{"gene":"TICAM2","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2003,"finding":"TICAM-2 physically bridges TLR4 and TICAM-1/TRIF: it binds directly to the TLR4 cytoplasmic domain and recruits TICAM-1, enabling IRF-3 activation and IFN-β induction downstream of LPS signaling. TICAM-2 itself exhibits minimal ability to activate NF-κB or the IFN-β promoter independently.","method":"Co-immunoprecipitation, overexpression, dominant-negative analysis, reporter assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP with functional reporter assays, replicated across independent labs in same year (PMID:14519765 and PMID:12721283)","pmids":["14519765"],"is_preprint":false},{"year":2003,"finding":"TICAM-2/TIRP interacts with IL-1 receptors and with kinase-inactive mutants of IRAK, IRAK-4, IRAK-2, IRAK-M, and TRAF6 via co-immunoprecipitation. Overexpression activates NF-κB and potentiates IL-1 receptor-mediated NF-κB activation. A dominant-negative TIRP mutant inhibits IL-1- but not TNF-triggered NF-κB activation, and TIRP-mediated NF-κB activation is blocked by dominant-negative IRAK, IRAK-2, TRAF6, and IKKβ, placing TICAM-2 upstream of these components.","method":"Co-immunoprecipitation, overexpression, dominant-negative epistasis, NF-κB reporter assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus epistasis via dominant-negative panel, single lab","pmids":["12721283"],"is_preprint":false},{"year":2015,"finding":"TICAM-2 membrane localization is governed by two distinct sequence elements: N-terminal myristoylation anchors it to membranes, and a conserved acidic motif D91/E92 is additionally required for correct membrane/endosomal localization. The D91A/E92A mutant is redistributed to the cytosol despite being myristoylated, cannot self-aggregate to activate TICAM-1 autonomously, but retains the ability to support TLR4-mediated IRF3 activation upon LPS stimulation in the context of assembled TLR4 complexes.","method":"Site-directed mutagenesis, subcellular fractionation/localization imaging, reporter assays in TICAM-2 knockout cell line reconstitution, colocalization analysis","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis combined with KO cell reconstitution and quantitative localization imaging in one study, multiple orthogonal methods","pmids":["26408662"],"is_preprint":false},{"year":2017,"finding":"By NMR structural analysis, TICAM-2 interacts with TICAM-1 through an acidic amino acid motif E87/D88/D89. The TIR domain of TICAM-2 couples with the TLR4 TIR domain dimer beneath the plasma membrane, and TICAM-2 itself dimerizes to constitute a binding site for TICAM-1. Endosomal localization of TICAM-2 (facilitated by N-terminal myristoylation and the D91/E92 motif) is essential for TLR4-mediated type I IFN induction from the endosome.","method":"NMR structural analysis, functional interface mapping, mutagenesis","journal":"Biochemical Society transactions","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structural data with functional validation of interface residues, single lab but orthogonal structural and mutagenesis methods","pmids":["28630139"],"is_preprint":false},{"year":2014,"finding":"The TRAM/TICAM-2 TIR domain physically interacts with the TLR4 TIR domain in vitro, and the BB-loop regions of both the TRAM TIR domain and the TLR4 TIR domain are essential for this physical interaction.","method":"Recombinant protein purification, in vitro GST pull-down, site-directed mutagenesis of BB-loop residues","journal":"Protein expression and purification","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted in vitro interaction with mutagenesis of critical BB-loop residues in both partners, single lab","pmids":["25306876"],"is_preprint":false},{"year":2017,"finding":"Ticam2-/- mice are highly susceptible to SARS-CoV infection (increased weight loss, pulmonary hemorrhage), demonstrating a critical protective role for TICAM2 in coronavirus disease. Allelic variation in Ticam2 was identified as the major-effect quantitative trait locus on chromosome 18 driving differential SARS-CoV pathogenesis between resistant and susceptible mouse strains.","method":"Ticam2 knockout mice, F2 genetic mapping (QTL analysis), SARS-CoV infection model with weight loss, viral titer, and hemorrhage phenotypes","journal":"G3 (Bethesda, Md.