{"gene":"TTLL4","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2007,"finding":"TTLL4 is a polyglutamylase enzyme that can modify a broad range of non-tubulin substrates in vitro, including nucleosome assembly proteins NAP1 and NAP2, and is the main polyglutamylase present in HeLa cells. Glutamate-rich stretches in substrate proteins are important for recognition by TTLL4.","method":"In vitro polyglutamylation assay with recombinant TTLL4 and proteomic identification of substrates; cellular context confirmed by overexpression and knockdown in HeLa cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic assay with multiple substrates, cellular validation, replicated across multiple substrates and methods in one study","pmids":["18045879"],"is_preprint":false},{"year":2010,"finding":"C. elegans TTLL-4 (ortholog of human TTLL4) is required for polyglutamylation of axonemal microtubules in sensory cilia; loss of ttll-4 reduces ciliary microtubule polyglutamylation.","method":"Genetic loss-of-function (ttll-4 mutants in C. elegans) with immunostaining for polyglutamylated tubulin","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic KO with defined molecular phenotype in a well-characterized model organism, single lab","pmids":["20519502"],"is_preprint":false},{"year":2010,"finding":"TTLL4 polyglutamylates PELP1 at its glutamate-rich stretch region in pancreatic cancer cells. PELP1 polyglutamylation influences its interaction with histone H3 and affects histone H3 acetylation, and PELP1 also interacts with LAS1L and SENP3, components of the MLL1-WDR5 chromatin remodeling supercomplex.","method":"shRNA knockdown of TTLL4 and measurement of PELP1 glutamylation levels; co-immunoprecipitation of PELP1 with histone H3 and chromatin remodeling complex components","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — knockdown with defined substrate and molecular readout, Co-IP for binding partners, single lab","pmids":["20442285"],"is_preprint":false},{"year":2011,"finding":"Loss of TTLL-4 (C. elegans ortholog) suppresses progressive ciliary degeneration and hyperglutamylation-induced defects in ccpp-1 (deglutamylase) mutants, placing TTLL-4 upstream as the glutamylase responsible for the damaging glutamate additions on ciliary microtubules.","method":"Genetic epistasis: double mutant analysis of ttll-4; ccpp-1 in C. elegans with ciliary dye-filling assay and immunostaining","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis replicated across multiple studies (PMID 21982591, 33064774), defines pathway position clearly","pmids":["21982591","33064774"],"is_preprint":false},{"year":2016,"finding":"TTLL4-mediated monoglutamylation of cGAS blocks its synthase activity (cGAMP production), thereby suppressing antiviral innate immune signaling. CCP5 removes this monoglutamylation to restore cGAS activity.","method":"Overexpression and knockdown of TTLL4 in cells with measurement of cGAMP synthesis; biochemical glutamylation assay; genetic rescue experiments","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — direct enzymatic activity shown, substrate (cGAS) identified, functional consequence (synthase inhibition) measured, CCP5 eraser identified, multiple orthogonal methods","pmids":["26829768"],"is_preprint":false},{"year":2016,"finding":"TTLL4 is required for proper cytoskeletal organization in red blood cells. The sole target of TTLL4 glutamylation in RBCs is nucleosome assembly protein 1 (NAP1). Glutamylation of NAP1 by TTLL4 promotes NAP1 binding to the RBC membrane; loss of TTLL4 leads to reduced membrane-associated glutamylated NAP1 and macromolecular aggregation of cytoskeletal components.","method":"Ttll4 knockout mice; immunoprecipitation with anti-glutamylation antibody GT335; ultrastructural analysis; phenylhydrazine hemolysis assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse model with multiple orthogonal methods (ultrastructure, biochemistry, functional assay), single lab but rigorous","pmids":["27974641"],"is_preprint":false},{"year":2017,"finding":"TTLL4 and TTLL13 polyglutamylate IL-7Rα in common helper-like innate lymphoid progenitors; IL-7Rα polyglutamylation triggers STAT5 activation and Sall3 expression to drive ILC3 differentiation. Ttll4-/- mice show reduced IL-7Rα polyglutamylation and Sall3 expression.","method":"Ttll4 knockout mice; measurement of IL-7Rα glutamylation; STAT5 activation assays; Sall3 expression analysis; IL-7Rα E446A knockin mouse","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse model, substrate identified (IL-7Rα), downstream signaling (STAT5/Sall3) defined, knockin mutation corroborates mechanism, multiple orthogonal methods","pmids":["28794449"],"is_preprint":false},{"year":2018,"finding":"TTLL4 polyglutamylates Klf4 at Glu381 during cell reprogramming; this polyglutamylation blocks Lys48-linked ubiquitination of Klf4 and stabilizes the protein, promoting iPSC induction and pluripotency. TTLL4 deletion abrogates cell reprogramming and early embryogenesis.","method":"TTLL4 knockout/knockdown; Klf4-E381A knockin mice; ubiquitination assays; iPSC reprogramming efficiency measurements","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — site-specific knockin mutation, KO mouse, ubiquitination assay establishing mechanistic crosstalk between glutamylation and ubiquitination, multiple methods","pmids":["29593216"],"is_preprint":false},{"year":2018,"finding":"In C. elegans sensory cilia, TTLL-4 glutamylase activity is activated by p38 MAPK signaling. A specific Thr residue in TTLL-4 (a putative MAPK phosphorylation site) is required for enhanced tubulin glutamylation and accelerated intraflagellar transport in response to starvation.","method":"Amino acid substitution mutagenesis of TTLL-4 phosphorylation site; IFT velocity measurement; behavioral assays in C. elegans","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-directed mutagenesis with functional readout (IFT velocity, behavior), C. elegans ortholog, single lab","pmids":["29849065"],"is_preprint":false},{"year":2020,"finding":"TTLL4 is exclusively a branch-initiating (initiase) glutamylase: it adds the first glutamate via an isopeptide bond to internal glutamates in tubulin C-terminal tails but does not elongate the chain. This was shown by co-crystal structures of TTLL6 and phylogenetic/mutational engineering, which also defined active-site residues distinguishing initiases from