was used to develop a cell-based ELISA assay for high-throughput screening.\",\n      \"method\": \"TTLL12 overexpression in cells, cell-based ELISA for nitrotyrosinated α-tubulin, high-throughput compound screen\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — cell-based overexpression assay with ELISA readout for tubulin PTM, single lab, no direct in vitro reconstitution of suppression mechanism\",\n      \"pmids\": [\"38394155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TTLL12 overexpression enhances SCC-25 cell survival in the presence of nitrotyrosine by disrupting/reducing nitration of the tyrosine residues of tubulin. TTLL12 silencing significantly inhibits cell proliferation.\",\n      \"method\": \"TTLL12-overexpressing and TTLL12-silenced cell lines, western blot, immunofluorescence, MTT proliferation assay\",\n      \"journal\": \"Oncology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — gain- and loss-of-function in cell lines with proliferation readout and tubulin nitration detection, but limited mechanistic depth; single lab\",\n      \"pmids\": [\"33123251\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Tumor-intrinsic TTLL12 drives immunosuppression by promoting secretion of chemokine CCL9, likely through binding to the promoter region of CCL9 (shown by chromatin immunoprecipitation and mass spectrometry), leading to recruitment and expansion of myeloid-derived suppressor cells (MDSCs) and resulting in a suppressive tumor immune microenvironment.\",\n      \"method\": \"Gain- and loss-of-function in syngeneic mouse models, flow cytometry, MDSC migration/proliferation assays in vitro, mass spectrometry, chromatin immunoprecipitation\",\n      \"journal\": \"Journal for immunotherapy of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and MS to identify CCL9 promoter binding, in vivo syngeneic models with immune readouts, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"40461158\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"TTLL12 counteracts BPOZ-2-mediated degradation of eEF1A1 by competing with BPOZ-2 for eEF1A1 binding, thereby preventing BPOZ-2 from recruiting eEF1A1 to CULLIN3 (CUL3) for ubiquitin-proteasome degradation. This stabilization of eEF1A1 promotes hepatocellular carcinoma cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitin-proteasome degradation assays, TTLL12 knockdown/overexpression with proliferation readout, in vivo tumor growth models\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP to define TTLL12/BPOZ-2/eEF1A1 interactions, proteasome inhibition assays, in vivo models; single lab, not yet independently replicated\",\n      \"pmids\": [\"42014684\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TTLL12 is an atypical, pseudo-enzyme member of the TTLL family that lacks conventional enzymatic activities but mechanistically acts as: (1) a cytosolic negative regulator of RIG-I/VISA antiviral innate immune signaling via direct protein–protein interactions with VISA, TBK1, and IKKε; (2) a regulator of primary ciliogenesis and microtubule dynamics in polarized epithelial cells through direct α/β-tubulin binding and promotion of lysine acetylation and arginine methylation; (3) a suppressor of tubulin nitrotyrosination; (4) a driver of tumor immune evasion by promoting CCL9 transcription and MDSC recruitment; and (5) an oncogenic stabilizer of eEF1A1 by competing with BPOZ-2 to prevent CUL3-mediated ubiquitin-proteasome degradation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TTLL12 is an atypical member of the tubulin tyrosine ligase-like family that functions as a pseudo-enzyme, exerting its cellular roles through direct protein–protein interactions rather than the methyltransferase or tubulin tyrosine ligase activities predicted from its sequence [#0, #2]. In innate immunity, cytosolic TTLL12 acts as a negative regulator of RIG-I-mediated antiviral signaling by binding directly to VISA/MAVS, TBK1, and IKKε and disrupting VISA's association with downstream signaling partners, dampening interferon-β induction independently of any catalytic activity [#0]. At the cytoskeleton, TTLL12 binds the α/β-tubulin heterodimer directly and localizes to the base of primary cilia, where it is required for ciliogenesis in polarized renal epithelial cells; rather than adding glutamate or glycine to tubulin tails like canonical TTLL enzymes, it promotes microtubule lysine acetylation and arginine methylation and modulates microtubule dynamics and stability [#1]. TTLL12 additionally suppresses ligation of nitrotyrosine onto detyrosinated α-tubulin, reducing tubulin nitrotyrosination in cells [#2]. These tubulin-related and signaling activities converge on pro-tumorigenic functions: TTLL12 enhances cancer cell survival and proliferation [#3], drives an immunosuppressive tumor microenvironment by promoting CCL9 transcription and recruitment of myeloid-derived suppressor cells [#4], and stabilizes eEF1A1 by competing with BPOZ-2 to block CUL3-mediated ubiquitin-proteasome degradation, thereby promoting hepatocellular carcinoma cell proliferation [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2016,\n      \"claim\": \"Established the first defined molecular function for TTLL12 as a cytosolic brake on antiviral innate immune signaling, answering whether this predicted enzyme has a non-cytoskeletal role.