{"gene":"TES","run_date":"2026-06-10T10:51:54","timeline":{"discoveries":[{"year":2003,"finding":"TES protein localizes to focal adhesions, actin stress fibres, and areas of cell-cell contact; its three C-terminal LIM domains are important for focal adhesion targeting, while the N-terminal region targets TES to actin stress fibres. Yeast two-hybrid and biochemical analyses identified interactions with mena, zyxin, and talin. Fibroblasts stably overexpressing TES showed increased spreading on fibronectin.","method":"Immunofluorescence localization, yeast two-hybrid, biochemical pull-down, stable overexpression with cell spreading assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (localization, Y2H, biochemical pulldown, functional overexpression assay), replicated across subsequent studies","pmids":["12571287"],"is_preprint":false},{"year":2003,"finding":"Tes is a focal adhesion component that negatively regulates proliferation of T47D breast carcinoma cells. In vivo co-immunoprecipitation demonstrated Tes is complexed with actin, Mena, and VASP. The isolated N-terminal half of Tes pulls down alpha-actinin and paxillin from cell extracts, while the C-terminal half recruits zyxin, Mena, and VASP. The two halves of Tes interact with each other in vitro and in vivo, suggesting a conformational (open/closed) state. Using RNAi ablation, zyxin is required to recruit Tes (as well as Mena and VASP) to focal adhesions, but not vinculin or paxillin; fibroblasts lacking Mena and VASP still recruit Tes to focal adhesions.","method":"Co-immunoprecipitation, GST pull-down from cell extracts, RNAi knockdown, fibroblasts lacking Mena/VASP (genetic), yeast two-hybrid","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, multiple orthogonal methods (pulldown, RNAi, genetic cell lines), replicated by subsequent studies","pmids":["12695497"],"is_preprint":false},{"year":2005,"finding":"RNAi-mediated knockdown of TES in HeLa cells results in loss of actin stress fibres and reduced RhoA activity, indicating TES is required for RhoA-dependent stress fibre maintenance. A hierarchy of recruitment was established: zyxin is recruited first, followed by VASP, then TES.","method":"RNAi knockdown, immunofluorescence, RhoA activity assay","journal":"Cell motility and the cytoskeleton","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi knockdown with defined cellular phenotype and pathway placement (RhoA), single lab, two orthogonal readouts","pmids":["15662727"],"is_preprint":false},{"year":2007,"finding":"The LIM3 domain of Tes binds to the EVH1 domain of Mena (but not VASP or Evl) by occluding the canonical FPPPP-binding site, despite lacking an FPPPP motif. Crystal structure of the LIM3:EVH1 complex was determined. Structure-based gain-of-function mutations defined molecular basis for Tes-Mena specificity. The LIM3 domain displaces Mena, but not VASP, from the leading edge and focal adhesions, and regulates cell migration through a Mena-dependent mechanism.","method":"Crystal structure determination, in vitro binding assay, gain-of-function mutagenesis, cell biology (Mena displacement, migration assay)","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation by mutagenesis and cell-based assays, multiple orthogonal methods","pmids":["18158903"],"is_preprint":false},{"year":2011,"finding":"Arp7A forms a complex with Tes and Mena in the subacrosomal layer of round spermatids. The crystal structure of the Arp7A(1-65)·LIM2-3(Tes)·EVH1(Mena) complex revealed that residues 28-49 of Arp7A contact LIM2-3 domains of Tes; two alanine residues occupy apolar pockets in both LIM domains and a GPAK linker binds the LIM2-3 junction, which are critical for the Arp7A-Tes interaction.","method":"Crystal structure, in vitro binding assay, alanine mutagenesis, co-immunoprecipitation, localization in spermatids","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with mutagenesis validation and in vivo localization, multiple orthogonal methods","pmids":["21278383"],"is_preprint":false},{"year":2005,"finding":"AlphaII-spectrin interacts with Tes; the interaction is mediated by the LIM domain of Tes and the alpha10 repeat of alphaII-spectrin, as shown by yeast two-hybrid screening and co-immunoprecipitation. An in vitro interaction between Tes and EVL was also identified, and both proteins co-localize at focal adhesions.","method":"Yeast two-hybrid screening, co-immunoprecipitation, in vitro binding, co-localization by immunofluorescence","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid confirmed by Co-IP, single lab, two orthogonal methods","pmids":["15656790"],"is_preprint":false},{"year":2008,"finding":"TES exists in different conformational states in different cellular compartments. The N-terminus of TES interacts with its third LIM domain (C-terminus) as demonstrated by GST pull-down, suggesting a 'closed' conformation. TES co-localizes with nucleolar marker B23 and co-immunoprecipitates with B23, indicating TES is also present in the nucleolus in a 'closed' conformational state.","method":"GST pull-down, co-immunoprecipitation, immunofluorescence co-localization with B23","journal":"Molecular and cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and pulldown, single lab, two orthogonal methods but limited functional follow-up","pmids":["18696217"],"is_preprint":false},{"year":2011,"finding":"Testin and actin-myosin are molecular targets of adjudin (an anti-spermatogenic agent) at the apical ectoplasmic specialization. Co-immunoprecipitation with an adjudin-specific antibody on affinity resin, combined with mass spectrometry and immunoblotting, identified testin as a direct binding partner of adjudin at the apical ES.","method":"Co-immunoprecipitation, mass spectrometry, immunoblotting","journal":"Spermatogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with MS confirmation, single lab, two orthogonal methods","pmids":["22319662"],"is_preprint":false},{"year":2011,"finding":"Testin interacts with the calcium-sensing receptor (CaR) intracellular tail; the interaction was identified by yeast two-hybrid screening and confirmed by co-immunoprecipitation in HEK293 cells. The interaction is mapped to the membrane proximal region of the CaR tail and the second zinc-finger of LIM domain 1 of testin. Ectopic expression of testin in HEK293 cells stably expressing CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling.","method":"Yeast two-hybrid, co-immunoprecipitation, confocal microscopy, Rho activity assay (functional ectopic expression)","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid confirmed by Co-IP and functional assay, single lab, multiple methods","pmids":["21843504"],"is_preprint":false},{"year":2015,"finding":"TES (along with VASP and zyxin) is a mechanosensitive component of Focal Adherens Junctions (FAJ) at cadherin-based adhesions. TES recruitment to FAJ is tension-dependent. Its recruitment is independent of the alpha-catenin/vinculin module. Structured Illumination Microscopy showed TES concentrates at locations distinct from the core cadherin complex. VASP and zyxin require binding to each other to localize to FAJs.","method":"Live imaging, Structured Illumination Microscopy, tension manipulation, fluorescence localization of mutants","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization with tension manipulation and SIM imaging, single lab, two orthogonal methods","pmids":["26611125"],"is_preprint":false},{"year":2015,"finding":"The Tes-binding site in zyxin was mapped to four highly conserved amino acids using pull-down assays and ectopic recruitment experiments. Zyxin recruits Tes to focal adhesions and modulates its turnover (shown by FRAP). Loss of zyxin-Tes interaction affects cell spreading, but zyxin regulates focal adhesion dynamics independent of Tes.","method":"Pull-down assay, ectopic recruitment experiments, FRAP, cell spreading quantification","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (pulldown, FRAP, functional assay), single lab","pmids":["26509500"],"is_preprint":false},{"year":2017,"finding":"The PET domain region of testin (amino acids 52-233) interacts with the C-terminal LIM1-2 domains in vitro and in cells. Testin can form an antiparallel homodimer. Tyrosine 288 has a critical role in the PET-LIM1-2 interaction.","method":"In vitro binding assay, cellular interaction assay, site-directed mutagenesis (Y288)","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and cellular assays with