elongases.","method":"Co-crystal structures with tetrahedral intermediate analogs; in vitro glutamylation assays; active-site mutagenesis to convert TTLL6 elongase into initiase; phylogenetic analysis","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures, in vitro reconstitution, mutagenesis, multiple orthogonal methods establishing mechanistic basis for initiase activity","pmids":["32747782"],"is_preprint":false},{"year":2020,"finding":"TTLL4 overexpression in breast cancer cells increases polyglutamylation of β-tubulin, enhances trafficking of secretory vesicles and multivesicular bodies, and alters exosome biogenesis, resulting in EVs that promote adhesion of cancer cells to brain endothelium and increase blood-brain barrier permeability.","method":"TTLL4 overexpression in MDA-MB231/MDA-MB468 cells; live-cell imaging of vesicle trafficking; nanoparticle tracking analysis; transmission electron microscopy; blood-brain barrier permeability assays","journal":"Journal of experimental & clinical cancer research : CR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — overexpression with multiple orthogonal readouts (live imaging, TEM, functional BBB assay), single lab, mechanism partially inferred","pmids":["32998758"],"is_preprint":false},{"year":2021,"finding":"NEK5 kinase interacts with TTLL4 (confirmed by yeast two-hybrid and immunoprecipitation) and negatively regulates its polyglutamylase activity through phosphorylation at Y815 and S1136. NEK5 silencing increases TTLL4-dependent polyglutamylation levels; catalytically inactive NEK5 has the same effect.","method":"Yeast two-hybrid screening; co-immunoprecipitation; mass spectrometry identification of phosphorylation sites; point mutagenesis; NEK5 siRNA knockdown with polyglutamylation level measurement","journal":"World journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal binding confirmed, phosphorylation sites identified by MS and mutagenesis, functional activity measured, single lab","pmids":["34084286"],"is_preprint":false},{"year":2022,"finding":"TTLL4 deficiency attenuates Purkinje cell degeneration and mitral cell death in pcd (Nna1/CCP1-mutant) mice, and partially rescues photoreceptor degeneration and impaired rhodopsin trafficking. TTLL4 loss produces a distinct polyglutamylation profile change compared to TTLL1 loss in pcd mice.","method":"Ttll4 knockout crossed into pcd mice; histological analysis of Purkinje cells, mitral cells, and photoreceptors; rhodopsin trafficking assays","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic rescue in mouse model, multiple neuronal cell types assessed, multiple orthogonal phenotypic readouts, rigorous genetic epistasis","pmids":["35404950"],"is_preprint":false},{"year":2023,"finding":"TTLL4 acts as a branch-initiating glutamylase on brain tubulin, preferentially modifying α-tubulin isoforms (producing stronger glutamylation immunosignals than for β-tubulin), with distinct modification sites compared to TTLL7. TTLL7 elongates less efficiently on microtubules pre-modified by TTLL4, suggesting TTLL4-initiated sites regulate TTLL7 elongation. Kinesin behavior differs on microtubules modified by TTLL4 versus TTLL7.","method":"In vitro glutamylation assay with purified recombinant TTLL4; tandem mass spectrometry of modification sites on synthetic peptides and recombinant tubulin; antibody-based detection; kinesin motility assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — purified recombinant enzyme, mass spectrometry site mapping, functional kinesin assay, multiple orthogonal methods in single study","pmids":["37321451"],"is_preprint":false},{"year":2024,"finding":"TTLL4 catalyzes post-translational glutamylation of acidic intrinsically disordered regions of histone chaperones Npm2 and Nap1 (Xenopus laevis). This glutamylation enhances DNA electrostatic mimicry of the chaperone IDRs, promoting binding and stabilization of H2A/H2B heterodimers and facilitating nucleosome assembly.","method":"In vitro TTLL4 glutamylation assay with Npm2 and Nap1; biochemical binding assays; computational modeling; biophysical studies; nucleosome assembly assay","journal":"iScience","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of enzymatic activity on defined substrates, multiple orthogonal methods (biochemical, biophysical, computational), functional nucleosome assembly readout","pmids":["38571760"],"is_preprint":false},{"year":2025,"finding":"TTLL4 mono-glutamylates NPM1c (mutant NPM1) at E126, stabilizing its cytoplasmic localization and promoting a differentiation block in AML cells. Genetic inactivation of TTLL4 reduces NPM1c glutamylation, triggers myeloid differentiation, impairs proliferation, and prolongs survival in an NPM1c/NRAS-driven mouse AML model. A small molecule EN7 selectively inhibits TTLL4 and recapitulates these phenotypes.","method":"Multiple genetic TTLL4 inactivation approaches in human NPM1c cell lines; Ttll4 KO mouse AML model; transcriptomic analysis; small molecule inhibitor (EN7) treatment","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic approaches and in vivo mouse model, substrate site identified, functional phenotype replicated by small molecule; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2025.04.07.647605"],"is_preprint":true},{"year":2026,"finding":"TTLL4 exhibits strong chain initiation (monoglutamylation) activity in human HEK293T cells, distinct from TTLL11 which shows elongation activity. TTLL4 overexpression increases polyglutamylation but does not disrupt microtubule dynamics (unlike TTLL6). TTLL4 activity is blocked by the small molecule inhibitor LDC10.","method":"Overexpression of TTLL4 in HEK293T cells; live-cell imaging of EB3 (microtubule dynamics); immunofluorescence for glutamylation patterns; LDC10 inhibitor treatment","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cellular overexpression with live imaging and functional readout, pharmacological inhibition, single lab","pmids":["41770829"],"is_preprint":false}],"current_model":"TTLL4 is a branch-initiating (initiase) polyglutamylase that catalyzes the addition of single glutamate residues via isopeptide bonds to internal glutamates on diverse substrates — including tubulin (preferentially α-isoform on brain microtubules), the histone chaperones NAP1 and Npm2, the immune sensor cGAS, the transcription factors Klf4 and NPM1c, and the signaling receptor IL-7Rα — with functional consequences including inhibition of cGAS synthase activity, stabilization of Klf4 and NPM1c via blocking ubiquitination, enhancement of histone chaperone DNA mimicry, and promotion of ILC3 differentiation; its activity is negatively regulated by NEK5-mediated phosphorylation at Y815 and S1136, and by p38 MAPK signaling in cilia; loss of TTLL4 rescues neurodegeneration in hyperglutamylation mouse models, while its overactivity drives pathological states including leukemic differentiation block and altered exosome biogenesis."