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse infection model with multiple disease phenotypes plus genetic mapping, single study","pmids":["28592648"],"is_preprint":false},{"year":2020,"finding":"TICAM2 is required for LPS-induced neutrophil exhaustion: TICAM2-deficient neutrophils show decreased expression of ICAM1, CD11b, and PD-L1 and reduced aggregation after prolonged LPS challenge. Mechanistically, LPS drives exhaustion through TICAM2-mediated activation of Src family kinases (SFK) and STAT1, as the SFK inhibitor Dasatinib blocks this process. TICAM2-deficient mice are protected from severe systemic inflammation and multi-organ injury after chemical-induced mucosal damage.","method":"TICAM2 knockout primary murine neutrophils, flow cytometry, pharmacological inhibition (Dasatinib), in vivo mucosal damage model","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO cells with defined cellular phenotype plus pharmacological epistasis (SFK inhibitor), single lab","pmids":["32873853"],"is_preprint":false},{"year":2019,"finding":"Phospho-AKT (T308/S473) phosphorylates STING and its adaptor TICAM2, augmenting downstream IRF3-dependent IDO1 transcription in Toxoplasma gondii infection. This places TICAM2 in a STING-TICAM2-IRF3-IDO1 signalosome regulated by AKT and β-catenin, functioning in a TBK-independent manner.","method":"Phosphosite mutagenesis, β-catenin knockout cells, co-immunoprecipitation, transcriptional reporter assays, infection model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis and KO cells with defined signaling readouts, single lab, multiple orthogonal methods","pmids":["30770800"],"is_preprint":false},{"year":2020,"finding":"SET8 interacts with ATF2 to regulate TICAM-2 promoter activity: H4K20me1 (downstream of SET8) and ATF2 both occupy the TICAM-2 promoter, and loss of SET8 increases TICAM-2 promoter activity and expression, thereby promoting LPS-mediated BV2 microglial inflammation.","method":"ChIP, luciferase reporter assays, shRNA knockdown, overexpression, co-immunoprecipitation","journal":"Canadian journal of physiology and pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus luciferase reporter plus Co-IP, multiple orthogonal methods, single lab","pmids":["32176860"],"is_preprint":false},{"year":2025,"finding":"TICAM2 ablation facilitates recovery from monocyte exhaustion after sepsis: Ticam2-/- bone marrow and splenic monocytes resemble healthy controls after one week of recovery from cecal slurry sepsis, whereas wild-type monocytes remain exhausted. Genome-wide DNA methylation profiling shows Ticam2-/- monocytes have altered methylation at CEBPE binding sites and regulatory regions of immune genes (Dmkn, Btg1), indicating TICAM2 contributes to epigenetic exhaustion memory.","method":"Ticam2 knockout mice, cecal slurry sepsis model, flow cytometry, genome-wide DNA methylation profiling","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse model with defined cellular phenotype and genome-wide epigenetic profiling, single lab","pmids":["39814939"],"is_preprint":false}],"current_model":"TICAM2 is a TIR domain-containing bridging adaptor that anchors to endosomal membranes via N-terminal myristoylation and an acidic D91/E92 motif; its TIR domain physically docks onto the TLR4 TIR domain dimer through BB-loop contacts, and recruits TICAM-1/TRIF via an E87/D88/D89 acidic interface, thereby transmitting LPS–TLR4 signals to IRF-3-dependent IFN-β production in a MyD88-independent manner; additionally, TICAM2 participates in a phospho-AKT–STING–TICAM2–IRF3–IDO1 signalosome during intracellular infection, drives neutrophil and monocyte exhaustion during sepsis through SFK/STAT1 activation and epigenetic programming, and is subject to transcriptional regulation by the SET8–ATF2–H4K20me1 axis."},"narrative":{"mechanistic_narrative":"TICAM2 (TRAM/TIRP) is a TIR domain-containing bridging adaptor that couples activated TLR4 to the downstream adaptor TICAM-1/TRIF, enabling IRF-3 activation and type I IFN-β induction during LPS signaling while having minimal capacity to activate NF-κB or the IFN-β promoter on its own [PMID:14519765]. Structurally, its TIR domain docks onto the TLR4 TIR domain dimer through reciprocal BB-loop contacts [PMID:25306876], and TICAM2 then self-dimerizes to form a binding surface that recruits TICAM-1 via an acidic E87/D88/D89 interface [PMID:28630139]. Correct subcellular positioning is required for this relay: N-terminal myristoylation anchors TICAM2 to membranes and a conserved acidic D91/E92 motif directs endosomal localization, which is essential for TLR4-driven type I IFN induction from the endosome; loss of D91/E92 redistributes the protein to the cytosol and abolishes autonomous self-aggregation while preserving its capacity to support IRF3 activation within assembled TLR4 complexes [PMID:26408662, PMID:28630139]. Beyond canonical TLR4 signaling, TICAM2 acts as a node in an AKT-driven STING-TICAM2-IRF3-IDO1 signalosome during intracellular infection [PMID:30770800] and drives myeloid exhaustion programs—promoting LPS-induced neutrophil exhaustion through Src-family-kinase and STAT1 activation [PMID:32873853] and contributing to epigenetic exhaustion memory in monocytes after sepsis [PMID:39814939]. TICAM2 expression is itself constrained by a SET8-ATF2-H4K20me1 axis acting at its promoter [PMID:32176860], and Ticam2 is the major-effect locus governing protective responses to SARS-CoV infection in mice [PMID:28592648].","teleology":[{"year":2003,"claim":"Established that TICAM2 is a dedicated bridging adaptor physically linking TLR4 to TRIF/TICAM-1, defining the MyD88-independent route to IRF-3 and IFN-β.","evidence":"Co-immunoprecipitation, overexpression, dominant-negative analysis and reporter assays in mammalian cells","pmids":["14519765"],"confidence":"High","gaps":["Does not resolve the structural basis of the TLR4 or TRIF contacts","Does not address subcellular site of signaling"]},{"year":2003,"claim":"Probed whether TICAM2 also feeds IL-1 receptor/NF-κB signaling, placing it upstream of IRAK/TRAF6/IKKβ in that branch.","evidence":"Co-immunoprecipitation with IL-1R and IRAK family members plus dominant-negative epistasis and NF-κB reporter assays, single lab","pmids":["12721283"],"confidence":"Medium","gaps":["Relies on overexpression and dominant-negative panels rather than endogenous loss-of-function","Apparent NF-κB role conflicts with later finding of minimal autonomous NF-κB activation"]},{"year":2014,"claim":"Defined the physical TLR4-TICAM2 interface, showing BB-loop residues of both TIR domains mediate the direct contact.","evidence":"Recombinant protein purification, in vitro GST pull-down, and BB-loop mutagenesis in both partners","pmids":["25306876"],"confidence":"High","gaps":["In vitro reconstitution does not establish stoichiometry in cells","Does not show how the contact triggers downstream TRIF recruitment"]},{"year":2015,"claim":"Resolved how TICAM2 is targeted to its signaling compartment, separating membrane anchoring (myristoylation) from endosomal localization (D91/E92) and showing localization is needed for autonomous activation.","evidence":"Site-directed mutagenesis, fractionation/imaging, and reporter assays in TICAM2-knockout reconstituted cells","pmids":["26408662"],"confidence":"High","gaps":["Does not identify the machinery reading the D91/E92 motif","Does not define how localization couples to self-aggregation mechanistically"]},{"year":2017,"claim":"Provided structural detail of the relay: TICAM2 dimerizes beneath the TLR4 TIR dimer and engages TICAM-1 through an acidic E87/D88/D89 motif at the endosome.","evidence":"NMR structural analysis with functional interface mapping and mutagenesis","pmids":["28630139"],"confidence":"High","gaps":["No full-length complex structure","Dynamics of dimerization upon TLR4 engagement not captured"]},{"year":2017,"claim":"Demonstrated organismal importance by showing Ticam2 is the major QTL governing protection against SARS-CoV pathogenesis.","evidence":"Ticam2 knockout mice, F2 QTL mapping, and SARS-CoV infection phenotyping","pmids":["28592648"],"confidence":"Medium","gaps":["Does not define which downstream TICAM2 output confers protection","Single study in mouse strains"]},{"year":2019,"claim":"Extended TICAM2 beyond TLR4 to an AKT-regulated STING-TICAM2-IRF3-IDO1 signalosome during Toxoplasma infection, acting TBK-independently.","evidence":"Phosphosite mutagenesis, β-catenin knockout cells, Co-IP, reporter assays, infection model","pmids":["30770800"],"confidence":"Medium","gaps":["Direct AKT phosphorylation sites on TICAM2 not mapped to function","Generality beyond Toxoplasma not established"]},{"year":2020,"claim":"Identified a TICAM2-dependent neutrophil exhaustion program driven by Src-family kinases and STAT1 during prolonged LPS challenge.","evidence":"TICAM2-knockout primary neutrophils, flow cytometry, Dasatinib epistasis, in vivo mucosal damage model","pmids":["32873853"],"confidence":"Medium","gaps":["Direct link from TICAM2 to SFK activation not biochemically defined","Pharmacological inhibitor lacks SFK selectivity"]},{"year":2020,"claim":"Showed TICAM2 expression is repressed by a SET8-ATF2-H4K20me1 axis at its promoter, linking chromatin regulation to TLR4-pathway output in microglia.","evidence":"ChIP, luciferase reporters, shRNA knockdown, overexpression, Co-IP in BV2 cells","pmids":["32176860"],"confidence":"Medium","gaps":["Does not establish direct ATF2 binding versus indirect effects","Cell-type generality not tested"]},{"year":2025,"claim":"Linked TICAM2 to durable epigenetic exhaustion memory, showing its ablation allows monocyte recovery and alters DNA methylation at immune-gene regulatory regions after sepsis.","evidence":"Ticam2 knockout mice, cecal slurry sepsis model, flow cytometry, genome-wide DNA methylation profiling","pmids":["39814939"],"confidence":"Medium","gaps":["Causal chain from TICAM2 signaling to methylation changes unresolved","CEBPE-site methylation