mutagenesis, single lab","pmids":["28542564"],"is_preprint":false},{"year":2021,"finding":"The full-length testin protein appears diffuse in the cytoplasm, while its C-terminal LIM domains alone recognize focal adhesions and strained actin, and the N-terminal domains alone recognize stress fibres. Phosphorylation mutations in the dimerization regions reveal testin's mechanosensitivity, causing relocation to focal adhesions and strained actin. Activated RhoA causes testin to relocate to stress fibres and become mechanosensitive.","method":"Fluorescence imaging of domain truncations/mutants, phosphorylation mutagenesis, RhoA activation (constitutively active RhoA expression)","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional domain dissection with mutagenesis and live imaging, single lab","pmids":["34038160"],"is_preprint":false},{"year":2013,"finding":"Testin (TES-1) and ZYX-1/Zyxin in C. elegans are recruited to apical junctions during embryonic elongation when junctions are under tension. In genetic backgrounds where elongation fails, junctional recruitment is severely compromised. Loss of tes-1 or zyx-1 results in junctional F-actin defects. Loss of either protein strongly enhances morphogenetic defects in hypomorphic cadherin/catenin complex mutants, establishing genetic interaction and epistasis.","method":"Genetic loss-of-function (mutant analysis), fluorescence imaging, genetic epistasis (double mutant analysis with cadherin/catenin complex hypomorphs)","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with defined phenotypic readout (junctional F-actin), replicated in C. elegans ortholog","pmids":["36384139"],"is_preprint":false},{"year":2006,"finding":"Depletion of Xenopus Tes (Xtes) leads to foreshortened head and severe defects in axis elongation. The anterior defect is due in part to inhibition of cranial neural crest migration. Simultaneous depletion of Xtes and Xenopus Prickle results in axial defects more severe than either alone, suggesting the two proteins act together to control axial elongation (genetic interaction).","method":"Morpholino-mediated knockdown in Xenopus embryos, phenotypic analysis, double knockdown epistasis","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with specific phenotypic readouts and genetic interaction, single lab","pmids":["16554046"],"is_preprint":false},{"year":2013,"finding":"Testin was identified as a Vangl2-interacting protein by yeast two-hybrid screen with cochlea cDNA library. Testin is enriched to cell-cell boundaries in the presence of Vangl2. Genetic inactivation of Testin leads to abnormal hair cell orientation in vestibule and cochlea. Testin genetically interacts with Vangl2 to regulate hair cell orientation in the cochlea and opening of the vaginal tract (double mutant analysis).","method":"Yeast two-hybrid, cell localization assay (Vangl2 co-expression), knockout mouse analysis, genetic epistasis (Testin/Vangl2 double mutants)","journal":"Developmental dynamics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid interaction confirmed by localization and genetic epistasis in mouse KO, single lab","pmids":["23996638"],"is_preprint":false},{"year":2005,"finding":"Adenoviral re-expression of TES in TES-negative breast cancer (T47D) and uterine sarcoma (MES-SA) cell lines induced apoptosis via caspase-dependent and caspase-independent mechanisms, respectively, and significantly reduced tumorigenic potential in nude mice. TES-positive MCF-7 cells were unaffected by Ad-TES infection.","method":"Adenoviral gene transfer, apoptosis assay (caspase activity), xenograft tumor assay in nude mice","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function (TES-negative lines) and gain-of-function with in vivo tumor assay, single lab","pmids":["15701871"],"is_preprint":false},{"year":2005,"finding":"Tes knockout mice show significantly increased susceptibility to NMBA-induced gastric carcinogenesis compared to wild-type mice, with 88% of heterozygous and 81% of homozygous knockout mice developing papillomas, atypical glandular metaplasia, and carcinomas vs. 25% benign lesions in wild-type, demonstrating in vivo tumor suppressor function.","method":"Knockout mouse model, carcinogen-induced tumor assay (NMBA + zinc-deficient diet), histopathological analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetically defined KO mouse with rigorous in vivo carcinogenesis assay and statistically significant dose-dependent phenotype","pmids":["16033868"],"is_preprint":false},{"year":2019,"finding":"TES interacts with Mena (identified by immunoprecipitation-based mass spectrometry) and inhibits the interaction of Mena with Lamellipodin (Lpd). TES suppresses migration and invasion of gastric cancer cells in a Mena-dependent manner (Transwell assays with Mena manipulation).","method":"Immunoprecipitation-mass spectrometry, co-immunoprecipitation, Transwell migration/invasion assay with Mena manipulation","journal":"Cancer communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP/MS identification with functional validation by Mena-dependent migration assay, single lab","pmids":["30728082"],"is_preprint":false},{"year":2018,"finding":"TES and filamin-C (FLN-C) interact with the E3 ubiquitin ligase Siah2 and are proteasomally degraded. K139-acetylated Siah2 (induced by H. pylori infection) significantly potentiates TES and FLN-C degradation. Acetylated Siah2-mediated downregulation of TES disrupts filopodia but promotes lamellipodia formation and enhances invasiveness of gastric cancer cells.","method":"Co-immunoprecipitation, proteasome inhibition assay, site-specific acetylation analysis, cell invasion/migration assay","journal":"The international journal of biochemistry & cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with functional consequence (proteasomal degradation, invasion assay), single lab","pmids":["30063986"],"is_preprint":false},{"year":2020,"finding":"Testin RNAi knockdown in Sertoli cells causes damage to the Sertoli cell barrier, causes diffusion of tight junction proteins occludin and ZO-1 away from the cell surface into the cytoplasm, reduces ZO-1/occludin association (shown by Co-IP), disrupts actin filament bundle arrangement, and reduces F-actin/G-actin ratio. ARP3 redistributes to the Sertoli cell interface after testin RNAi.","method":"RNAi knockdown, Co-immunoprecipitation, co-immunofluorescence, F-actin/G-actin ratio assay, barrier function assay","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi with multiple orthogonal readouts (Co-IP, immunofluorescence, actin assay), single lab","pmids":["31975378"],"is_preprint":false},{"year":2018,"finding":"TES directly interacts with calcineurin (shown by co-immunoprecipitation) and suppresses calcineurin downstream signalling. Adeno-associated virus-driven cardiac TES overexpression attenuated ventricular dilation, cardiac hypertrophy, dysfunction, and fibrosis induced by aortic banding in mice. TES knockdown exacerbated hypertrophy; cyclosporin A (calcineurin inhibitor) reversed this, confirming calcineurin-dependence.","method":"Co-immunoprecipitation, adeno-associated viral overexpression, adenoviral shRNA knockdown, aortic banding mouse model, pharmacological rescue with cyclosporin A","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP interaction with pharmacological and genetic rescue in vivo, single lab","pmids":["30467953"],"is_preprint":false},{"year":2021,"finding":"Testin (encoded by TES) interacts with KCNE2 potassium channel β-subunit (identified by yeast two-hybrid and confirmed by co-immunoprecipitation). Ectopic expression of Testin nullifies KCNE2 effects on Kv1.5 voltage dependence and gating kinetics, but does not prevent KCNE2 regulation of KCNQ1. Testin alone did not alter Kv1.5 function.","method":"Yeast two-hybrid, co-immunoprecipitation, whole-cell patch clamp electrophysiology","journal":"Channels (Austin, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid confirmed by Co-IP and functional electrophysiology, single lab","pmids":["33464998"],"is_preprint":false},{"year":2016,"finding":"TES overexpression in colorectal cancer cells inhibits cell proliferation, migration, and invasion while increasing apoptosis, and activates p38 MAPK signalling (upregulation of pro-apoptotic proteins, downregulation of anti-apoptotic proteins). TES knockdown by shRNA elicited opposite effects.","method":"Overexpression and shRNA knockdown, proliferation/migration/invasion