},"narrative":{"mechanistic_narrative":"TTLL4 is a branch-initiating (initiase) protein glutamylase that catalyzes the addition of a single glutamate via an isopeptide bond to internal glutamate residues on diverse substrates, without elongating the resulting branch [PMID:32747782, PMID:41770829]. Its substrate range extends well beyond tubulin: it is the predominant cellular polyglutamylase in HeLa cells and recognizes glutamate-rich stretches in target proteins, modifying the nucleosome assembly proteins NAP1/NAP2 in vitro [PMID:18045879]. On microtubules, TTLL4 acts preferentially on α-tubulin isoforms in brain and deposits initiator sites that constrain downstream elongation by TTLL7, with distinct effects on kinesin motility [PMID:37321451]. Through monoglutamylation of non-tubulin substrates, TTLL4 controls multiple cellular programs: it monoglutamylates the DNA sensor cGAS to block its cGAMP-synthase activity and suppress antiviral signaling [PMID:26829768]; it modifies the histone chaperones Nap1 and Npm2 within acidic disordered regions to enhance their DNA electrostatic mimicry and promote nucleosome assembly [PMID:38571760]; it glutamylates IL-7Rα to trigger STAT5/Sall3 signaling and ILC3 differentiation [PMID:28794449]; and it glutamylates Klf4 at Glu381 to block Lys48-linked ubiquitination, stabilizing Klf4 and enabling reprogramming and early embryogenesis [PMID:29593216]. In red blood cells its sole target is NAP1, whose glutamylation drives membrane association and cytoskeletal organization [PMID:27974641]. TTLL4 activity is negatively regulated by NEK5 kinase, which binds TTLL4 and phosphorylates it at Y815 and S1136 to dampen polyglutamylation [PMID:34084286]. Genetic loss of TTLL4 suppresses ciliary degeneration in C. elegans deglutamylase mutants and attenuates Purkinje cell, mitral cell, and photoreceptor degeneration in pcd mice, identifying TTLL4 as a source of pathological hyperglutamylation [PMID:21982591, PMID:33064774, PMID:35404950].","teleology":[{"year":2007,"claim":"Established that TTLL4 is an enzymatically active polyglutamylase with a broad non-tubulin substrate range, answering whether the TTLL family member acted only on tubulin.","evidence":"In vitro polyglutamylation assay with recombinant TTLL4 plus proteomic substrate identification and HeLa knockdown/overexpression","pmids":["18045879"],"confidence":"High","gaps":["Did not define the chemistry of the modification (initiation vs elongation)","Physiological relevance of NAP1/NAP2 modification not established in vivo"]},{"year":2010,"claim":"Genetic loss-of-function placed the TTLL4 ortholog as a glutamylase of axonemal microtubules in sensory cilia and identified PELP1 as a chromatin-linked substrate, extending substrate scope to cilia and transcriptional machinery.","evidence":"C. elegans ttll-4 mutants with anti-glutamylation immunostaining; shRNA knockdown and Co-IP of PELP1 in pancreatic cancer cells","pmids":["20519502","20442285"],"confidence":"Medium","gaps":["Direct enzyme-substrate catalysis on PELP1 not reconstituted in vitro","Functional consequence of ciliary glutamylation not yet defined"]},{"year":2011,"claim":"Genetic epistasis defined TTLL4 as the glutamylase upstream of CCP-mediated deglutamylation, showing its activity is the source of damaging hyperglutamylation on ciliary microtubules.","evidence":"ttll-4; ccpp-1 double mutant analysis with ciliary dye-filling and immunostaining in C. elegans","pmids":["21982591","33064774"],"confidence":"High","gaps":["Mammalian equivalent of the ciliary degeneration axis not directly tested here","Glutamate chain length on damaging sites not quantified"]},{"year":2016,"claim":"Identified two distinct physiological substrate axes — cGAS for innate immune control and NAP1 for erythrocyte cytoskeleton — demonstrating TTLL4 monoglutamylation as a tunable regulatory switch with dedicated erasers.","evidence":"Overexpression/knockdown with cGAMP and glutamylation assays plus CCP5 rescue; Ttll4 KO mice with GT335 IP, ultrastructure, and hemolysis assays","pmids":["26829768","27974641"],"confidence":"High","gaps":["Structural basis for substrate selection across cGAS vs NAP1 unknown","How upstream signals direct TTLL4 to specific substrates not defined"]},{"year":2017,"claim":"Linked TTLL4 glutamylation of IL-7Rα to STAT5/Sall3 signaling and ILC3 differentiation, establishing a receptor-modifying role in immune cell fate.","evidence":"Ttll4 KO mice, IL-7Rα glutamylation measurement, STAT5 assays, and IL-7Rα E446A knockin","pmids":["28794449"],"confidence":"High","gaps":["Mechanism by which receptor glutamylation potentiates STAT5 activation not resolved","Relative contributions of TTLL4 vs TTLL13 to the modification unclear"]},{"year":2018,"claim":"Revealed glutamylation-ubiquitination crosstalk: TTLL4 modification of Klf4 at Glu381 blocks K48 ubiquitination and stabilizes the factor, connecting the enzyme to reprogramming and embryogenesis; concurrently showed p38 MAPK activates the ciliary ortholog.","evidence":"Klf4-E381A knockin mice, KO/knockdown, ubiquitination and iPSC assays; phosphosite mutagenesis with IFT velocity readout in C. elegans","pmids":["29593216","29849065"],"confidence":"High","gaps":["Whether ubiquitination blocking generalizes to other substrates unknown","Direct kinase-to-TTLL4 phosphorylation in mammals not mapped here"]},{"year":2020,"claim":"Structural and engineering work defined TTLL4 mechanistically as a pure branch-initiase that adds the first isopeptide-linked glutamate but cannot elongate, while overexpression studies tied it to vesicle/exosome biogenesis in cancer.","evidence":"Co-crystal structures with tetrahedral intermediate analogs and initiase/elongase active-site mutagenesis; TTLL4 overexpression with live imaging, NTA, TEM, and BBB permeability assays","pmids":["32747782","32998758"],"confidence":"High","gaps":["Initiase chemistry shown via