\",\n      \"evidence\": \"Reciprocal Co-IP, cell fractionation, CRISPR/RNAi knockdown with IFN-β phenotype, and Sendai virus infection assays, plus mutagenesis testing enzymatic dependency\",\n      \"pmids\": [\"28011935\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis of how TTLL12 disrupts VISA's interactions is undefined\",\n        \"Whether this immune role is shared across cell types is not addressed\",\n        \"No direct demonstration of catalytic inactivity, only mutagenesis inference\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Resolved whether TTLL12 acts on microtubules like other TTLL family members, showing it binds tubulin directly but promotes atypical PTMs and is required for ciliogenesis.\",\n      \"evidence\": \"In vitro tubulin-binding assay, localization imaging, loss-of-function in polarized renal epithelial cells, and acetylation/arginine-methylation PTM readouts\",\n      \"pmids\": [\"38177908\", \"37546873\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether TTLL12 directly catalyzes acetylation/methylation or recruits other enzymes is unresolved\",\n        \"Mechanism linking microtubule PTM changes to cilium assembly not detailed\",\n        \"Connection to the immune-signaling role is not established\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linked TTLL12 to cancer cell survival, showing it protects cells from nitrotyrosine stress and supports proliferation.\",\n      \"evidence\": \"Gain- and loss-of-function SCC-25 cell lines with western blot, immunofluorescence, and MTT proliferation assay\",\n      \"pmids\": [\"33123251\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single lab, limited mechanistic depth on how nitration protection drives survival\",\n        \"No in vivo confirmation in this study\",\n        \"Tubulin nitration detection not reconstituted biochemically\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined a specific biochemical output of TTLL12's pseudo-enzymatic action—suppression of tubulin nitrotyrosination—and converted it into a screening readout.\",\n      \"evidence\": \"TTLL12 overexpression with cell-based ELISA for nitrotyrosinated α-tubulin and high-throughput compound screening\",\n      \"pmids\": [\"38394155\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No direct in vitro reconstitution of the suppression mechanism\",\n        \"Whether suppression is via competition for tubulin C-terminus or another route is unclear\",\n        \"Single-lab overexpression assay only\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected tumor-intrinsic TTLL12 to immune evasion via a transcriptional/chemokine axis recruiting suppressive myeloid cells.\",\n      \"evidence\": \"Gain/loss-of-function in syngeneic mouse models, flow cytometry, MDSC migration/proliferation assays, ChIP and mass spectrometry implicating CCL9 promoter binding\",\n      \"pmids\": [\"40461158\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct DNA-binding versus indirect promoter recruitment not firmly distinguished\",\n        \"How a cytosolic/microtubule-associated protein engages chromatin is mechanistically unexplained\",\n        \"Single lab, not independently replicated\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identified a protein-stabilization mechanism whereby TTLL12 promotes oncogenesis by shielding eEF1A1 from ubiquitin-proteasome degradation.\",\n      \"evidence\": \"Co-IP mapping TTLL12/BPOZ-2/eEF1A1 interactions, ubiquitin-proteasome degradation assays, knockdown/overexpression proliferation readouts, and in vivo tumor models\",\n      \"pmids\": [\"42014684\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single lab, not yet independently replicated\",\n        \"Competition stoichiometry and binding interface with BPOZ-2 undefined\",\n        \"Relationship to TTLL12's tubulin and immune roles unknown\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether TTLL12's diverse activities (immune signaling, microtubule PTMs, transcription, protein stabilization) reflect one unifying biochemical mechanism or independent moonlighting functions remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No structural model explaining how a single pseudo-enzyme engages tubulin, signaling adaptors, chromatin, and eEF1A1\",\n        \"No reconciliation of cytosolic versus ciliary-base versus nuclear localizations\",\n        \"No direct evidence of any catalytic activity for TTLL12\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"MAVS\", \"TBK1\", \"IKBKE\", \"TUBA\", \"TUBB\", \"EEF1A1\", \"BPOZ-2\", \"CUL3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}