{"gene":"TIFA","run_date":"2026-04-28T21:42:59","timeline":{"discoveries":[{"year":2000,"finding":"T6BP (later called TIFA) was identified as a protein that specifically associates with TRAF6 via the coiled-coil region of T6BP interacting with the N-terminal ring finger and zinc finger domains of TRAF6. IL-1, but not TNF, induces TRAF6-T6BP complex formation in a ligand-dependent manner requiring IRAK.","method":"Yeast two-hybrid screen, co-immunoprecipitation, ligand-stimulation assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal co-IP with domain mapping, single lab","pmids":["10920205"],"is_preprint":false},{"year":2002,"finding":"T2BP (TIFA) was identified as a TRAF2-binding protein via a mammalian two-hybrid screen. The TRAF domain of TRAF2 is required for binding to T2BP, and almost the entire T2BP protein is needed for TRAF2 interaction. Overexpression of T2BP activates NF-κB and AP-1 in a dose-dependent manner.","method":"Mammalian two-hybrid screening, co-immunoprecipitation, NF-κB/AP-1 reporter assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, co-IP with functional reporter assay","pmids":["11798190"],"is_preprint":false},{"year":2003,"finding":"TIFA was identified as an adaptor protein linking TRAF6 to IRAK-1 in IL-1 receptor signaling. TIFA binds TRAF6 constitutively and associates with IRAK-1 in an IL-1 stimulation-dependent manner. Mutations abolishing TRAF6 binding or FHA domain phosphopeptide binding prevented NF-κB and JNK activation. TIFA overexpression significantly enhanced the IRAK-1/TRAF6 interaction.","method":"Co-immunoprecipitation, transient transfection, NF-κB/JNK reporter assays, FHA domain mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP, mutagenesis, IL-1 stimulation-dependent interaction confirmed with endogenous proteins","pmids":["12566447"],"is_preprint":false},{"year":2004,"finding":"TIFA activates IKK by promoting oligomerization and K63-linked polyubiquitination of TRAF6, leading to TAK1 and IKK activation. Only high-molecular-weight oligomeric forms of TIFA (not monomer/dimer) activate IKK. TIFA requires a functional TRAF6-binding site for this activity. This was reconstituted in vitro with purified TIFA, TRAF6, TAK1 complex, and Ubc13-Uev1A, independently of the proteasome.","method":"In vitro IKK reconstitution with purified proteins, crude cytosolic extract assays, gel filtration to separate oligomeric forms, ubiquitination assays, TRAF6-binding-defective mutant","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with purified proteins plus mutagenesis, mechanistically rigorous","pmids":["15492226"],"is_preprint":false},{"year":2004,"finding":"TIFAB, a TIFA-related protein lacking TRAF family binding, was identified as a negative regulator of TIFA-mediated NF-κB activation. TIFAB binds TIFA but not TRAF proteins, and increases the amount of TRAF6 co-precipitated with TIFA, suggesting TIFAB inhibits TIFA-mediated TRAF6 activation possibly by inducing a conformational change in TIFA.","method":"Co-immunoprecipitation, transient transfection, NF-κB reporter assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, co-IP plus reporter assay, mechanism partially inferred","pmids":["15047173"],"is_preprint":false},{"year":2005,"finding":"Xenopus laevis homologs of TRAF6 (XTRAF6) and TIFA (XTIFA) were identified. Overexpression of XTIFA activated NF-κB by binding XTRAF6, and a mutation abolishing XTRAF6 binding abolished NF-κB activation, confirming conservation of the TIFA-TRAF6 signaling mechanism in vertebrate development.","method":"Xenopus overexpression, NF-κB reporter assay, binding-defective mutant","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 3 — ortholog functional validation, single lab, mutagenesis confirms mechanism","pmids":["16023795"],"is_preprint":false},{"year":2006,"finding":"ZCCHC11, a novel zinc finger protein, was identified as a TIFA-interacting partner. ZCCHC11L translocates from nucleus to cytoplasm in response to LPS and binds TIFA. ZCCHC11 functions as a negative regulator of TLR-mediated NF-κB activation in a TRAF6-dependent manner, as shown by overexpression and siRNA knockdown experiments.","method":"GST-TIFA