TTLL6 surrogate structures, not TTLL4 crystal directly","Causal link between tubulin glutamylation and exosome cargo selection partially inferred"]},{"year":2021,"claim":"Defined NEK5 as a direct binding partner and negative regulator of TTLL4 via phosphorylation at Y815 and S1136, establishing post-translational control of glutamylase output.","evidence":"Yeast two-hybrid, reciprocal Co-IP, MS phosphosite mapping, point mutagenesis, and NEK5 siRNA with glutamylation readout","pmids":["34084286"],"confidence":"Medium","gaps":["Single lab; structural basis of inhibition unknown","Physiological contexts where NEK5 controls TTLL4 not defined"]},{"year":2022,"claim":"Genetic rescue in pcd mice established TTLL4 as a clinically relevant driver of neurodegenerative hyperglutamylation, with a substrate/site profile distinct from TTLL1.","evidence":"Ttll4 KO crossed into pcd mice with histology of Purkinje, mitral, and photoreceptor cells and rhodopsin trafficking assays","pmids":["35404950"],"confidence":"High","gaps":["Specific tubulin sites responsible for neuronal toxicity not pinpointed","Only partial rescue of photoreceptor phenotype"]},{"year":2023,"claim":"Resolved how TTLL4 cooperates with elongases on neuronal microtubules — preferentially initiating on α-tubulin and constraining TTLL7 elongation — with consequences for kinesin behavior.","evidence":"In vitro glutamylation with purified recombinant TTLL4, tandem MS site mapping, and kinesin motility assays","pmids":["37321451"],"confidence":"High","gaps":["In vivo significance of the TTLL4/TTLL7 division of labor not tested","Effect on other microtubule motors beyond kinesin unknown"]},{"year":2025,"claim":"Extended the Klf4 stabilization paradigm to oncogenic NPM1c: TTLL4 monoglutamylation at E126 maintains cytoplasmic NPM1c and a differentiation block, nominating TTLL4 as an AML target druggable by EN7.","evidence":"Multiple genetic TTLL4 inactivation approaches in NPM1c cell lines, Ttll4 KO NPM1c/NRAS mouse AML model, transcriptomics, and EN7 inhibitor (preprint)","pmids":["bio_10.1101_2025.04.07.647605"],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","EN7 selectivity and off-target profile not fully established","Mechanism by which E126 glutamylation enforces cytoplasmic retention not resolved"]},{"year":2026,"claim":"Confirmed in human cells that TTLL4 is a chain-initiator whose overexpression raises glutamylation without disrupting microtubule dynamics, and is pharmacologically blockable by LDC10.","evidence":"TTLL4 overexpression in HEK293T with EB3 live imaging, glutamylation immunofluorescence, and LDC10 inhibitor treatment","pmids":["41770829"],"confidence":"Medium","gaps":["Single lab; endogenous TTLL4 not tested","Inhibitor specificity across the TTLL family not characterized"]},{"year":null,"claim":"How TTLL4 selects among its many substrates (tubulin, cGAS, Klf4, NPM1c, IL-7Rα, NAP1, Npm2) in a given cellular context, and how upstream signaling reroutes this selectivity, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of TTLL4 bound to a non-tubulin substrate","Determinants of substrate choice beyond glutamate-rich stretches unknown","Cell-type-specific regulation of substrate repertoire undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,9,13,14,16]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,4,6,7,14,15]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[9,14]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[5,13,16]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[1,3,8]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,14]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[4,6]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,7,15]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[2,14]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[12,15]}],"complexes":[],"partners":["NEK5","CGAS","KLF4","IL7R","NAP1","PELP1","NPM1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q14679","full_name":"Tubulin monoglutamylase TTLL4","aliases":["Protein monoglutamylase TTLL4","Tubulin--tyrosine ligase-like protein 4"],"length_aa":1199,"mass_kda":133.4,"function":"Monoglutamylase which modifies both tubulin and non-tubulin proteins, adding a single glutamate on the gamma-carboxyl group of specific glutamate residues of target proteins. Involved in the side-chain initiation step of the polyglutamylation reaction but not in the elongation step. Preferentially modifies beta-tail tubulin over the alpha-tubulin. Monoglutamylates nucleosome assembly proteins NAP1L1 and NAP1L4. Monoglutamylates nucleotidyltransferase CGAS, leading to inhibition of CGAS catalytic activity, thereby preventing antiviral defense function. Involved in KLF4 glutamylation which impedes its ubiquitination, thereby leading to somatic cell reprogramming, pluripotency maintenance and embryogenesis","subcellular_location":"Cytoplasm; Cell projection, cilium; Cytoplasm, cytoskeleton, cilium basal body","url":"https://www.uniprot.org/uniprotkb/Q14679/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TTLL4","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"PSME3","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TTLL4","total_profiled":1310},"omim":[{"mim_id":"620485","title":"TUBULIN TYROSINE LIGASE-LIKE 13; TTLL13","url":"https://www.omim.org/entry/620485"},{"mim_id":"618738","title":"TUBULIN TYROSINE LIGASE-LIKE 4; TTLL4","url":"https://www.omim.org/entry/618738"},{"mim_id":"617345","title":"ATP/GTP-BINDING PROTEIN-LIKE 2; AGBL2","url":"https://www.omim.org/entry/617345"},{"mim_id":"610849","title":"TUBULIN TYROSINE LIGASE-LIKE 6; TTLL6","url":"https://www.omim.org/entry/610849"},{"mim_id":"609455","title":"PROLINE-, GLUTAMIC ACID-, AND LEUCINE-RICH PROTEIN 1; PELP1","url":"https://www.omim.org/entry/609455"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Mitochondria","reliability":"Uncertain"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TTLL4"},"hgnc":{"alias_symbol":["KIAA0173"],"prev_symbol":[]},"alphafold":{"accession":"Q14679","domains":[{"cath_id":"-","chopping":"972-1087","consensus_level":"high","plddt":91.6721,"start":972,"end":1087}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14679","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q14679-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q14679-F1-predicted_aligned_error_v6.png","plddt_mean":57.