assays, apoptosis assay, p38 MAPK pathway analysis by western blot","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — bidirectional manipulation (OE and KD) with pathway analysis, single lab","pmids":["27323777"],"is_preprint":false},{"year":2024,"finding":"Elevated Testin expression in colonic epithelial cells inactivates the JNK/P38 signalling pathway, preventing depletion of tight junction proteins ZO-1 and Claudin-1 and protecting intestinal barrier integrity in a Crohn's disease mouse model. Testin overexpression ameliorated colitis symptoms and reduced inflammatory cytokines; effects were demonstrated in colonic organoids treated with LPS.","method":"Adenoviral overexpression in vivo (TNBS colitis model), colonic organoids, western blotting of TJ proteins and JNK/P38 pathway, barrier permeability assay","journal":"Chemico-biological interactions","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo gain-of-function with organoid validation and defined signalling pathway, single lab","pmids":["39237074"],"is_preprint":false},{"year":2004,"finding":"Chicken TES (cTES) localizes to focal adhesions, actin stress fibres, and cell-cell contact sites; GST-cTES pulls down zyxin and actin. Overexpression of cTES increases cell spreading on fibronectin and decreases cell motility.","method":"Immunofluorescence localization, GST pulldown, overexpression with cell spreading and motility assay","journal":"Cell motility and the cytoskeleton","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — pulldown and functional overexpression, replicated findings from human TES study","pmids":["14743347"],"is_preprint":false},{"year":2016,"finding":"Re-expression of TESTIN protein in ALL cell lines causes rapid cell death or cell cycle arrest independent of TP53 activity, as shown by transfection of TES expression plasmids followed by cell proliferation, cell death, and cell cycle assays.","method":"Plasmid transfection (TES re-expression), cell death and cell cycle assays, TP53-independence established using TP53-null lines","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function with cell cycle and death assays, TP53-independence tested, single lab","pmids":["26985820"],"is_preprint":false}],"current_model":"TES/Testin is a multidomain LIM-domain scaffold protein (PET domain + 3 C-terminal LIM domains) that localizes to focal adhesions, actin stress fibres, and cell-cell junctions via its LIM domains, adopts open and closed (intramolecular) conformations that regulate its interactions, recruits to focal adhesions in a zyxin-dependent and tension-sensitive manner, directly binds cytoskeletal partners including Mena (via an atypical LIM3–EVH1 interaction that competes with FPPPP-motif proteins), zyxin, VASP, talin, alpha-actinin, paxillin, actin, and alphaII-spectrin, regulates actin stress fibre integrity through RhoA signalling, suppresses cell migration and invasion partly by blocking Mena–Lamellipodin interaction, acts as an in vivo tumour suppressor (knockout mice show enhanced carcinogenesis), and also modulates specific signalling pathways including calcineurin/NFAT in cardiac hypertrophy, p38 MAPK in colorectal cancer, and JNK/P38 in intestinal barrier protection."},"narrative":{"mechanistic_narrative":"TES (Testin) is a multidomain LIM-domain scaffold protein that organizes the actin cytoskeleton at focal adhesions, stress fibres, and cell-cell junctions, where it acts as a mechanosensitive adaptor and tumour suppressor [PMID:12571287, PMID:12695497, PMID:16033868]. Its three C-terminal LIM domains target focal adhesions and strained actin while its N-terminal PET-containing region directs it to stress fibres; an intramolecular interaction between the N-terminus and LIM3 (involving Tyr288 and dimerization-region phosphorylation) holds TES in a 'closed' conformation that gates its localization and partner binding, with active RhoA driving relocation to stress fibres and conferring mechanosensitivity [PMID:12571287, PMID:18696217, PMID:28542564, PMID:34038160]. TES is recruited to focal adhesions and tension-bearing cadherin-based junctions in a zyxin-dependent, tension-sensitive hierarchy (zyxin → VASP → TES), and it is required for RhoA-dependent stress fibre integrity [PMID:12695497, PMID:15662727, PMID:26611125, PMID:26509500]. Through an atypical LIM3–EVH1 interaction that occludes the canonical FPPPP-binding site, TES binds Mena (but not VASP or Evl) and displaces Mena from the leading edge, blocking the Mena–Lamellipodin interaction to suppress cell migration and invasion [PMID:18158903, PMID:30728082]. TES additionally engages cytoskeletal and junctional partners including alphaII-spectrin, actin, alpha-actinin, paxillin, and Arp7A [PMID:12695497, PMID:21278383, PMID:15656790]. As an in vivo tumour suppressor, Tes loss increases carcinogen-induced gastric tumorigenesis in knockout mice, while re-expression of TES in TES-negative carcinoma and leukemia cells induces apoptosis or cell-cycle arrest and reduces tumorigenicity [PMID:15701871, PMID:16033868, PMID:26985820]; TES protein levels are themselves downregulated via Siah2-mediated proteasomal degradation potentiated by H. pylori-induced acetylation [PMID:30063986]. Beyond cytoskeletal control, TES interacts with diverse signalling components—calcineurin (suppressing NFAT signalling in cardiac hypertrophy), the calcium-sensing receptor, and KCNE2—and modulates p38 MAPK and JNK/p38 pathways governing colorectal cancer cell fate and intestinal/Sertoli barrier integrity [PMID:30467953, PMID:21843504, PMID:33464998, PMID:27323777, PMID:39237074, PMID:31975378].","teleology":[{"year":2003,"claim":"Established TES as a focal-adhesion and cytoskeletal protein with a modular domain logic, answering where it localizes and which partners it uses, defining it as a scaffold rather than an enzyme.","evidence":"Immunofluorescence, yeast two-hybrid, biochemical pull-down, and overexpression spreading assays in fibroblasts and breast carcinoma cells","pmids":["12571287","12695497"],"confidence":"High","gaps":["Stoichiometry and order of multi-partner assembly not resolved","Functional consequence of each individual interaction not separated"]},{"year":2005,"claim":"Defined a recruitment hierarchy and pathway placement by showing TES is required for RhoA-dependent stress fibre maintenance and is recruited downstream of zyxin and VASP.","evidence":"RNAi knockdown in HeLa cells with RhoA activity assay and immunofluorescence; additional Y2H/Co-IP identifying alphaII-spectrin and EVL partners","pmids":["15662727","15656790"],"confidence":"Medium","gaps":["Mechanism by which TES feeds back on RhoA activity not defined","Single-lab knockdown phenotype"]},{"year":2005,"claim":"Demonstrated TES tumour-suppressor function in vivo and in cancer cell lines, establishing biological significance of its loss.","evidence":"Tes knockout mice with carcinogen-induced gastric tumorigenesis; adenoviral re-expression in TES-negative carcinoma lines with apoptosis and xenograft assays","pmids":["16033868","15701871"],"confidence":"High","gaps":["Molecular pathway linking TES loss to tumorigenesis not defined in these studies","Tissue specificity of tumour suppression unclear"]},{"year":2008,"claim":"Provided structural-mechanistic basis for TES specificity by solving how its LIM3 domain captures the Mena EVH1 domain through an atypical, FPPPP-independent mode that selectively excludes VASP/Evl, linking the interaction to migration control.","evidence":"Crystal structure of LIM3:EVH1 complex with structure-based gain-of-function mutagenesis and Mena-displacement/migration assays; subsequent structure of the Arp7A·LIM2-3·Mena ternary complex","pmids":["18158903","21278383"],"confidence":"High","gaps":["In vivo relevance of LIM3-Mena competition in tissue contexts not fully established","How conformational gating regulates EVH1 availability not structurally resolved"]},{"year":2008,"claim":"Defined the open/closed conformational switch by mapping an N-terminus–LIM3 intramolecular interaction and showing compartment-specific conformational states, including unexpected nucleolar localization.","evidence":"GST pull-down, Co-IP, and B23 co-localization; later PET–LIM1-2 mapping with Tyr288 mutagenesis and homodimer detection","pmids":["18696217","28542564"],"confidence":"Medium","gaps":["Functional role of nucleolar TES unknown","Physiological trigger toggling open/closed states not defined"]},{"year":2015,"claim":"Established