affinity purification, mass spectrometry, co-immunoprecipitation, siRNA knockdown, NF-κB reporter assay, immunofluorescence","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — affinity purification/MS identification, co-IP confirmation, functional siRNA knockdown","pmids":["16643855"],"is_preprint":false},{"year":2007,"finding":"TIFA (T6BP) was identified as a myosin VI binding partner by yeast two-hybrid screen, confirmed in vitro and in vivo. T6BP and myosin VI localize to the trans-Golgi complex and perinuclear vesicles. RNAi knockdown of T6BP reduced membrane ruffling, increased stress fibres and focal adhesions, upregulated constitutive secretion, and T6BP was found to inhibit NF-κB activation.","method":"Yeast two-hybrid screen, in vitro binding, co-immunoprecipitation, immunofluorescence, electron microscopy, RNAi knockdown, secretion assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods, localization with functional consequence via RNAi","pmids":["17635994"],"is_preprint":false},{"year":2012,"finding":"TIFA contains a novel phosphorylation site at Thr9, and phosphorylated Thr9 (pThr9) binds the FHA domain of a different TIFA molecule, driving intermolecular oligomerization. Unphosphorylated TIFA exists as an intrinsic dimer; FHA-pThr9 binding occurs between different dimers. TIFA silencing attenuates TNF-α-mediated NF-κB downstream signaling, establishing a link between TNF-α stimulation and NF-κB activation.","method":"Phosphorylation site identification, FHA-pT binding assay, size-exclusion chromatography, NMR/biochemical analysis, siRNA silencing, NF-κB reporter assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 — novel PTM identified, intermolecular FHA-pT mechanism demonstrated biochemically, functional silencing confirmed","pmids":["22566686"],"is_preprint":false},{"year":2013,"finding":"TIFA expression is upregulated after hypoxia-reoxygenation in a TLR4- and MyD88-dependent manner. Under these conditions, TIFA associates with TRAF6 constitutively and with IRAK-1 only after hypoxia-reoxygenation. siRNA knockdown of TIFA reduced NF-κB activation and HMGB1 upregulation/release, placing TIFA in a TLR4/MyD88-dependent feed-forward signaling loop.","method":"qRT-PCR, western blotting, co-immunoprecipitation, siRNA knockdown, EMSA, TLR4−/− and MyD88−/− cells","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 — genetic knockouts plus siRNA with defined signaling readouts, single lab","pmids":["23722163"],"is_preprint":false},{"year":2015,"finding":"Crystal structures of TIFA revealed that the FHA domain forms an intrinsic dimer in solution, and the pThr9 peptide binds at a site that can only interact with a different dimer (not within the same dimer), providing the structural mechanism for phosphorylation-dependent TIFA oligomerization. This oligomerization is essential for NF-κB activation in immune responses.","method":"X-ray crystallography of TIFA and TIFA-pThr9 peptide complex, small angle X-ray scattering, functional mutagenesis","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with functional validation, explains oligomerization mechanism at atomic level","pmids":["26389808"],"is_preprint":false},{"year":2015,"finding":"Cytosolic detection of the bacterial metabolite heptose-1,7-bisphosphate (HBP), a conserved Gram-negative LPS biosynthetic intermediate absent from eukaryotes, triggers TIFA-dependent NF-κB activation. A genome-wide RNAi screen identified TIFA as essential for this response. HBP induces phosphorylation-dependent TIFA oligomerization, activating innate and adaptive immune responses in vivo.","method":"Genome-wide RNAi screen, HBP cytosolic delivery assay, NF-κB reporter, in vivo mouse model, TIFA phosphorylation analysis","journal":"Science","confidence":"High","confidence_rationale":"Tier 1–2 — genome-wide screen plus mechanistic follow-up, in vitro and in vivo, replicated by multiple labs","pmids":["26068852"],"is_preprint":false},{"year":2015,"finding":"TIFA reconstitution in hepatocellular carcinoma cells promotes two independent apoptosis pathways (p53-dependent cell cycle arrest and caspase-8/caspase-3 activation). A