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TTLL4","jax_strain_url":"https://www.jax.org/strain/search?query=TTLL4"},"sequence":{"accession":"Q14679","fasta_url":"https://rest.uniprot.org/uniprotkb/Q14679.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q14679/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14679"}},"corpus_meta":[{"pmid":"26829768","id":"PMC_26829768","title":"Glutamylation of the DNA sensor cGAS regulates its binding and synthase activity in antiviral immunity.","date":"2016","source":"Nature immunology","url":"https://pubmed.ncbi.nlm.nih.gov/26829768","citation_count":191,"is_preprint":false},{"pmid":"21982591","id":"PMC_21982591","title":"The tubulin deglutamylase CCPP-1 regulates the function and stability of sensory cilia in C. elegans.","date":"2011","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/21982591","citation_count":102,"is_preprint":false},{"pmid":"20519502","id":"PMC_20519502","title":"Identification of tubulin deglutamylase among Caenorhabditis elegans and mammalian cytosolic carboxypeptidases (CCPs).","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20519502","citation_count":96,"is_preprint":false},{"pmid":"18045879","id":"PMC_18045879","title":"Polyglutamylation is a post-translational modification with a broad range of substrates.","date":"2007","source":"The Journal of biological 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broad range of non-tubulin substrates in vitro, including nucleosome assembly proteins NAP1 and NAP2, and is the main polyglutamylase present in HeLa cells. Glutamate-rich stretches in substrate proteins are important for recognition by TTLL4.\",\n      \"method\": \"In vitro polyglutamylation assay with recombinant TTLL4 and proteomic identification of substrates; cellular context confirmed by overexpression and knockdown in HeLa cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic assay with multiple substrates, cellular validation, replicated across multiple substrates and methods in one study\",\n      \"pmids\": [\"18045879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"C. elegans TTLL-4 (ortholog of human TTLL4) is required for polyglutamylation of axonemal microtubules in sensory cilia; loss of ttll-4 reduces ciliary microtubule polyglutamylation.\",\n      \"method\": \"Genetic loss-of-function (ttll-4 mutants in C. elegans) with immunostaining for polyglutamylated tubulin\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic KO with defined molecular phenotype in a well-characterized model organism, single lab\",\n      \"pmids\": [\"20519502\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TTLL4 polyglutamylates PELP1 at its glutamate-rich stretch region in pancreatic cancer cells. PELP1 polyglutamylation influences its interaction with histone H3 and affects histone H3 acetylation, and PELP1 also interacts with LAS1L and SENP3, components of the MLL1-WDR5 chromatin remodeling supercomplex.\",\n      \"method\": \"shRNA knockdown of TTLL4 and measurement of PELP1 glutamylation levels; co-immunoprecipitation of PELP1 with histone H3 and chromatin remodeling complex components\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — knockdown with defined substrate and molecular readout, Co-IP for binding partners, single lab\",\n      \"pmids\": [\"20442285\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Loss of TTLL-4 (C. elegans ortholog) suppresses progressive ciliary degeneration and hyperglutamylation-induced defects in ccpp-1 (deglutamylase) mutants, placing TTLL-4 upstream as the glutamylase responsible for the damaging glutamate additions on ciliary microtubules.\",\n      \"method\": \"Genetic epistasis: double mutant analysis of ttll-4; ccpp-1 in C. elegans with ciliary dye-filling assay and immunostaining\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis replicated across multiple studies (PMID 21982591, 33064774), defines pathway position clearly\",\n      \"pmids\": [\"21982591\", \"33064774\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TTLL4-mediated monoglutamylation of cGAS blocks its synthase activity (cGAMP production), thereby suppressing antiviral innate immune signaling. CCP5 removes this monoglutamylation to restore cGAS activity.\",\n      \"method\": \"Overexpression and knockdown of TTLL4 in cells with measurement of cGAMP synthesis; biochemical glutamylation assay; genetic rescue experiments\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — direct enzymatic activity shown, substrate (cGAS) identified, functional consequence (synthase inhibition) measured, CCP5 eraser identified, multiple orthogonal methods\",\n      \"pmids\": [\"26829768\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TTLL4 is required for proper cytoskeletal organization in red blood cells. The sole target of TTLL4 glutamylation in RBCs is nucleosome assembly protein 1 (NAP1). Glutamylation of NAP1 by TTLL4 promotes NAP1 binding to the RBC membrane; loss of TTLL4 leads to reduced membrane-associated glutamylated NAP1 and macromolecular aggregation of cytoskeletal components.\",\n      \"method\": \"Ttll4 knockout mice; immunoprecipitation with anti-glutamylation antibody GT335; ultrastructural analysis; phenylhydrazine hemolysis assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse model with multiple orthogonal methods (ultrastructure, biochemistry, functional assay), single lab but rigorous\",\n      \"pmids\": [\"27974641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TTLL4 and TTLL13 polyglutamylate IL-7Rα in common helper-like innate lymphoid progenitors; IL-7Rα polyglutamylation triggers STAT5 activation and Sall3 expression to drive ILC3 differentiation. Ttll4-/- mice show reduced IL-7Rα polyglutamylation and Sall3 expression.\",\n      \"method\": \"Ttll4 knockout mice; measurement of IL-7Rα glutamylation; STAT5 activation assays; Sall3 expression analysis; IL-7Rα E446A knockin mouse\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse model, substrate identified (IL-7Rα), downstream signaling (STAT5/Sall3) defined, knockin mutation corroborates mechanism, multiple orthogonal methods\",\n      \"pmids\": [\"28794449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TTLL4 polyglutamylates Klf4 at Glu381 during cell reprogramming; this polyglutamylation blocks Lys48-linked ubiquitination of Klf4 and stabilizes the protein, promoting iPSC induction and pluripotency. TTLL4 deletion abrogates cell reprogramming and early embryogenesis.