TES as a tension-sensitive mechanosensor by mapping its zyxin-binding determinants and showing tension-dependent recruitment to cadherin-based junctions distinct from the core cadherin complex.","evidence":"Pull-down/FRAP zyxin-binding mapping plus SIM imaging and tension manipulation at focal adherens junctions","pmids":["26509500","26611125"],"confidence":"Medium","gaps":["Direct force-sensing element within TES not identified","Single-lab imaging studies"]},{"year":2021,"claim":"Resolved the mechanosensitive behaviour of TES at the domain level, showing conformation and phosphorylation control relocation to strained actin and that RhoA activation drives mechanosensitivity.","evidence":"Fluorescence imaging of domain truncations and phosphorylation mutants with constitutively active RhoA expression","pmids":["34038160"],"confidence":"Medium","gaps":["Kinases responsible for the relevant phosphorylation not identified","Quantitative force thresholds not defined"]},{"year":2013,"claim":"Demonstrated conserved developmental roles in tension-dependent junctional actin organization and planar polarity through orthologs and genetic interactions.","evidence":"C. elegans tes-1/zyx-1 loss-of-function and epistasis with cadherin/catenin; Xenopus Xtes morpholino knockdown with Prickle interaction; mouse Testin/Vangl2 knockout epistasis","pmids":["36384139","16554046","23996638"],"confidence":"Medium","gaps":["Molecular link between TES and PCP machinery not defined","Whether developmental and adhesion roles share a mechanism unresolved"]},{"year":2019,"claim":"Connected TES tumour suppression to a defined molecular mechanism—blocking Mena–Lamellipodin—and to its regulated turnover by an acetylated Siah2 E3 ligase in gastric cancer.","evidence":"IP-MS, Co-IP, Mena-dependent Transwell assays; Siah2 Co-IP with proteasome inhibition and invasion assays under H. pylori-induced acetylation","pmids":["30728082","30063986"],"confidence":"Medium","gaps":["Whether Mena-Lpd blockade accounts for in vivo tumour suppression not shown","Single-lab mechanistic studies"]},{"year":2024,"claim":"Extended TES function to signalling modulation across tissues, implicating it in calcineurin/NFAT-dependent cardiac hypertrophy, p38 MAPK-driven apoptosis in colorectal cancer, and JNK/p38-dependent epithelial barrier protection.","evidence":"Co-IP plus in vivo overexpression/knockdown with pharmacological rescue (cardiac); bidirectional manipulation with pathway western blots (colorectal); in vivo overexpression and organoids (intestinal barrier); RNAi barrier disruption in Sertoli cells; CaR and KCNE2 interaction with functional electrophysiology","pmids":["30467953","27323777","39237074","31975378","21843504","33464998"],"confidence":"Medium","gaps":["How a cytoskeletal scaffold mechanistically tunes calcineurin, p38, and JNK signalling not resolved","Whether these signalling roles reflect direct or cytoskeleton-mediated effects unclear"]},{"year":null,"claim":"It remains unresolved how the conformational/mechanosensitive switch of TES is integrated with its diverse signalling outputs (calcineurin, p38/JNK, ion-channel regulation) to produce tissue-specific tumour-suppressor and barrier functions.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying mechanism linking mechanosensing to signalling modulation","No structural model of full-length TES in its active state","Endogenous upstream regulators of conformational switching unidentified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,3]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,5]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[9,12]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,18,21]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,2,12]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1,9]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[12]},{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,8,21]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[16,17,18,19]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[16,23,26]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[13,14,15]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[9,13]}],"complexes":["focal adhesion","focal adherens junction"],"partners":["ENAH","ZYX","VASP","TLN1","ACTN1","SPTAN1","PXN","SIAH2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UGI8","full_name":"Testin","aliases":["TESS"],"length_aa":421,"mass_kda":48.0,"function":"Scaffold protein that may play a role in cell adhesion, cell spreading and in the reorganization of the actin cytoskeleton. Plays a role in the regulation of cell proliferation. May act as a tumor suppressor. 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genetics","url":"https://pubmed.ncbi.nlm.nih.gov/28146551","citation_count":10,"is_preprint":false},{"pmid":"32862383","id":"PMC_32862383","title":"Cancer-testis Antigen OY-TES-1 Expression and Immunogenicity in Hepatocellular Carcinoma.","date":"2020","source":"Current medical science","url":"https://pubmed.ncbi.nlm.nih.gov/32862383","citation_count":9,"is_preprint":false},{"pmid":"35743291","id":"PMC_35743291","title":"Safety of Special Waveform of Transcranial Electrical Stimulation (TES): In Vivo Assessment.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35743291","citation_count":9,"is_preprint":false},{"pmid":"12606342","id":"PMC_12606342","title":"Mouse testin: complementary DNA cloning, genomic organization, and characterization of its proximal promoter region.","date":"2002","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/12606342","citation_count":8,"is_preprint":false},{"pmid":"14579264","id":"PMC_14579264","title":"Identification of a CTL-directed epitope encoded by an intron of the putative tumor suppressor gene Testin of the common fragile site 7G region: a peptide vaccine candidate for HLA-B52+ and HLA-62+ cancer patients.","date":"2003","source":"European journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/14579264","citation_count":8,"is_preprint":false},{"pmid":"34686213","id":"PMC_34686213","title":"Differential regulation of transposable elements (TEs) during the murine submandibular gland development.","date":"2021","source":"Mobile DNA","url":"https://pubmed.ncbi.nlm.nih.gov/34686213","citation_count":8,"is_preprint":false},{"pmid":"27622437","id":"PMC_27622437","title":"Production and evaluation of the recombinant antigen TES-30 of Toxocara canis for the immunodiagnosis of toxocariasis.","date":"2016","source":"Biomedica : revista del Instituto Nacional de Salud","url":"https://pubmed.ncbi.nlm.nih.gov/27622437","citation_count":8,"is_preprint":false},{"pmid":"29425867","id":"PMC_29425867","title":"The effects of biological buffers TRIS, TAPS, TES on the stability of lysozyme.","date":"2018","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/29425867","citation_count":8,"is_preprint":false},{"pmid":"32170669","id":"PMC_32170669","title":"Chitinase 3-Like 1, Nestin, and Testin Proteins as Novel Biomarkers of Potential Clinical Use in Colorectal Cancer: A Review.","date":"2020","source":"Advances in experimental medicine and biology","url":"https://pubmed.ncbi.nlm.nih.gov/32170669","citation_count":7,"is_preprint":false},{"pmid":"36384139","id":"PMC_36384139","title":"TES-1/Tes and ZYX-1/Zyxin protect junctional actin networks under tension during epidermal morphogenesis in the C. elegans embryo.","date":"2022","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/36384139","citation_count":7,"is_preprint":false},{"pmid":"28542564","id":"PMC_28542564","title":"The PET and LIM1-2 domains of testin contribute to intramolecular and homodimeric interactions.","date":"2017","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/28542564","citation_count":7,"is_preprint":false},{"pmid":"25119600","id":"PMC_25119600","title":"TES was epigenetically silenced and suppressed the epithelial-mesenchymal transition in breast cancer.","date":"2014","source":"Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine","url":"https://pubmed.ncbi.nlm.nih.gov/25119600","citation_count":7,"is_preprint":false},{"pmid":"33464998","id":"PMC_33464998","title":"The focal adhesion protein Testin modulates KCNE2 potassium channel β subunit activity.","date":"2021","source":"Channels (Austin, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/33464998","citation_count":6,"is_preprint":false},{"pmid":"30467953","id":"PMC_30467953","title":"Testin protects against cardiac hypertrophy by targeting a calcineurin-dependent signalling pathway.","date":"2018","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/30467953","citation_count":6,"is_preprint":false},{"pmid":"30870519","id":"PMC_30870519","title":"The serodiagnostic potential of recombinant proteins TES-30 and TES-120 in an indirect ELISA in the diagnosis of toxocariasis in cattle, horses, and sheep.","date":"2019","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/30870519","citation_count":5,"is_preprint":false},{"pmid":"26509500","id":"PMC_26509500","title":"Delineating the Tes Interaction Site in Zyxin and Studying Cellular Effects of Its Disruption.