non-oligomerizing TIFA mutant had minimal effect, demonstrating that TIFA oligomerization is required for its tumor-suppressive apoptotic function.","method":"TIFA reconstitution in HCC cell lines, non-oligomerizing mutant expression, caspase assays, p53 pathway analysis, xenograft mouse model, TUNEL staining","journal":"Oncogenesis","confidence":"Medium","confidence_rationale":"Tier 2 — gain-of-function reconstitution plus mutant controls, in vivo xenograft, single lab","pmids":["26501855"],"is_preprint":false},{"year":2016,"finding":"TIFA is phosphorylated at Thr9 by Aurora A kinase, triggering NF-κB survival pathway activation in acute myeloid leukemia. Silencing TIFA decreased leukemic cell growth and chemoresistance by downregulating Bcl-2 and Bcl-XL. In vivo delivery of TIFA-inhibitory fragments potentiated myeloblast clearance in xenograft mice.","method":"Aurora A kinase assay for Thr9 phosphorylation, siRNA silencing in AML lines and primary patient cells, Bcl-2/Bcl-XL western blotting, xenograft mouse model","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — kinase-substrate relationship established, functional silencing in patient cells and xenograft, multiple readouts","pmids":["28069801"],"is_preprint":false},{"year":2016,"finding":"TIFA acts as a crucial mediator of NLRP3 inflammasome in vascular endothelial cells, regulating both priming (signal 1) and activation (signal 2). For signal 1, TIFA is transactivated by SREBP2 and induces NF-κB to upregulate NLRP3 components. For signal 2, Akt phosphorylates TIFA at Thr9, enabling TIFA-TIFA homophilic oligomerization that facilitates higher-order NLRP3 assembly and TIFA-caspase-1 interaction.","method":"SREBP2 transactivation assay, Akt kinase assay, TIFA Thr9 phosphorylation, co-immunoprecipitation (TIFA-caspase-1), oligomerization assays, atheroprone flow model, hyperlipidemia mouse model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — multiple mechanisms defined with orthogonal methods, in vitro and in vivo, Akt as upstream kinase identified","pmids":["27965388"],"is_preprint":false},{"year":2016,"finding":"TIFA suppresses hepatocellular carcinoma via MALT1-dependent and MALT1-independent signaling. MALT1 competes with TIFA and its silencing enhances TIFA-induced apoptosis. Long-duration TIFA reconstitution activates JNK and p38 via TRAF6 binding: JNK activation drives apoptosis while p38 governs cell cycle arrest via p53-p21 signaling.","method":"MALT1 shRNA silencing, JNK/p38 activation assays, TRAF6 co-immunoprecipitation, caspase assays, in vitro and xenograft in vivo models","journal":"Signal transduction and targeted therapy","confidence":"Medium","confidence_rationale":"Tier 2 — multiple signaling pathway readouts, in vivo confirmation, single lab","pmids":["29263897"],"is_preprint":false},{"year":2017,"finding":"In H. pylori-infected gastric epithelial cells, ALPK1 kinase phosphorylates TIFA in response to the T4SS-delivered bacterial metabolite β-HBP (D-glycero-β-D-manno-heptose 1,7-bisphosphate), leading to formation of large TIFA complexes (TIFAsomes) that include TRAF2. NF-κB activation, TIFA phosphorylation, and TIFAsome formation all depend on functional ALPK1, establishing ALPK1 as the upstream kinase in this innate immune axis.","method":"Genome-wide RNAi screen, CRISPR/Cas9 knockout, recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry, mutant H. pylori strains","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods including CRISPR KO, MS, and recombinant proteins; replicated across labs","pmids":["28877472"],"is_preprint":false},{"year":2017,"finding":"HBP delivered to the host cytosol via the H. pylori cag-T4SS activates the TIFA-dependent cytosolic surveillance pathway independently of NOD1, driving NF-κB-dependent inflammation within hours of infection and preceding NOD1 activation. CagA toxin contributes to NF-κB responses subsequent to TIFA and NOD1 activation, establishing sequential activation: TIFA → NOD1 → CagA.","method":"Genetic epistasis with TIFA and NOD1 knockdown/knockout, timed NF-κB activation assays, cag-T4SS mutant bacteria, HBP delivery assays","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 — epistasis established with genetic tools, pathway ordering defined, replicated across labs","pmids":["28811347"],"is_preprint":false},{"year":2017,"finding":"TIFA threonine 9 and the FHA domain are required for TIFA oligomerization in both infected and bystander cells during Shigella and Salmonella infection. TIFA oligomerization triggers TRAF6 oligomerization and NF-κB activation. ALPK1 is the critical kinase responsible for TIFA oligomerization and IL-8 expression in response to invasive and extracellular Gram-negative bacteria.","method":"Genome-wide RNAi screen, T9A and FHA domain mutants, NF-κB/IL-8 reporter assays, bacterial infection models, ALPK1 siRNA","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 — genome-wide screen, domain mutagenesis, multiple bacterial pathogens tested; replicated across labs","pmids":["28222186"],"is_preprint":false},{"year":2017,"finding":"TIFA senses HBP released during intracellular bacterial replication in the host cytosol, assembling into large signaling complexes to drive dynamic NF-κB activation proportional to bacterial proliferation rate. IECs lacking TIFA could not discriminate between proliferating and stagnant intracellular bacteria despite intact NOD1/2 pathways, defining TIFA as a rheostat for intracellular bacterial replication.","method":"TIFA-knockout IECs, Shigella intracellular replication assay, NF-κB activation kinetics, HBP detection assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — TIFA KO with specific phenotypic readout, epistasis with NOD1/2, defined bacterial sensing function","pmids":["28514661"],"is_preprint":false},{"year":2018,"finding":"TIFA undergoes nuclear translocation and accumulates on damaged chromatin following genotoxic stress. DNA damage induces TIFA phosphorylation at Thr9, and this pThr9-FHA interaction is required for chromatin enrichment. TIFA then partners with TRAF2 to stimulate ubiquitination of NEMO (whose sumoylation, phosphorylation, and ubiquitination relay DNA damage to NF-κB). TRAF2 knockdown suppressed TIFA-enhanced NEMO ubiquitination under genotoxic stress.","method":"Nuclear fractionation, chromatin immunoprecipitation, TIFA overexpression and T9A mutant, TRAF2 knockdown, NEMO ubiquitination assay, NF-κB target gene expression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — localization linked to function, multiple orthogonal methods including ChIP, mutagenesis, and TRAF2 KD","pmids":["29581234"],"is_preprint":false},{"year":2018,"finding":"Direct binding between TIFA and the TRAF domain of TRAF6 was demonstrated biochemically, and the crystal structures of TRAF6-TRAF domain complexed with wild-type and S174Q/M179D mutant TIFA C-terminal peptides were solved. The structures revealed salt-bridge formation between TIFA residues 177-181 and TRAF6 binding-pocket residues. A rationally designed TIFA double mutant (S174Q/M179D) showed enhanced binding to endogenous full-length TRAF6.","method":"In vitro binding assays, X-ray crystallography of TRAF domain-TIFA peptide complexes, co-immunoprecipitation with full-length TRAF6","journal":"Chembiochem","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with mutagenesis and biochemical validation of direct TIFA-TRAF6 interaction","pmids":["30378729"],"is_preprint":false},{"year":2020,"finding":"H. pylori-induced DNA double-strand breaks occur co-transcriptionally in S-phase cells and depend on ALPK1/TIFA/NF-κB signaling triggered by β-ADP-heptose. DNA damage requires co-transcriptional RNA/DNA hybrids (R-loops) that form as a consequence of β-ADP-heptose/ALPK1/TIFA/NF-κB signaling, linking bacterial innate immune activation to replication stress.","method":"DNA damage markers (γH2AX), R-loop immunofluorescence (S9.6 antibody), ALPK1/TIFA pathway inhibition, gastric organoid primary cells, replication fork stalling assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, primary organoid cells, mechanistic pathway linking TIFA to R-loop-dependent DNA damage","pmids":["33037203"],"is_preprint":false},{"year":2020,"finding":"Crystal structures of mouse TIFA and phosphomimetic mutants (T9D and T9E) confirmed the dimeric