correlation not functionally validated"]},{"year":null,"claim":"How the endosomal TLR4 adaptor function of TICAM2 mechanistically connects to its roles in STING signaling and myeloid exhaustion/epigenetic memory remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified mechanism linking adaptor activity to exhaustion programs","No structure of TICAM2 in non-TLR4 complexes","Direct post-translational modification sites controlling pathway choice undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,3]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[4,3]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[2,3]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,6,9]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,7]}],"complexes":["STING-TICAM2-IRF3-IDO1 signalosome","TLR4 signaling complex"],"partners":["TLR4","TICAM1","STING","AKT","TRAF6","IRAK"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q86XR7","full_name":"TIR domain-containing adapter molecule 2","aliases":["Putative NF-kappa-B-activating protein 502","TRIF-related adapter molecule","Toll-like receptor adaptor protein 3","Toll/interleukin-1 receptor domain-containing protein","MyD88-4"],"length_aa":235,"mass_kda":26.9,"function":"Functions as a sorting adapter in different signaling pathways to facilitate downstream signaling leading to type I interferon induction (PubMed:16603631, PubMed:16757566, PubMed:25385819, PubMed:25825441). In TLR4 signaling, physically bridges TLR4 and TICAM1 and functionally transmits signal to TICAM1 in early endosomes after endocytosis of TLR4. In TLR2 signaling, physically bridges TLR2 and MYD88 and is required for the TLR2-dependent movement of MYD88 to endosomes following ligand engagement (PubMed:25385819). Involved in IL-18 signaling and is proposed to function as a sorting adapter for MYD88 in IL-18 signaling during adaptive immune response (PubMed:22685567). Forms a complex with RAB11FIP2 that is recruited to the phagosomes to promote the activation of the actin-regulatory GTPases RAC1 and CDC42 and subsequent phagocytosis of Gram-negative bacteria (PubMed:30883606) Proposed to inhibit LPS-TLR4 signaling at the late endosome by interaction with isoform 1 thereby disrupting the association of isoform 1 with TICAM1. May be involved in TLR4 degradation in late endosomes","subcellular_location":"Endoplasmic reticulum; Early endosome membrane; Late endosome membrane","url":"https://www.uniprot.org/uniprotkb/Q86XR7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TICAM2","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TICAM2","total_profiled":1310},"omim":[{"mim_id":"619990","title":"TRANSMEMBRANE p24 TRAFFICKING PROTEIN 7; TMED7","url":"https://www.omim.org/entry/619990"},{"mim_id":"608321","title":"TIR DOMAIN-CONTAINING ADAPTOR MOLECULE 2; TICAM2","url":"https://www.omim.org/entry/608321"},{"mim_id":"606270","title":"TOLL-LIKE RECEPTOR 10; TLR10","url":"https://www.omim.org/entry/606270"},{"mim_id":"176878","title":"PROTEIN-TYROSINE PHOSPHATASE, NONRECEPTOR-TYPE, 4; PTPN4","url":"https://www.omim.org/entry/176878"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TICAM2"},"hgnc":{"alias_symbol":["TRAM","TICAM-2","TIRP"],"prev_symbol":[]},"alphafold":{"accession":"Q86XR7","domains":[{"cath_id":"3.40.50.10140","chopping":"80-232","consensus_level":"high","plddt":92.1106,"start":80,"end":232}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86XR7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q86XR7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q86XR7-F1-predicted_aligned_error_v6.png","plddt_mean":74.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TICAM2","jax_strain_url":"https://www.jax.org/strain/search?query=TICAM2"},"sequence":{"accession":"Q86XR7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q86XR7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q86XR7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86XR7"}},"corpus_meta":[{"pmid":"14519765","id":"PMC_14519765","title":"TIR-containing adapter molecule (TICAM)-2, a bridging adapter recruiting to toll-like receptor 4 TICAM-1 that induces interferon-beta.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/14519765","citation_count":303,"is_preprint":false},{"pmid":"12721283","id":"PMC_12721283","title":"TIRP, a novel Toll/interleukin-1 receptor (TIR) domain-containing adapter protein involved in TIR signaling.