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26509500","citation_count":5,"is_preprint":false},{"pmid":"16554046","id":"PMC_16554046","title":"Tes regulates neural crest migration and axial elongation in Xenopus.","date":"2006","source":"Developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/16554046","citation_count":5,"is_preprint":false},{"pmid":"35463964","id":"PMC_35463964","title":"Structural Basis of Human Dimeric α-Amino-β-Carboxymuconate-ε-Semialdehyde Decarboxylase Inhibition With TES-1025.","date":"2022","source":"Frontiers in molecular biosciences","url":"https://pubmed.ncbi.nlm.nih.gov/35463964","citation_count":5,"is_preprint":false},{"pmid":"29938803","id":"PMC_29938803","title":"Reactivity of recombinant Toxocara canis TES-30/120 in experimentally infected mice.","date":"2018","source":"Parasite immunology","url":"https://pubmed.ncbi.nlm.nih.gov/29938803","citation_count":5,"is_preprint":false},{"pmid":"36299519","id":"PMC_36299519","title":"Evaluation of new Toxocara canis chimeric antigens as an alternative to conventional TES-Ag for anti-Toxocara antibodies detection.","date":"2022","source":"Heliyon","url":"https://pubmed.ncbi.nlm.nih.gov/36299519","citation_count":5,"is_preprint":false},{"pmid":"39237074","id":"PMC_39237074","title":"The JNK/P38 signalling pathway activated by testin protects the intestinal epithelial barrier against Crohn's disease-like colitis.","date":"2024","source":"Chemico-biological interactions","url":"https://pubmed.ncbi.nlm.nih.gov/39237074","citation_count":4,"is_preprint":false},{"pmid":"18799041","id":"PMC_18799041","title":"[Expression and clinical significance of TESTIN in primary gastric cancer].","date":"2008","source":"Ai zheng = Aizheng = Chinese journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/18799041","citation_count":4,"is_preprint":false},{"pmid":"17416990","id":"PMC_17416990","title":"Using RNA interference to knock down the adhesion protein TES.","date":"2007","source":"Methods in molecular biology (Clifton, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/17416990","citation_count":3,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":48713,"output_tokens":6884,"usd":0.1247,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":15887,"output_tokens":4248,"usd":0.092817,"stage2_stop_reason":"end_turn"},"total_usd":0.217517,"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\": 2003,\n      \"finding\": \"TES protein localizes to focal adhesions, actin stress fibres, and areas of cell-cell contact; its three C-terminal LIM domains are important for focal adhesion targeting, while the N-terminal region targets TES to actin stress fibres. Yeast two-hybrid and biochemical analyses identified interactions with mena, zyxin, and talin. Fibroblasts stably overexpressing TES showed increased spreading on fibronectin.\",\n      \"method\": \"Immunofluorescence localization, yeast two-hybrid, biochemical pull-down, stable overexpression with cell spreading assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (localization, Y2H, biochemical pulldown, functional overexpression assay), replicated across subsequent studies\",\n      \"pmids\": [\"12571287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Tes is a focal adhesion component that negatively regulates proliferation of T47D breast carcinoma cells. In vivo co-immunoprecipitation demonstrated Tes is complexed with actin, Mena, and VASP. The isolated N-terminal half of Tes pulls down alpha-actinin and paxillin from cell extracts, while the C-terminal half recruits zyxin, Mena, and VASP. The two halves of Tes interact with each other in vitro and in vivo, suggesting a conformational (open/closed) state. Using RNAi ablation, zyxin is required to recruit Tes (as well as Mena and VASP) to focal adhesions, but not vinculin or paxillin; fibroblasts lacking Mena and VASP still recruit Tes to focal adhesions.\",\n      \"method\": \"Co-immunoprecipitation, GST pull-down from cell extracts, RNAi knockdown, fibroblasts lacking Mena/VASP (genetic), yeast two-hybrid\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, multiple orthogonal methods (pulldown, RNAi, genetic cell lines), replicated by subsequent studies\",\n      \"pmids\": [\"12695497\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"RNAi-mediated knockdown of TES in HeLa cells results in loss of actin stress fibres and reduced RhoA activity, indicating TES is required for RhoA-dependent stress fibre maintenance. A hierarchy of recruitment was established: zyxin is recruited first, followed by VASP, then TES.\",\n      \"method\": \"RNAi knockdown, immunofluorescence, RhoA activity assay\",\n      \"journal\": \"Cell motility and the cytoskeleton\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi knockdown with defined cellular phenotype and pathway placement (RhoA), single lab, two orthogonal readouts\",\n      \"pmids\": [\"15662727\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The LIM3 domain of Tes binds to the EVH1 domain of Mena (but not VASP or Evl) by occluding the canonical FPPPP-binding site, despite lacking an FPPPP motif. Crystal structure of the LIM3:EVH1 complex was determined. Structure-based gain-of-function mutations defined molecular basis for Tes-Mena specificity. The LIM3 domain displaces Mena, but not VASP, from the leading edge and focal adhesions, and regulates cell migration through a Mena-dependent mechanism.\",\n      \"method\": \"Crystal structure determination, in vitro binding assay, gain-of-function mutagenesis, cell biology (Mena displacement, migration assay)\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation by mutagenesis and cell-based assays, multiple orthogonal methods\",\n      \"pmids\": [\"18158903\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Arp7A forms a complex with Tes and Mena in the subacrosomal layer of round spermatids. The crystal structure of the Arp7A(1-65)·LIM2-3(Tes)·EVH1(Mena) complex revealed that residues 28-49 of Arp7A contact LIM2-3 domains of Tes; two alanine residues occupy apolar pockets in both LIM domains and a GPAK linker binds the LIM2-3 junction, which are critical for the Arp7A-Tes interaction.\",\n      \"method\": \"Crystal structure, in vitro binding assay, alanine mutagenesis, co-immunoprecipitation, localization in spermatids\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with mutagenesis validation and in vivo localization, multiple orthogonal methods\",\n      \"pmids\": [\"21278383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"AlphaII-spectrin interacts with Tes; the interaction is mediated by the LIM domain of Tes and the alpha10 repeat of alphaII-spectrin, as shown by yeast two-hybrid screening and co-immunoprecipitation. An in vitro interaction between Tes and EVL was also identified, and both proteins co-localize at focal adhesions.\",\n      \"method\": \"Yeast two-hybrid screening, co-immunoprecipitation, in vitro binding, co-localization by immunofluorescence\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid confirmed by Co-IP, single lab, two orthogonal methods\",\n      \"pmids\": [\"15656790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TES exists in different conformational states in different cellular compartments. The N-terminus of TES interacts with its third LIM domain (C-terminus) as demonstrated by GST pull-down, suggesting a 'closed' conformation. TES co-localizes with nucleolar marker B23 and co-immunoprecipitates with B23, indicating TES is also present in the nucleolus in a 'closed' conformational state.