structure similar to human TIFA. Size-exclusion chromatography analysis of TIFA and TIFA-TRAF6 complexes suggested a model for the TIFA-TRAF6 signaling complex assembly wherein TIFA oligomers recruit TRAF6.","method":"X-ray crystallography, small angle X-ray scattering, size-exclusion chromatography, phosphomimetic mutagenesis","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 — crystal structures with multiple mutants, SAXS, biochemical complex analysis","pmids":["32198460"],"is_preprint":false},{"year":2021,"finding":"TIFA has dual functions in H. pylori-infected gastric epithelial cells: (1) TIFA-TRAF6 interaction enables TAK1 binding, activating classical NF-κB signaling; (2) TIFA-TRAF2 interaction causes transient displacement of cIAP1 from TRAF2 and proteasomal degradation of cIAP1, facilitating alternative NF-κB pathway activation. Both interactions contribute to TIFAsome formation.","method":"Co-immunoprecipitation (TIFA-TRAF6-TAK1 and TIFA-TRAF2-cIAP1 complexes), TIFA knockout, proteasome inhibitor experiments, classical and alternative NF-κB pathway reporters","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 — multiple co-IP complexes, TIFA KO, two distinct NF-κB pathway mechanisms defined, single lab with strong mechanistic evidence","pmids":["34328245"],"is_preprint":false},{"year":2022,"finding":"TIFA expression in intestinal epithelial cells is required for NF-κB activation by ADP-heptose released from the commensal bacterium Akkermansia muciniphila, via the ALPK1/TIFA/TRAF6 axis. This signaling induces MUC2, BIRC3, and TNFAIP3 expression involved in intestinal barrier maintenance.","method":"Drug inhibitors, CRISPR gene editing (ALPK1, TIFA, TRAF6 KO), ADP-heptose characterization, NF-κB reporter, gene expression assays","journal":"Gut microbes","confidence":"Medium","confidence_rationale":"Tier 2 — CRISPR KO, multiple pathway components validated, functional gene expression readouts","pmids":["36036242"],"is_preprint":false},{"year":2022,"finding":"TIFA promotes colorectal cancer cell proliferation via TRAF6 binding and oligomerization: mutants at the oligomerization site (T9A) or TRAF6-binding site (D6 deletion) abolished TIFA-mediated proliferation enhancement. The mechanism involves RSK and PRAS40 kinase activation downstream of TIFA.","method":"TIFA knockdown, T9A and D6 mutant overexpression, cell proliferation assays in vitro and xenograft in vivo, RSK/PRAS40 phosphorylation assays","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 — mutagenesis of key functional residues, in vivo xenograft, downstream kinase pathway identified","pmids":["35639239"],"is_preprint":false},{"year":2023,"finding":"H. pylori promotes TIFA turnover in gastric epithelial cells via both proteasomal and lysosomal degradation pathways following ALPK1-dependent TIFA activation. H. pylori infection promotes TIFA interaction with free polyubiquitin and with autophagy/trafficking adaptors optineurin, TAX1BP1, and LAMP1, implicating selective autophagy in TIFA degradation. TRAF2, TRAF6, TAK1, and NEMO are not required for TIFA degradation.","method":"Proteasome and lysosome inhibitors, co-immunoprecipitation of TIFA with optineurin/TAX1BP1/LAMP1, H. pylori infection assays, western blotting for TIFA levels","journal":"European journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple inhibitors plus co-IP of degradation complex, genetic dissection of required factors","pmids":["36965415"],"is_preprint":false},{"year":2024,"finding":"TIFAB forms a stable heterodimer with monomeric TIFA (not the intrinsic TIFA dimer), inhibiting TIFA dimer formation and suppressing TIFA-TRAF6 signaling. Structural analysis showed the TIFA/TIFAB complex is a 'pseudo-TIFA dimer' lacking the phosphorylation site and TRAF6-binding motif contributed by TIFAB, preventing formation of the orderly phosphorylated TIFA oligomer required for NF-κB activation.","method":"X-ray crystallography of TIFA/TIFAB complex, biochemical binding assays, cell-based NF-κB activation assays, size-exclusion chromatography","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — crystal structure of inhibitory complex with functional