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12721283","citation_count":80,"is_preprint":false},{"pmid":"28592648","id":"PMC_28592648","title":"Allelic Variation in the Toll-Like Receptor Adaptor Protein Ticam2 Contributes to SARS-Coronavirus Pathogenesis in Mice.","date":"2017","source":"G3 (Bethesda, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/28592648","citation_count":68,"is_preprint":false},{"pmid":"25876455","id":"PMC_25876455","title":"MiR-27a ameliorates inflammatory damage to the blood-spinal cord barrier after spinal cord ischemia: reperfusion injury in rats by downregulating TICAM-2 of the TLR4 signaling pathway.","date":"2015","source":"Journal of neuroinflammation","url":"https://pubmed.ncbi.nlm.nih.gov/25876455","citation_count":68,"is_preprint":false},{"pmid":"15618008","id":"PMC_15618008","title":"TICAM-1 and TICAM-2: toll-like receptor adapters that participate in induction of type 1 interferons.","date":"2005","source":"The international journal of biochemistry & cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/15618008","citation_count":46,"is_preprint":false},{"pmid":"28630139","id":"PMC_28630139","title":"Functional interfaces between TICAM-2/TRAM and TICAM-1/TRIF in TLR4 signaling.","date":"2017","source":"Biochemical Society transactions","url":"https://pubmed.ncbi.nlm.nih.gov/28630139","citation_count":39,"is_preprint":false},{"pmid":"30770800","id":"PMC_30770800","title":"Tryptophan-kynurenine pathway attenuates β-catenin-dependent pro-parasitic role of STING-TICAM2-IRF3-IDO1 signalosome in Toxoplasma gondii infection.","date":"2019","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/30770800","citation_count":32,"is_preprint":false},{"pmid":"32873853","id":"PMC_32873853","title":"TICAM2-related pathway mediates neutrophil exhaustion.","date":"2020","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/32873853","citation_count":31,"is_preprint":false},{"pmid":"39814939","id":"PMC_39814939","title":"Ticam2 ablation facilitates monocyte exhaustion recovery after sepsis.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/39814939","citation_count":6,"is_preprint":false},{"pmid":"26408662","id":"PMC_26408662","title":"Identification of a Regulatory Acidic Motif as the Determinant of Membrane Localization of TICAM-2.","date":"2015","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/26408662","citation_count":4,"is_preprint":false},{"pmid":"32176860","id":"PMC_32176860","title":"SET8 participates in lipopolysaccharide-mediated BV2 cell inflammation via modulation of TICAM-2 expression.","date":"2020","source":"Canadian journal of physiology and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/32176860","citation_count":3,"is_preprint":false},{"pmid":"25306876","id":"PMC_25306876","title":"Recombinant production of functional full-length and truncated human TRAM/TICAM-2 adaptor protein involved in Toll-like receptor and interferon signaling.","date":"2014","source":"Protein expression and purification","url":"https://pubmed.ncbi.nlm.nih.gov/25306876","citation_count":3,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9091,"output_tokens":2892,"usd":0.035326,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10151,"output_tokens":3421,"usd":0.06814,"stage2_stop_reason":"end_turn"},"total_usd":0.103466,"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\": \"TICAM-2 physically bridges TLR4 and TICAM-1/TRIF: it binds directly to the TLR4 cytoplasmic domain and recruits TICAM-1, enabling IRF-3 activation and IFN-β induction downstream of LPS signaling. TICAM-2 itself exhibits minimal ability to activate NF-κB or the IFN-β promoter independently.\",\n      \"method\": \"Co-immunoprecipitation, overexpression, dominant-negative analysis, reporter assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP with functional reporter assays, replicated across independent labs in same year (PMID:14519765 and PMID:12721283)\",\n      \"pmids\": [\"14519765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"TICAM-2/TIRP interacts with IL-1 receptors and with kinase-inactive mutants of IRAK, IRAK-4, IRAK-2, IRAK-M, and TRAF6 via co-immunoprecipitation. Overexpression activates NF-κB and potentiates IL-1 receptor-mediated NF-κB activation. A dominant-negative TIRP mutant inhibits IL-1- but not TNF-triggered NF-κB activation, and TIRP-mediated NF-κB activation is blocked by dominant-negative IRAK, IRAK-2, TRAF6, and IKKβ, placing TICAM-2 upstream of these components.\",\n      \"method\": \"Co-immunoprecipitation, overexpression, dominant-negative epistasis, NF-κB reporter assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus epistasis via dominant-negative panel, single lab\",\n      \"pmids\": [\"12721283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TICAM-2 membrane localization is governed by two distinct sequence elements: N-terminal myristoylation anchors it to membranes, and a conserved acidic motif D91/E92 is additionally required for correct membrane/endosomal localization. The D91A/E92A mutant is redistributed to the cytosol despite being myristoylated, cannot self-aggregate to activate TICAM-1 autonomously, but retains the ability to support TLR4-mediated IRF3 activation upon LPS stimulation in the context of assembled TLR4 complexes.