\",\n      \"method\": \"GST pull-down, co-immunoprecipitation, immunofluorescence co-localization with B23\",\n      \"journal\": \"Molecular and cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and pulldown, single lab, two orthogonal methods but limited functional follow-up\",\n      \"pmids\": [\"18696217\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Testin and actin-myosin are molecular targets of adjudin (an anti-spermatogenic agent) at the apical ectoplasmic specialization. Co-immunoprecipitation with an adjudin-specific antibody on affinity resin, combined with mass spectrometry and immunoblotting, identified testin as a direct binding partner of adjudin at the apical ES.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, immunoblotting\",\n      \"journal\": \"Spermatogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with MS confirmation, single lab, two orthogonal methods\",\n      \"pmids\": [\"22319662\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Testin interacts with the calcium-sensing receptor (CaR) intracellular tail; the interaction was identified by yeast two-hybrid screening and confirmed by co-immunoprecipitation in HEK293 cells. The interaction is mapped to the membrane proximal region of the CaR tail and the second zinc-finger of LIM domain 1 of testin. Ectopic expression of testin in HEK293 cells stably expressing CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, confocal microscopy, Rho activity assay (functional ectopic expression)\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid confirmed by Co-IP and functional assay, single lab, multiple methods\",\n      \"pmids\": [\"21843504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TES (along with VASP and zyxin) is a mechanosensitive component of Focal Adherens Junctions (FAJ) at cadherin-based adhesions. TES recruitment to FAJ is tension-dependent. Its recruitment is independent of the alpha-catenin/vinculin module. Structured Illumination Microscopy showed TES concentrates at locations distinct from the core cadherin complex. VASP and zyxin require binding to each other to localize to FAJs.\",\n      \"method\": \"Live imaging, Structured Illumination Microscopy, tension manipulation, fluorescence localization of mutants\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization with tension manipulation and SIM imaging, single lab, two orthogonal methods\",\n      \"pmids\": [\"26611125\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The Tes-binding site in zyxin was mapped to four highly conserved amino acids using pull-down assays and ectopic recruitment experiments. Zyxin recruits Tes to focal adhesions and modulates its turnover (shown by FRAP). Loss of zyxin-Tes interaction affects cell spreading, but zyxin regulates focal adhesion dynamics independent of Tes.\",\n      \"method\": \"Pull-down assay, ectopic recruitment experiments, FRAP, cell spreading quantification\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (pulldown, FRAP, functional assay), single lab\",\n      \"pmids\": [\"26509500\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The PET domain region of testin (amino acids 52-233) interacts with the C-terminal LIM1-2 domains in vitro and in cells. Testin can form an antiparallel homodimer. Tyrosine 288 has a critical role in the PET-LIM1-2 interaction.\",\n      \"method\": \"In vitro binding assay, cellular interaction assay, site-directed mutagenesis (Y288)\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and cellular assays with mutagenesis, single lab\",\n      \"pmids\": [\"28542564\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The full-length testin protein appears diffuse in the cytoplasm, while its C-terminal LIM domains alone recognize focal adhesions and strained actin, and the N-terminal domains alone recognize stress fibres. Phosphorylation mutations in the dimerization regions reveal testin's mechanosensitivity, causing relocation to focal adhesions and strained actin. Activated RhoA causes testin to relocate to stress fibres and become mechanosensitive.\",\n      \"method\": \"Fluorescence imaging of domain truncations/mutants, phosphorylation mutagenesis, RhoA activation (constitutively active RhoA expression)\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional domain dissection with mutagenesis and live imaging, single lab\",\n      \"pmids\": [\"34038160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Testin (TES-1) and ZYX-1/Zyxin in C. elegans are recruited to apical junctions during embryonic elongation when junctions are under tension. In genetic backgrounds where elongation fails, junctional recruitment is severely compromised. Loss of tes-1 or zyx-1 results in junctional F-actin defects. Loss of either protein strongly enhances morphogenetic defects in hypomorphic cadherin/catenin complex mutants, establishing genetic interaction and epistasis.\",\n      \"method\": \"Genetic loss-of-function (mutant analysis), fluorescence imaging, genetic epistasis (double mutant analysis with cadherin/catenin complex hypomorphs)\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with defined phenotypic readout (junctional F-actin), replicated in C. elegans ortholog\",\n      \"pmids\": [\"36384139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Depletion of Xenopus Tes (Xtes) leads to foreshortened head and severe defects in axis elongation. The anterior defect is due in part to inhibition of cranial neural crest migration. Simultaneous depletion of Xtes and Xenopus Prickle results in axial defects more severe than either alone, suggesting the two proteins act together to control axial elongation (genetic interaction).\",\n      \"method\": \"Morpholino-mediated knockdown in Xenopus embryos, phenotypic analysis, double knockdown epistasis\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with specific phenotypic readouts and genetic interaction, single lab\",\n      \"pmids\": [\"16554046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Testin was identified as a Vangl2-interacting protein by yeast two-hybrid screen with cochlea cDNA library. Testin is enriched to cell-cell boundaries in the presence of Vangl2. Genetic inactivation of Testin leads to abnormal hair cell orientation in vestibule and cochlea. Testin genetically interacts with Vangl2 to regulate hair cell orientation in the cochlea and opening of the vaginal tract (double mutant analysis).\",\n      \"method\": \"Yeast two-hybrid, cell localization assay (Vangl2 co-expression), knockout mouse analysis, genetic epistasis (Testin/Vangl2 double mutants)\",\n      \"journal\": \"Developmental dynamics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid interaction confirmed by localization and genetic epistasis in mouse KO, single lab\",\n      \"pmids\": [\"23996638\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Adenoviral re-expression of TES in TES-negative breast cancer (T47D) and uterine sarcoma (MES-SA) cell lines induced apoptosis via caspase-dependent and caspase-independent mechanisms, respectively, and significantly reduced tumorigenic potential in nude mice. TES-positive MCF-7 cells were unaffected by Ad-TES infection.\",\n      \"method\": \"Adenoviral gene transfer, apoptosis assay (caspase activity), xenograft tumor assay in nude mice\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function (TES-negative lines) and gain-of-function with in vivo tumor assay, single lab\",\n      \"pmids\": [\"15701871\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Tes knockout mice show significantly increased susceptibility to NMBA-induced gastric carcinogenesis compared to wild-type mice, with 88% of heterozygous and 81% of homozygous knockout mice developing papillomas, atypical glandular metaplasia, and carcinomas vs. 25% benign lesions in wild-type, demonstrating in vivo tumor suppressor function.\",\n      \"method\": \"Knockout mouse model, carcinogen-induced tumor assay (NMBA + zinc-deficient diet), histopathological analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetically defined KO mouse with rigorous in vivo carcinogenesis assay and statistically significant dose-dependent phenotype\",\n      \"pmids\": [\"16033868\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TES interacts with Mena (identified by immunoprecipitation-based mass spectrometry) and inhibits the interaction of Mena with Lamellipodin (Lpd). TES suppresses migration and invasion of gastric cancer cells in a Mena-dependent manner (Transwell assays with Mena manipulation).