validation, mechanistic basis for TIFAB negative regulation established","pmids":["38442163"],"is_preprint":false},{"year":2024,"finding":"ALPK1-induced TIFA phosphorylation at Thr9 drives liquid-liquid phase separation (LLPS) of TIFA, mediated by the pT9-FHA domain interaction and an intrinsically disordered region. TRAF6 is subsequently recruited into TIFA condensates, where K63-linked polyubiquitin chain synthesis occurs and downstream effectors are enriched and activated. Chemical inhibition of LLPS (compound 22) blocked the ALPK1-TIFA-TRAF6 pathway.","method":"Phase separation assays, fluorescence microscopy of condensates, chemical probe inhibitor, K63-ubiquitination assays in condensates, intrinsically disordered region analysis","journal":"Research (Washington, D.C.)","confidence":"High","confidence_rationale":"Tier 1–2 — direct demonstration of LLPS with functional validation, chemical inhibitor confirmation, mechanistic link to TRAF6 ubiquitination","pmids":["38357697"],"is_preprint":false},{"year":2024,"finding":"IFN-γ licenses the ALPK1/TIFA pathway in human monocytes by upregulating TIFA expression; TIFA induction alone is sufficient to recapitulate the licensing effect of IFN-γ. This licensing was blocked by JAK inhibitors. B cells did not require IFN-γ for ALPK1/TIFA responsiveness.","method":"ADP-heptose stimulation of human mononuclear cell subsets, IFN-γ treatment, TIFA overexpression, JAK inhibitor blockade, NF-κB activation assays","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 — cell-type-specific pathway licensing, TIFA overexpression rescue, pharmacological inhibition, single lab","pmids":["39868044"],"is_preprint":false},{"year":2025,"finding":"TRAF2 interacts with TIFA via two conserved sequence motifs, one of which (Pro159-Xaa-Xaa-Glu162) is a novel TRAF2-binding motif. ADP-heptose induces TIFA degradation by autophagy, and both TRAF2 and TRAF6 contribute to this autophagic degradation process.","method":"Mutagenesis of TIFA motifs, co-immunoprecipitation, autophagy flux assays, TRAF2/TRAF6 knockdown","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 — novel binding motif identified by mutagenesis, autophagy degradation mechanism established with genetic tools","pmids":["40696502"],"is_preprint":false},{"year":2025,"finding":"NSUN3 stabilizes TIFA mRNA through m5C modification recognized by IGF2BP2, increasing TIFA expression. Knockdown of NSUN3 reduced TIFA expression and alleviated LPS-induced kidney injury, placing NSUN3 as an upstream regulator of TIFA at the post-transcriptional level.","method":"MeRIP-seq, methylation RNA immunoprecipitation-qPCR, actinomycin D mRNA stability assay, NSUN3 knockdown, TIFA western blotting","journal":"Clinical and experimental pharmacology & physiology","confidence":"Medium","confidence_rationale":"Tier 2 — m5C modification mapped by MeRIP-seq, mRNA stability assay, functional knockdown, single lab","pmids":["39924309"],"is_preprint":false},{"year":2025,"finding":"METTL3-mediated m6A modification of TIFA mRNA is recognized by IGF2BP2 to enhance TIFA mRNA stability. Increased TIFA promotes NLRP3 transcription via NF-κB signaling, activating the NLRP3 inflammasome and Caspase-1, driving pyroptosis in tubular epithelial cells during ischemic AKI.","method":"MeRIP-seq, RNA-seq, m6A-RIP, METTL3 conditional KO mice, TIFA knockdown, NLRP3/Caspase-1/GSDMD western blot, pyroptosis assays","journal":"Free radical biology & medicine","confidence":"High","confidence_rationale":"Tier 1–2 — MeRIP-seq identifies modification site, conditional KO mouse, multiple mechanistic readouts along METTL3→TIFA→NF-κB→NLRP3→pyroptosis axis","pmids":["41320097"],"is_preprint":false},{"year":2025,"finding":"ALPK1 activation enhances STING pathway outputs including canonical NF-κB/interferon responses and noncanonical STING proton channel-dependent LC3B lipidation and NLRP3 inflammasome activation. Conversely, STING activation increases ALPK1 protein expression and triggers TIFA Thr9 phosphorylation, establishing bidirectional crosstalk between the ALPK1-TIFA and STING innate immune pathways.","method":"ALPK1 gain-of-function mutations, STING