\",\n      \"method\": \"Site-directed mutagenesis, subcellular fractionation/localization imaging, reporter assays in TICAM-2 knockout cell line reconstitution, colocalization analysis\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis combined with KO cell reconstitution and quantitative localization imaging in one study, multiple orthogonal methods\",\n      \"pmids\": [\"26408662\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"By NMR structural analysis, TICAM-2 interacts with TICAM-1 through an acidic amino acid motif E87/D88/D89. The TIR domain of TICAM-2 couples with the TLR4 TIR domain dimer beneath the plasma membrane, and TICAM-2 itself dimerizes to constitute a binding site for TICAM-1. Endosomal localization of TICAM-2 (facilitated by N-terminal myristoylation and the D91/E92 motif) is essential for TLR4-mediated type I IFN induction from the endosome.\",\n      \"method\": \"NMR structural analysis, functional interface mapping, mutagenesis\",\n      \"journal\": \"Biochemical Society transactions\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structural data with functional validation of interface residues, single lab but orthogonal structural and mutagenesis methods\",\n      \"pmids\": [\"28630139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The TRAM/TICAM-2 TIR domain physically interacts with the TLR4 TIR domain in vitro, and the BB-loop regions of both the TRAM TIR domain and the TLR4 TIR domain are essential for this physical interaction.\",\n      \"method\": \"Recombinant protein purification, in vitro GST pull-down, site-directed mutagenesis of BB-loop residues\",\n      \"journal\": \"Protein expression and purification\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted in vitro interaction with mutagenesis of critical BB-loop residues in both partners, single lab\",\n      \"pmids\": [\"25306876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Ticam2-/- mice are highly susceptible to SARS-CoV infection (increased weight loss, pulmonary hemorrhage), demonstrating a critical protective role for TICAM2 in coronavirus disease. Allelic variation in Ticam2 was identified as the major-effect quantitative trait locus on chromosome 18 driving differential SARS-CoV pathogenesis between resistant and susceptible mouse strains.\",\n      \"method\": \"Ticam2 knockout mice, F2 genetic mapping (QTL analysis), SARS-CoV infection model with weight loss, viral titer, and hemorrhage phenotypes\",\n      \"journal\": \"G3 (Bethesda, Md.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse infection model with multiple disease phenotypes plus genetic mapping, single study\",\n      \"pmids\": [\"28592648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TICAM2 is required for LPS-induced neutrophil exhaustion: TICAM2-deficient neutrophils show decreased expression of ICAM1, CD11b, and PD-L1 and reduced aggregation after prolonged LPS challenge. Mechanistically, LPS drives exhaustion through TICAM2-mediated activation of Src family kinases (SFK) and STAT1, as the SFK inhibitor Dasatinib blocks this process. TICAM2-deficient mice are protected from severe systemic inflammation and multi-organ injury after chemical-induced mucosal damage.\",\n      \"method\": \"TICAM2 knockout primary murine neutrophils, flow cytometry, pharmacological inhibition (Dasatinib), in vivo mucosal damage model\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO cells with defined cellular phenotype plus pharmacological epistasis (SFK inhibitor), single lab\",\n      \"pmids\": [\"32873853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Phospho-AKT (T308/S473) phosphorylates STING and its adaptor TICAM2, augmenting downstream IRF3-dependent IDO1 transcription in Toxoplasma gondii infection. This places TICAM2 in a STING-TICAM2-IRF3-IDO1 signalosome regulated by AKT and β-catenin, functioning in a TBK-independent manner.\",\n      \"method\": \"Phosphosite mutagenesis, β-catenin knockout cells, co-immunoprecipitation, transcriptional reporter assays, infection model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis and KO cells with defined signaling readouts, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"30770800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SET8 interacts with ATF2 to regulate TICAM-2 promoter activity: H4K20me1 (downstream of SET8) and ATF2 both occupy the TICAM-2 promoter, and loss of SET8 increases TICAM-2 promoter activity and expression, thereby promoting LPS-mediated BV2 microglial inflammation.