\",\n      \"method\": \"TTLL4 knockout/knockdown; Klf4-E381A knockin mice; ubiquitination assays; iPSC reprogramming efficiency measurements\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — site-specific knockin mutation, KO mouse, ubiquitination assay establishing mechanistic crosstalk between glutamylation and ubiquitination, multiple methods\",\n      \"pmids\": [\"29593216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In C. elegans sensory cilia, TTLL-4 glutamylase activity is activated by p38 MAPK signaling. A specific Thr residue in TTLL-4 (a putative MAPK phosphorylation site) is required for enhanced tubulin glutamylation and accelerated intraflagellar transport in response to starvation.\",\n      \"method\": \"Amino acid substitution mutagenesis of TTLL-4 phosphorylation site; IFT velocity measurement; behavioral assays in C. elegans\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-directed mutagenesis with functional readout (IFT velocity, behavior), C. elegans ortholog, single lab\",\n      \"pmids\": [\"29849065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TTLL4 is exclusively a branch-initiating (initiase) glutamylase: it adds the first glutamate via an isopeptide bond to internal glutamates in tubulin C-terminal tails but does not elongate the chain. This was shown by co-crystal structures of TTLL6 and phylogenetic/mutational engineering, which also defined active-site residues distinguishing initiases from elongases.\",\n      \"method\": \"Co-crystal structures with tetrahedral intermediate analogs; in vitro glutamylation assays; active-site mutagenesis to convert TTLL6 elongase into initiase; phylogenetic analysis\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures, in vitro reconstitution, mutagenesis, multiple orthogonal methods establishing mechanistic basis for initiase activity\",\n      \"pmids\": [\"32747782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TTLL4 overexpression in breast cancer cells increases polyglutamylation of β-tubulin, enhances trafficking of secretory vesicles and multivesicular bodies, and alters exosome biogenesis, resulting in EVs that promote adhesion of cancer cells to brain endothelium and increase blood-brain barrier permeability.\",\n      \"method\": \"TTLL4 overexpression in MDA-MB231/MDA-MB468 cells; live-cell imaging of vesicle trafficking; nanoparticle tracking analysis; transmission electron microscopy; blood-brain barrier permeability assays\",\n      \"journal\": \"Journal of experimental & clinical cancer research : CR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — overexpression with multiple orthogonal readouts (live imaging, TEM, functional BBB assay), single lab, mechanism partially inferred\",\n      \"pmids\": [\"32998758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NEK5 kinase interacts with TTLL4 (confirmed by yeast two-hybrid and immunoprecipitation) and negatively regulates its polyglutamylase activity through phosphorylation at Y815 and S1136. NEK5 silencing increases TTLL4-dependent polyglutamylation levels; catalytically inactive NEK5 has the same effect.\",\n      \"method\": \"Yeast two-hybrid screening; co-immunoprecipitation; mass spectrometry identification of phosphorylation sites; point mutagenesis; NEK5 siRNA knockdown with polyglutamylation level measurement\",\n      \"journal\": \"World journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding confirmed, phosphorylation sites identified by MS and mutagenesis, functional activity measured, single lab\",\n      \"pmids\": [\"34084286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TTLL4 deficiency attenuates Purkinje cell degeneration and mitral cell death in pcd (Nna1/CCP1-mutant) mice, and partially rescues photoreceptor degeneration and impaired rhodopsin trafficking. TTLL4 loss produces a distinct polyglutamylation profile change compared to TTLL1 loss in pcd mice.\",\n      \"method\": \"Ttll4 knockout crossed into pcd mice; histological analysis of Purkinje cells, mitral cells, and photoreceptors; rhodopsin trafficking assays\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic rescue in mouse model, multiple neuronal cell types assessed, multiple orthogonal phenotypic readouts, rigorous genetic epistasis\",\n      \"pmids\": [\"35404950\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TTLL4 acts as a branch-initiating glutamylase on brain tubulin, preferentially modifying α-tubulin isoforms (producing stronger glutamylation immunosignals than for β-tubulin), with distinct modification sites compared to TTLL7. TTLL7 elongates less efficiently on microtubules pre-modified by TTLL4, suggesting TTLL4-initiated sites regulate TTLL7 elongation. Kinesin behavior differs on microtubules modified by TTLL4 versus TTLL7.\",\n      \"method\": \"In vitro glutamylation assay with purified recombinant TTLL4; tandem mass spectrometry of modification sites on synthetic peptides and recombinant tubulin; antibody-based detection; kinesin motility assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — purified recombinant enzyme, mass spectrometry site mapping, functional kinesin assay, multiple orthogonal methods in single study\",\n      \"pmids\": [\"37321451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TTLL4 catalyzes post-translational glutamylation of acidic intrinsically disordered regions of histone chaperones Npm2 and Nap1 (Xenopus laevis). This glutamylation enhances DNA electrostatic mimicry of the chaperone IDRs, promoting binding and stabilization of H2A/H2B heterodimers and facilitating nucleosome assembly.\",\n      \"method\": \"In vitro TTLL4 glutamylation assay with Npm2 and Nap1; biochemical binding assays; computational modeling; biophysical studies; nucleosome assembly assay\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of enzymatic activity on defined substrates, multiple orthogonal methods (biochemical, biophysical, computational), functional nucleosome assembly readout\",\n      \"pmids\": [\"38571760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TTLL4 mono-glutamylates NPM1c (mutant NPM1) at E126, stabilizing its cytoplasmic localization and promoting a differentiation block in AML cells. Genetic inactivation of TTLL4 reduces NPM1c glutamylation, triggers myeloid differentiation, impairs proliferation, and prolongs survival in an NPM1c/NRAS-driven mouse AML model. A small molecule EN7 selectively inhibits TTLL4 and recapitulates these phenotypes.