pathway reporters, LC3B lipidation assay, NLRP3 inflammasome activation, TIFA pThr9 immunoblot, eIF2α phosphorylation assay","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — multiple pathway readouts, bidirectional signaling demonstrated, preprint not yet peer-reviewed","pmids":[],"is_preprint":true}],"current_model":"TIFA is a small adaptor protein with an FHA domain that, upon phosphorylation at Thr9 by ALPK1 (in response to bacterial ADP-heptose/HBP) or by Aurora A/Akt (in response to cellular stresses), undergoes FHA-pThr9-mediated intermolecular oligomerization and liquid-liquid phase separation to form TIFAsomes; these condensates recruit and activate TRAF6 (via a C-terminal binding motif) and TRAF2 (via a novel Pro-X-X-Glu motif), promoting K63-linked TRAF6 polyubiquitination and downstream TAK1/IKK/NF-κB activation (both classical and alternative pathways), NLRP3 inflammasome priming and assembly, and DNA damage-induced NF-κB signaling via NEMO ubiquitination, while TIFAB negatively regulates this pathway by forming an inhibitory heterodimer with monomeric TIFA."},"narrative":{"teleology":[{"year":2000,"claim":"Identification of TIFA (T6BP) as a TRAF6-interacting adaptor established the first molecular link between this protein and IL-1/IRAK-dependent NF-κB signaling, answering how TRAF6 receives upstream signals.","evidence":"Yeast two-hybrid screen and co-immunoprecipitation with domain mapping in IL-1-stimulated cells","pmids":["10920205"],"confidence":"Medium","gaps":["Single lab; endogenous interaction under physiological conditions not fully characterized","Upstream kinase unknown","TIFA's binding to other TRAF family members not tested"]},{"year":2002,"claim":"Discovery that TIFA also binds TRAF2 and activates both NF-κB and AP-1 expanded the adaptor's functional scope beyond the TRAF6 axis.","evidence":"Mammalian two-hybrid screen and NF-κB/AP-1 reporter assays with co-immunoprecipitation","pmids":["11798190"],"confidence":"Medium","gaps":["TRAF2-binding site on TIFA not mapped","Physiological stimulus driving TIFA-TRAF2 interaction unknown","Relationship between TRAF2 and TRAF6 binding not tested"]},{"year":2003,"claim":"Demonstration that TIFA bridges IRAK-1 and TRAF6, and that both the FHA domain and TRAF6-binding motif are required for NF-κB/JNK activation, defined TIFA as a scaffolding adaptor rather than a passive binder.","evidence":"Reciprocal co-IP with endogenous proteins, IL-1 stimulation, FHA and TRAF6-binding mutants in reporter assays","pmids":["12566447"],"confidence":"High","gaps":["FHA domain ligand (phosphopeptide source) not identified","In vivo relevance not tested","Stoichiometry of the IRAK-1/TIFA/TRAF6 complex undefined"]},{"year":2004,"claim":"In vitro reconstitution proved that only oligomeric TIFA activates TRAF6 K63-linked polyubiquitination and downstream TAK1/IKK, establishing oligomerization as the activating switch and ruling out proteasome dependence.","evidence":"Purified protein reconstitution with TIFA, TRAF6, TAK1 complex, and Ubc13-Uev1A; gel filtration separation of oligomeric forms","pmids":["15492226"],"confidence":"High","gaps":["What triggers TIFA oligomerization in cells was unknown","Structural basis of oligomerization not resolved","TIFAB's inhibitory mechanism only partially characterized"]},{"year":2004,"claim":"Identification of TIFAB as a TIFA-binding negative regulator that lacks TRAF-binding capacity revealed the first endogenous brake on TIFA-mediated signaling.","evidence":"Co-immunoprecipitation and NF-κB reporter assays with TIFAB overexpression","pmids":["15047173"],"confidence":"Medium","gaps":["TIFAB inhibitory mechanism (conformational vs. competitive) unclear","Structural basis of TIFA-TIFAB interaction not solved","Physiological contexts of TIFAB regulation unknown"]},{"year":2012,"claim":"Discovery that Thr9 phosphorylation drives FHA-mediated intermolecular oligomerization between TIFA dimers solved the long-standing question of how TIFA oligomerization is triggered.","evidence":"Phosphosite identification, NMR/biochemical analysis, size-exclusion chromatography, FHA-pThr9 binding assays, siRNA