\",\n      \"method\": \"ChIP, luciferase reporter assays, shRNA knockdown, overexpression, co-immunoprecipitation\",\n      \"journal\": \"Canadian journal of physiology and pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus luciferase reporter plus Co-IP, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"32176860\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TICAM2 ablation facilitates recovery from monocyte exhaustion after sepsis: Ticam2-/- bone marrow and splenic monocytes resemble healthy controls after one week of recovery from cecal slurry sepsis, whereas wild-type monocytes remain exhausted. Genome-wide DNA methylation profiling shows Ticam2-/- monocytes have altered methylation at CEBPE binding sites and regulatory regions of immune genes (Dmkn, Btg1), indicating TICAM2 contributes to epigenetic exhaustion memory.\",\n      \"method\": \"Ticam2 knockout mice, cecal slurry sepsis model, flow cytometry, genome-wide DNA methylation profiling\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse model with defined cellular phenotype and genome-wide epigenetic profiling, single lab\",\n      \"pmids\": [\"39814939\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TICAM2 is a TIR domain-containing bridging adaptor that anchors to endosomal membranes via N-terminal myristoylation and an acidic D91/E92 motif; its TIR domain physically docks onto the TLR4 TIR domain dimer through BB-loop contacts, and recruits TICAM-1/TRIF via an E87/D88/D89 acidic interface, thereby transmitting LPS–TLR4 signals to IRF-3-dependent IFN-β production in a MyD88-independent manner; additionally, TICAM2 participates in a phospho-AKT–STING–TICAM2–IRF3–IDO1 signalosome during intracellular infection, drives neutrophil and monocyte exhaustion during sepsis through SFK/STAT1 activation and epigenetic programming, and is subject to transcriptional regulation by the SET8–ATF2–H4K20me1 axis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TICAM2 (TRAM/TIRP) is a TIR domain-containing bridging adaptor that couples activated TLR4 to the downstream adaptor TICAM-1/TRIF, enabling IRF-3 activation and type I IFN-\\u03b2 induction during LPS signaling while having minimal capacity to activate NF-\\u03baB or the IFN-\\u03b2 promoter on its own [#0]. Structurally, its TIR domain docks onto the TLR4 TIR domain dimer through reciprocal BB-loop contacts [#4], and TICAM2 then self-dimerizes to form a binding surface that recruits TICAM-1 via an acidic E87/D88/D89 interface [#3]. Correct subcellular positioning is required for this relay: N-terminal myristoylation anchors TICAM2 to membranes and a conserved acidic D91/E92 motif directs endosomal localization, which is essential for TLR4-driven type I IFN induction from the endosome; loss of D91/E92 redistributes the protein to the cytosol and abolishes autonomous self-aggregation while preserving its capacity to support IRF3 activation within assembled TLR4 complexes [#2, #3]. Beyond canonical TLR4 signaling, TICAM2 acts as a node in an AKT-driven STING-TICAM2-IRF3-IDO1 signalosome during intracellular infection [#7] and drives myeloid exhaustion programs\\u2014promoting LPS-induced neutrophil exhaustion through Src-family-kinase and STAT1 activation [#6] and contributing to epigenetic exhaustion memory in monocytes after sepsis [#9]. TICAM2 expression is itself constrained by a SET8-ATF2-H4K20me1 axis acting at its promoter [#8], and Ticam2 is the major-effect locus governing protective responses to SARS-CoV infection in mice [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established that TICAM2 is a dedicated bridging adaptor physically linking TLR4 to TRIF/TICAM-1, defining the MyD88-independent route to IRF-3 and IFN-\\u03b2.\",\n      \"evidence\": \"Co-immunoprecipitation, overexpression, dominant-negative analysis and reporter assays in mammalian cells\",\n      \"pmids\": [\"14519765\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not resolve the structural basis of the TLR4 or TRIF contacts\", \"Does not address subcellular site of signaling\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Probed whether TICAM2 also feeds IL-1 receptor/NF-\\u03baB signaling, placing it upstream of IRAK/TRAF6/IKK\\u03b2 in that branch.\",\n      \"evidence\": \"Co-immunoprecipitation with IL-1R and IRAK family members plus dominant-negative epistasis and NF-\\u03baB reporter assays, single lab\",\n      \"pmids\": [\"12721283\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relies on overexpression and dominant-negative panels rather than endogenous loss-of-function\", \"Apparent NF-\\u03baB role conflicts with later finding of minimal autonomous NF-\\u03baB activation\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined the physical TLR4-TICAM2 interface, showing BB-loop residues of both TIR domains mediate the direct contact.