\",\n      \"method\": \"Immunoprecipitation-mass spectrometry, co-immunoprecipitation, Transwell migration/invasion assay with Mena manipulation\",\n      \"journal\": \"Cancer communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP/MS identification with functional validation by Mena-dependent migration assay, single lab\",\n      \"pmids\": [\"30728082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TES and filamin-C (FLN-C) interact with the E3 ubiquitin ligase Siah2 and are proteasomally degraded. K139-acetylated Siah2 (induced by H. pylori infection) significantly potentiates TES and FLN-C degradation. Acetylated Siah2-mediated downregulation of TES disrupts filopodia but promotes lamellipodia formation and enhances invasiveness of gastric cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, proteasome inhibition assay, site-specific acetylation analysis, cell invasion/migration assay\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with functional consequence (proteasomal degradation, invasion assay), single lab\",\n      \"pmids\": [\"30063986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Testin RNAi knockdown in Sertoli cells causes damage to the Sertoli cell barrier, causes diffusion of tight junction proteins occludin and ZO-1 away from the cell surface into the cytoplasm, reduces ZO-1/occludin association (shown by Co-IP), disrupts actin filament bundle arrangement, and reduces F-actin/G-actin ratio. ARP3 redistributes to the Sertoli cell interface after testin RNAi.\",\n      \"method\": \"RNAi knockdown, Co-immunoprecipitation, co-immunofluorescence, F-actin/G-actin ratio assay, barrier function assay\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi with multiple orthogonal readouts (Co-IP, immunofluorescence, actin assay), single lab\",\n      \"pmids\": [\"31975378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TES directly interacts with calcineurin (shown by co-immunoprecipitation) and suppresses calcineurin downstream signalling. Adeno-associated virus-driven cardiac TES overexpression attenuated ventricular dilation, cardiac hypertrophy, dysfunction, and fibrosis induced by aortic banding in mice. TES knockdown exacerbated hypertrophy; cyclosporin A (calcineurin inhibitor) reversed this, confirming calcineurin-dependence.\",\n      \"method\": \"Co-immunoprecipitation, adeno-associated viral overexpression, adenoviral shRNA knockdown, aortic banding mouse model, pharmacological rescue with cyclosporin A\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP interaction with pharmacological and genetic rescue in vivo, single lab\",\n      \"pmids\": [\"30467953\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Testin (encoded by TES) interacts with KCNE2 potassium channel β-subunit (identified by yeast two-hybrid and confirmed by co-immunoprecipitation). Ectopic expression of Testin nullifies KCNE2 effects on Kv1.5 voltage dependence and gating kinetics, but does not prevent KCNE2 regulation of KCNQ1. Testin alone did not alter Kv1.5 function.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, whole-cell patch clamp electrophysiology\",\n      \"journal\": \"Channels (Austin, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid confirmed by Co-IP and functional electrophysiology, single lab\",\n      \"pmids\": [\"33464998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TES overexpression in colorectal cancer cells inhibits cell proliferation, migration, and invasion while increasing apoptosis, and activates p38 MAPK signalling (upregulation of pro-apoptotic proteins, downregulation of anti-apoptotic proteins). TES knockdown by shRNA elicited opposite effects.\",\n      \"method\": \"Overexpression and shRNA knockdown, proliferation/migration/invasion assays, apoptosis assay, p38 MAPK pathway analysis by western blot\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bidirectional manipulation (OE and KD) with pathway analysis, single lab\",\n      \"pmids\": [\"27323777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Elevated Testin expression in colonic epithelial cells inactivates the JNK/P38 signalling pathway, preventing depletion of tight junction proteins ZO-1 and Claudin-1 and protecting intestinal barrier integrity in a Crohn's disease mouse model. Testin overexpression ameliorated colitis symptoms and reduced inflammatory cytokines; effects were demonstrated in colonic organoids treated with LPS.\",\n      \"method\": \"Adenoviral overexpression in vivo (TNBS colitis model), colonic organoids, western blotting of TJ proteins and JNK/P38 pathway, barrier permeability assay\",\n      \"journal\": \"Chemico-biological interactions\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo gain-of-function with organoid validation and defined signalling pathway, single lab\",\n      \"pmids\": [\"39237074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Chicken TES (cTES) localizes to focal adhesions, actin stress fibres, and cell-cell contact sites; GST-cTES pulls down zyxin and actin. Overexpression of cTES increases cell spreading on fibronectin and decreases cell motility.\",\n      \"method\": \"Immunofluorescence localization, GST pulldown, overexpression with cell spreading and motility assay\",\n      \"journal\": \"Cell motility and the cytoskeleton\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — pulldown and functional overexpression, replicated findings from human TES study\",\n      \"pmids\": [\"14743347\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Re-expression of TESTIN protein in ALL cell lines causes rapid cell death or cell cycle arrest independent of TP53 activity, as shown by transfection of TES expression plasmids followed by cell proliferation, cell death, and cell cycle assays.\",\n      \"method\": \"Plasmid transfection (TES re-expression), cell death and cell cycle assays, TP53-independence established using TP53-null lines\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function with cell cycle and death assays, TP53-independence tested, single lab\",\n      \"pmids\": [\"26985820\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TES/Testin is a multidomain LIM-domain scaffold protein (PET domain + 3 C-terminal LIM domains) that localizes to focal adhesions, actin stress fibres, and cell-cell junctions via its LIM domains, adopts open and closed (intramolecular) conformations that regulate its interactions, recruits to focal adhesions in a zyxin-dependent and tension-sensitive manner, directly binds cytoskeletal partners including Mena (via an atypical LIM3–EVH1 interaction that competes with FPPPP-motif proteins), zyxin, VASP, talin, alpha-actinin, paxillin, actin, and alphaII-spectrin, regulates actin stress fibre integrity through RhoA signalling, suppresses cell migration and invasion partly by blocking Mena–Lamellipodin interaction, acts as an in vivo tumour suppressor (knockout mice show enhanced carcinogenesis), and also modulates specific signalling pathways including calcineurin/NFAT in cardiac hypertrophy, p38 MAPK in colorectal cancer, and JNK/P38 in intestinal barrier protection.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TES (Testin) is a multidomain LIM-domain scaffold protein that organizes the actin cytoskeleton at focal adhesions, stress fibres, and cell-cell junctions, where it acts as a mechanosensitive adaptor and tumour suppressor [#0, #1, #17]. Its three C-terminal LIM domains target focal adhesions and strained actin while its N-terminal PET-containing region directs it to stress fibres; an intramolecular interaction between the N-terminus and LIM3 (involving Tyr288 and dimerization-region phosphorylation) holds TES in a 'closed' conformation that gates its localization and partner binding, with active RhoA driving relocation to stress fibres and conferring mechanosensitivity [#0, #6, #11, #12]. TES is recruited to focal adhesions and tension-bearing cadherin-based junctions in a zyxin-dependent, tension-sensitive hierarchy (zyxin → VASP → TES), and it is required for RhoA-dependent stress fibre integrity [#1, #2, #9, #10]. Through an atypical LIM3–EVH1 interaction that occludes the canonical FPPPP-binding site, TES binds Mena (but not VASP or Evl) and displaces Mena from the leading edge, blocking the Mena–Lamellipodin interaction to suppress cell migration and invasion [#3, #18]. TES additionally engages cytoskeletal and junctional partners including alphaII-spectrin, actin, alpha-actinin, paxillin, and Arp7A [#1, #4, #5]. As an in vivo tumour suppressor, Tes loss increases carcinogen-induced gastric tumorigenesis in knockout mice, while re-expression of TES in TES-negative carcinoma and leukemia cells induces apoptosis or cell-cycle arrest and reduces tumorigenicity [#16, #17, #26]; TES protein levels are themselves downregulated via Siah2-mediated proteasomal degradation potentiated by H. pylori-induced acetylation [#19]. Beyond cytoskeletal control, TES interacts with diverse signalling components—calcineurin (suppressing NFAT signalling in cardiac hypertrophy), the calcium-sensing receptor, and KCNE2—and modulates p38 MAPK and JNK/p38 pathways governing colorectal cancer cell fate and intestinal/Sertoli barrier integrity [#21, #8, #22, #23, #24, #20].