\",\n      \"method\": \"Multiple genetic TTLL4 inactivation approaches in human NPM1c cell lines; Ttll4 KO mouse AML model; transcriptomic analysis; small molecule inhibitor (EN7) treatment\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic approaches and in vivo mouse model, substrate site identified, functional phenotype replicated by small molecule; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.04.07.647605\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"TTLL4 exhibits strong chain initiation (monoglutamylation) activity in human HEK293T cells, distinct from TTLL11 which shows elongation activity. TTLL4 overexpression increases polyglutamylation but does not disrupt microtubule dynamics (unlike TTLL6). TTLL4 activity is blocked by the small molecule inhibitor LDC10.\",\n      \"method\": \"Overexpression of TTLL4 in HEK293T cells; live-cell imaging of EB3 (microtubule dynamics); immunofluorescence for glutamylation patterns; LDC10 inhibitor treatment\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cellular overexpression with live imaging and functional readout, pharmacological inhibition, single lab\",\n      \"pmids\": [\"41770829\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TTLL4 is a branch-initiating (initiase) polyglutamylase that catalyzes the addition of single glutamate residues via isopeptide bonds to internal glutamates on diverse substrates — including tubulin (preferentially α-isoform on brain microtubules), the histone chaperones NAP1 and Npm2, the immune sensor cGAS, the transcription factors Klf4 and NPM1c, and the signaling receptor IL-7Rα — with functional consequences including inhibition of cGAS synthase activity, stabilization of Klf4 and NPM1c via blocking ubiquitination, enhancement of histone chaperone DNA mimicry, and promotion of ILC3 differentiation; its activity is negatively regulated by NEK5-mediated phosphorylation at Y815 and S1136, and by p38 MAPK signaling in cilia; loss of TTLL4 rescues neurodegeneration in hyperglutamylation mouse models, while its overactivity drives pathological states including leukemic differentiation block and altered exosome biogenesis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TTLL4 is a branch-initiating (initiase) protein glutamylase that catalyzes the addition of a single glutamate via an isopeptide bond to internal glutamate residues on diverse substrates, without elongating the resulting branch [#9, #16]. Its substrate range extends well beyond tubulin: it is the predominant cellular polyglutamylase in HeLa cells and recognizes glutamate-rich stretches in target proteins, modifying the nucleosome assembly proteins NAP1/NAP2 in vitro [#0]. On microtubules, TTLL4 acts preferentially on \\u03b1-tubulin isoforms in brain and deposits initiator sites that constrain downstream elongation by TTLL7, with distinct effects on kinesin motility [#13]. Through monoglutamylation of non-tubulin substrates, TTLL4 controls multiple cellular programs: it monoglutamylates the DNA sensor cGAS to block its cGAMP-synthase activity and suppress antiviral signaling [#4]; it modifies the histone chaperones Nap1 and Npm2 within acidic disordered regions to enhance their DNA electrostatic mimicry and promote nucleosome assembly [#14]; it glutamylates IL-7R\\u03b1 to trigger STAT5/Sall3 signaling and ILC3 differentiation [#6]; and it glutamylates Klf4 at Glu381 to block Lys48-linked ubiquitination, stabilizing Klf4 and enabling reprogramming and early embryogenesis [#7]. In red blood cells its sole target is NAP1, whose glutamylation drives membrane association and cytoskeletal organization [#5]. TTLL4 activity is negatively regulated by NEK5 kinase, which binds TTLL4 and phosphorylates it at Y815 and S1136 to dampen polyglutamylation [#11]. Genetic loss of TTLL4 suppresses ciliary degeneration in C. elegans deglutamylase mutants and attenuates Purkinje cell, mitral cell, and photoreceptor degeneration in pcd mice, identifying TTLL4 as a source of pathological hyperglutamylation [#3, #12].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established that TTLL4 is an enzymatically active polyglutamylase with a broad non-tubulin substrate range, answering whether the TTLL family member acted only on tubulin.\",\n      \"evidence\": \"In vitro polyglutamylation assay with recombinant TTLL4 plus proteomic substrate identification and HeLa knockdown/overexpression\",\n      \"pmids\": [\"18045879\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the chemistry of the modification (initiation vs elongation)\", \"Physiological relevance of NAP1/NAP2 modification not established in vivo\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Genetic loss-of-function placed the TTLL4 ortholog as a glutamylase of axonemal microtubules in sensory cilia and identified PELP1 as a chromatin-linked substrate, extending substrate scope to cilia and transcriptional machinery.\",\n      \"evidence\": \"C. elegans ttll-4 mutants with anti-glutamylation immunostaining; shRNA knockdown and Co-IP of PELP1 in pancreatic cancer cells\",\n      \"pmids\": [\"20519502\", \"20442285\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct enzyme-substrate catalysis on PELP1 not reconstituted in vitro\", \"Functional consequence of ciliary glutamylation not yet defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Genetic epistasis defined TTLL4 as the glutamylase upstream of CCP-mediated deglutamylation, showing its activity is the source of damaging hyperglutamylation on ciliary microtubules.\",\n      \"evidence\": \"ttll-4; ccpp-1 double mutant analysis with ciliary dye-filling and immunostaining in C. elegans\",\n      \"pmids\": [\"21982591\", \"33064774\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mammalian equivalent of the ciliary degeneration axis not directly tested here\", \"Glutamate chain length on damaging sites not quantified\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified two distinct physiological substrate axes \\u2014 cGAS for innate immune control and NAP1 for erythrocyte cytoskeleton \\u2014 demonstrating TTLL4 monoglutamylation as a tunable regulatory switch with dedicated erasers.