\",\n      \"evidence\": \"Recombinant protein purification, in vitro GST pull-down, and BB-loop mutagenesis in both partners\",\n      \"pmids\": [\"25306876\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro reconstitution does not establish stoichiometry in cells\", \"Does not show how the contact triggers downstream TRIF recruitment\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Resolved how TICAM2 is targeted to its signaling compartment, separating membrane anchoring (myristoylation) from endosomal localization (D91/E92) and showing localization is needed for autonomous activation.\",\n      \"evidence\": \"Site-directed mutagenesis, fractionation/imaging, and reporter assays in TICAM2-knockout reconstituted cells\",\n      \"pmids\": [\"26408662\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not identify the machinery reading the D91/E92 motif\", \"Does not define how localization couples to self-aggregation mechanistically\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Provided structural detail of the relay: TICAM2 dimerizes beneath the TLR4 TIR dimer and engages TICAM-1 through an acidic E87/D88/D89 motif at the endosome.\",\n      \"evidence\": \"NMR structural analysis with functional interface mapping and mutagenesis\",\n      \"pmids\": [\"28630139\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length complex structure\", \"Dynamics of dimerization upon TLR4 engagement not captured\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrated organismal importance by showing Ticam2 is the major QTL governing protection against SARS-CoV pathogenesis.\",\n      \"evidence\": \"Ticam2 knockout mice, F2 QTL mapping, and SARS-CoV infection phenotyping\",\n      \"pmids\": [\"28592648\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not define which downstream TICAM2 output confers protection\", \"Single study in mouse strains\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended TICAM2 beyond TLR4 to an AKT-regulated STING-TICAM2-IRF3-IDO1 signalosome during Toxoplasma infection, acting TBK-independently.\",\n      \"evidence\": \"Phosphosite mutagenesis, \\u03b2-catenin knockout cells, Co-IP, reporter assays, infection model\",\n      \"pmids\": [\"30770800\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct AKT phosphorylation sites on TICAM2 not mapped to function\", \"Generality beyond Toxoplasma not established\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified a TICAM2-dependent neutrophil exhaustion program driven by Src-family kinases and STAT1 during prolonged LPS challenge.\",\n      \"evidence\": \"TICAM2-knockout primary neutrophils, flow cytometry, Dasatinib epistasis, in vivo mucosal damage model\",\n      \"pmids\": [\"32873853\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct link from TICAM2 to SFK activation not biochemically defined\", \"Pharmacological inhibitor lacks SFK selectivity\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showed TICAM2 expression is repressed by a SET8-ATF2-H4K20me1 axis at its promoter, linking chromatin regulation to TLR4-pathway output in microglia.\",\n      \"evidence\": \"ChIP, luciferase reporters, shRNA knockdown, overexpression, Co-IP in BV2 cells\",\n      \"pmids\": [\"32176860\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not establish direct ATF2 binding versus indirect effects\", \"Cell-type generality not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked TICAM2 to durable epigenetic exhaustion memory, showing its ablation allows monocyte recovery and alters DNA methylation at immune-gene regulatory regions after sepsis.\",\n      \"evidence\": \"Ticam2 knockout mice, cecal slurry sepsis model, flow cytometry, genome-wide DNA methylation profiling\",\n      \"pmids\": [\"39814939\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal chain from TICAM2 signaling to methylation changes unresolved\", \"CEBPE-site methylation correlation not functionally validated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the endosomal TLR4 adaptor function of TICAM2 mechanistically connects to its roles in STING signaling and myeloid exhaustion/epigenetic memory remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified mechanism linking adaptor activity to exhaustion programs\", \"No structure of TICAM2 in non-TLR4 complexes\", \"Direct post-translational modification sites controlling pathway choice undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [4, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 6, 9]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 7]}\n    ],\n    \"complexes\": [\"STING-TICAM2-IRF3-IDO1 signalosome\", \"TLR4 signaling complex\"],\n    \"partners\": [\"TLR4\", \"TICAM1\", \"STING\", \"AKT\", \"TRAF6\", \"IRAK\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}