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established TES as a focal-adhesion and cytoskeletal protein with a modular domain logic, answering where it localizes and which partners it uses, defining it as a scaffold rather than an enzyme.\",\n      \"evidence\": \"Immunofluorescence, yeast two-hybrid, biochemical pull-down, and overexpression spreading assays in fibroblasts and breast carcinoma cells\",\n      \"pmids\": [\"12571287\", \"12695497\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and order of multi-partner assembly not resolved\", \"Functional consequence of each individual interaction not separated\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defined a recruitment hierarchy and pathway placement by showing TES is required for RhoA-dependent stress fibre maintenance and is recruited downstream of zyxin and VASP.\",\n      \"evidence\": \"RNAi knockdown in HeLa cells with RhoA activity assay and immunofluorescence; additional Y2H/Co-IP identifying alphaII-spectrin and EVL partners\",\n      \"pmids\": [\"15662727\", \"15656790\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which TES feeds back on RhoA activity not defined\", \"Single-lab knockdown phenotype\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Demonstrated TES tumour-suppressor function in vivo and in cancer cell lines, establishing biological significance of its loss.\",\n      \"evidence\": \"Tes knockout mice with carcinogen-induced gastric tumorigenesis; adenoviral re-expression in TES-negative carcinoma lines with apoptosis and xenograft assays\",\n      \"pmids\": [\"16033868\", \"15701871\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular pathway linking TES loss to tumorigenesis not defined in these studies\", \"Tissue specificity of tumour suppression unclear\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Provided structural-mechanistic basis for TES specificity by solving how its LIM3 domain captures the Mena EVH1 domain through an atypical, FPPPP-independent mode that selectively excludes VASP/Evl, linking the interaction to migration control.\",\n      \"evidence\": \"Crystal structure of LIM3:EVH1 complex with structure-based gain-of-function mutagenesis and Mena-displacement/migration assays; subsequent structure of the Arp7A·LIM2-3·Mena ternary complex\",\n      \"pmids\": [\"18158903\", \"21278383\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of LIM3-Mena competition in tissue contexts not fully established\", \"How conformational gating regulates EVH1 availability not structurally resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined the open/closed conformational switch by mapping an N-terminus–LIM3 intramolecular interaction and showing compartment-specific conformational states, including unexpected nucleolar localization.\",\n      \"evidence\": \"GST pull-down, Co-IP, and B23 co-localization; later PET–LIM1-2 mapping with Tyr288 mutagenesis and homodimer detection\",\n      \"pmids\": [\"18696217\", \"28542564\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional role of nucleolar TES unknown\", \"Physiological trigger toggling open/closed states not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Established TES as a tension-sensitive mechanosensor by mapping its zyxin-binding determinants and showing tension-dependent recruitment to cadherin-based junctions distinct from the core cadherin complex.\",\n      \"evidence\": \"Pull-down/FRAP zyxin-binding mapping plus SIM imaging and tension manipulation at focal adherens junctions\",\n      \"pmids\": [\"26509500\", \"26611125\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct force-sensing element within TES not identified\", \"Single-lab imaging studies\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Resolved the mechanosensitive behaviour of TES at the domain level, showing conformation and phosphorylation control relocation to strained actin and that RhoA activation drives mechanosensitivity.\",\n      \"evidence\": \"Fluorescence imaging of domain truncations and phosphorylation mutants with constitutively active RhoA expression\",\n      \"pmids\": [\"34038160\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Kinases responsible for the relevant phosphorylation not identified\", \"Quantitative force thresholds not defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated conserved developmental roles in tension-dependent junctional actin organization and planar polarity through orthologs and genetic interactions.\",\n      \"evidence\": \"C. elegans tes-1/zyx-1 loss-of-function and epistasis with cadherin/catenin; Xenopus Xtes morpholino knockdown with Prickle interaction; mouse Testin/Vangl2 knockout epistasis\",\n      \"pmids\": [\"36384139\", \"16554046\", \"23996638\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between TES and PCP machinery not defined\", \"Whether developmental and adhesion roles share a mechanism unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected TES tumour suppression to a defined molecular mechanism—blocking Mena–Lamellipodin—and to its regulated turnover by an acetylated Siah2 E3 ligase in gastric cancer.\",\n      \"evidence\": \"IP-MS, Co-IP, Mena-dependent Transwell assays; Siah2 Co-IP with proteasome inhibition and invasion assays under H. pylori-induced acetylation\",\n      \"pmids\": [\"30728082\", \"30063986\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Mena-Lpd blockade accounts for in vivo tumour suppression not shown\", \"Single-lab mechanistic studies\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended TES function to signalling modulation across tissues, implicating it in calcineurin/NFAT-dependent cardiac hypertrophy, p38 MAPK-driven apoptosis in colorectal cancer, and JNK/p38-dependent epithelial barrier protection.\",\n      \"evidence\": \"Co-IP plus in vivo overexpression/knockdown with pharmacological rescue (cardiac); bidirectional manipulation with pathway western blots (colorectal); in vivo overexpression and organoids (intestinal barrier); RNAi barrier disruption in Sertoli cells; CaR and KCNE2 interaction with functional electrophysiology\",\n      \"pmids\": [\"30467953\", \"27323777\", \"39237074\", \"31975378\", \"21843504\", \"33464998\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How a cytoskeletal scaffold mechanistically tunes calcineurin, p38, and JNK signalling not resolved\", \"Whether these signalling roles reflect direct or cytoskeleton-mediated effects unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how the conformational/mechanosensitive switch of TES is integrated with its diverse signalling outputs (calcineurin, p38/JNK, ion-channel regulation) to produce tissue-specific tumour-suppressor and barrier functions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying mechanism linking mechanosensing to signalling modulation\", \"No structural model of full-length TES in its active state\", \"Endogenous upstream regulators of conformational switching unidentified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 5]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [9, 12]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 18, 21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 2, 12]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 9]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 8, 21]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [16, 17, 18, 19]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [16, 23, 26]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [13, 14, 15]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [9, 13]}\n    ],\n    \"complexes\": [\"focal adhesion\", \"focal adherens junction\"],\n    \"partners\": [\"ENAH\", \"ZYX\", \"VASP\", \"TLN1\", \"ACTN1\", \"SPTAN1\", \"PXN\", \"SIAH2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}