\",\n      \"evidence\": \"Overexpression/knockdown with cGAMP and glutamylation assays plus CCP5 rescue; Ttll4 KO mice with GT335 IP, ultrastructure, and hemolysis assays\",\n      \"pmids\": [\"26829768\", \"27974641\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for substrate selection across cGAS vs NAP1 unknown\", \"How upstream signals direct TTLL4 to specific substrates not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Linked TTLL4 glutamylation of IL-7R\\u03b1 to STAT5/Sall3 signaling and ILC3 differentiation, establishing a receptor-modifying role in immune cell fate.\",\n      \"evidence\": \"Ttll4 KO mice, IL-7R\\u03b1 glutamylation measurement, STAT5 assays, and IL-7R\\u03b1 E446A knockin\",\n      \"pmids\": [\"28794449\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which receptor glutamylation potentiates STAT5 activation not resolved\", \"Relative contributions of TTLL4 vs TTLL13 to the modification unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Revealed glutamylation-ubiquitination crosstalk: TTLL4 modification of Klf4 at Glu381 blocks K48 ubiquitination and stabilizes the factor, connecting the enzyme to reprogramming and embryogenesis; concurrently showed p38 MAPK activates the ciliary ortholog.\",\n      \"evidence\": \"Klf4-E381A knockin mice, KO/knockdown, ubiquitination and iPSC assays; phosphosite mutagenesis with IFT velocity readout in C. elegans\",\n      \"pmids\": [\"29593216\", \"29849065\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ubiquitination blocking generalizes to other substrates unknown\", \"Direct kinase-to-TTLL4 phosphorylation in mammals not mapped here\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Structural and engineering work defined TTLL4 mechanistically as a pure branch-initiase that adds the first isopeptide-linked glutamate but cannot elongate, while overexpression studies tied it to vesicle/exosome biogenesis in cancer.\",\n      \"evidence\": \"Co-crystal structures with tetrahedral intermediate analogs and initiase/elongase active-site mutagenesis; TTLL4 overexpression with live imaging, NTA, TEM, and BBB permeability assays\",\n      \"pmids\": [\"32747782\", \"32998758\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Initiase chemistry shown via TTLL6 surrogate structures, not TTLL4 crystal directly\", \"Causal link between tubulin glutamylation and exosome cargo selection partially inferred\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined NEK5 as a direct binding partner and negative regulator of TTLL4 via phosphorylation at Y815 and S1136, establishing post-translational control of glutamylase output.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal Co-IP, MS phosphosite mapping, point mutagenesis, and NEK5 siRNA with glutamylation readout\",\n      \"pmids\": [\"34084286\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; structural basis of inhibition unknown\", \"Physiological contexts where NEK5 controls TTLL4 not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Genetic rescue in pcd mice established TTLL4 as a clinically relevant driver of neurodegenerative hyperglutamylation, with a substrate/site profile distinct from TTLL1.\",\n      \"evidence\": \"Ttll4 KO crossed into pcd mice with histology of Purkinje, mitral, and photoreceptor cells and rhodopsin trafficking assays\",\n      \"pmids\": [\"35404950\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific tubulin sites responsible for neuronal toxicity not pinpointed\", \"Only partial rescue of photoreceptor phenotype\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Resolved how TTLL4 cooperates with elongases on neuronal microtubules \\u2014 preferentially initiating on \\u03b1-tubulin and constraining TTLL7 elongation \\u2014 with consequences for kinesin behavior.\",\n      \"evidence\": \"In vitro glutamylation with purified recombinant TTLL4, tandem MS site mapping, and kinesin motility assays\",\n      \"pmids\": [\"37321451\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo significance of the TTLL4/TTLL7 division of labor not tested\", \"Effect on other microtubule motors beyond kinesin unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended the Klf4 stabilization paradigm to oncogenic NPM1c: TTLL4 monoglutamylation at E126 maintains cytoplasmic NPM1c and a differentiation block, nominating TTLL4 as an AML target druggable by EN7.\",\n      \"evidence\": \"Multiple genetic TTLL4 inactivation approaches in NPM1c cell lines, Ttll4 KO NPM1c/NRAS mouse AML model, transcriptomics, and EN7 inhibitor (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.04.07.647605\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"EN7 selectivity and off-target profile not fully established\", \"Mechanism by which E126 glutamylation enforces cytoplasmic retention not resolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Confirmed in human cells that TTLL4 is a chain-initiator whose overexpression raises glutamylation without disrupting microtubule dynamics, and is pharmacologically blockable by LDC10.\",\n      \"evidence\": \"TTLL4 overexpression in HEK293T with EB3 live imaging, glutamylation immunofluorescence, and LDC10 inhibitor treatment\",\n      \"pmids\": [\"41770829\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; endogenous TTLL4 not tested\", \"Inhibitor specificity across the TTLL family not characterized\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TTLL4 selects among its many substrates (tubulin, cGAS, Klf4, NPM1c, IL-7R\\u03b1, NAP1, Npm2) in a given cellular context, and how upstream signaling reroutes this selectivity, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of TTLL4 bound to a non-tubulin substrate\", \"Determinants of substrate choice beyond glutamate-rich stretches unknown\", \"Cell-type-specific regulation of substrate repertoire undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 9, 13, 14, 16]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 4, 6, 7, 14, 15]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [9, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [5, 13, 16]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [1, 3, 8]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [4, 6]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 7, 15]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [2, 14]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [12, 15]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NEK5\", \"cGAS\", \"Klf4\", \"IL7R\", \"NAP1\", \"PELP1\", \"NPM1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}