silencing","pmids":["22566686"],"confidence":"High","gaps":["Identity of the Thr9 kinase(s) in different signaling contexts unknown","Structural visualization of the oligomeric assembly not achieved","Contribution of pThr9-independent oligomerization mechanisms not excluded"]},{"year":2015,"claim":"Crystal structures of TIFA and the FHA-pThr9 peptide complex provided the atomic-level explanation for why pThr9 binding can only occur between different dimers, rationalizing phosphorylation-dependent polymerization.","evidence":"X-ray crystallography, SAXS, functional mutagenesis","pmids":["26389808"],"confidence":"High","gaps":["Full-length oligomeric structure not solved","In vivo stoichiometry of TIFAsomes unknown","Dynamics of assembly and disassembly not captured"]},{"year":2015,"claim":"A genome-wide RNAi screen identified TIFA as essential for the cytosolic innate immune response to bacterial HBP, establishing TIFA as a pattern recognition adaptor for a conserved Gram-negative metabolite.","evidence":"Genome-wide RNAi screen, HBP cytosolic delivery, NF-κB reporter, in vivo mouse model","pmids":["26068852"],"confidence":"High","gaps":["The direct sensor/kinase upstream of TIFA for HBP not yet identified","Whether other bacterial metabolites activate TIFA unknown","Contribution of TIFA to adaptive immunity only partially explored"]},{"year":2016,"claim":"Identification of Akt as a Thr9 kinase linking disturbed hemodynamic flow to TIFA oligomerization and NLRP3 inflammasome assembly in endothelial cells demonstrated that TIFA integrates metabolic and mechanical stress signals beyond pathogen detection.","evidence":"Akt kinase assay, TIFA oligomerization assays, TIFA-caspase-1 co-IP, atheroprone flow model, hyperlipidemia mouse model","pmids":["27965388"],"confidence":"High","gaps":["How TIFA physically bridges NLRP3 ASC specks not resolved","Whether TIFA directly interacts with NLRP3 components structurally unknown","Relative contribution of Akt vs. ALPK1 in different tissues not defined"]},{"year":2017,"claim":"Convergent studies identified ALPK1 as the kinase that phosphorylates TIFA at Thr9 in response to bacterial β-HBP/ADP-heptose delivered by T4SS and other secretion systems, completing the ALPK1→TIFA→TRAF6→NF-κB innate immune axis.","evidence":"Genome-wide RNAi screens, CRISPR knockout of ALPK1, recombinant protein technology, multiple Gram-negative bacterial pathogens (H. pylori, Shigella, Salmonella)","pmids":["28877472","28222186","28514661","28811347"],"confidence":"High","gaps":["Direct in vitro kinase assay of ALPK1 on TIFA not reported in all studies","How ADP-heptose activates ALPK1 enzymatically not resolved","Whether additional kinases act on Thr9 in infection contexts not excluded"]},{"year":2018,"claim":"Discovery that TIFA translocates to damaged chromatin and promotes TRAF2-dependent NEMO ubiquitination extended TIFA function to DNA damage-induced NF-κB signaling, a context independent of pathogen detection.","evidence":"Nuclear fractionation, chromatin enrichment, TIFA T9A mutant, TRAF2 knockdown, NEMO ubiquitination assay","pmids":["29581234"],"confidence":"High","gaps":["Kinase responsible for Thr9 phosphorylation in genotoxic stress not identified","Whether TIFA forms TIFAsome-like condensates on chromatin untested","Relationship to ATM/ATR signaling not explored"]},{"year":2018,"claim":"Crystal structures of the TRAF6-TIFA C-terminal peptide complex defined the salt-bridge network mediating TIFA-TRAF6 binding and enabled rational design of enhanced-affinity mutants.","evidence":"X-ray crystallography of TRAF6 TRAF domain with WT and S174Q/M179D TIFA peptides, binding assays","pmids":["30378729"],"confidence":"High","gaps":["Full-length TIFA-TRAF6 complex structure not achieved","Whether enhanced-affinity mutants alter signaling output in cells not fully tested","Structural basis for TIFA-TRAF2 interaction not resolved"]},{"year":2021,"claim":"Dissection of TIFA's dual engagement of TRAF6 and TRAF2 revealed that TIFA-TRAF6 activates classical NF-κB via TAK1, while TIFA-TRAF2 triggers cIAP1 displacement and degradation to activate the alternative NF-κB pathway.","evidence":"Co-IP of TIFA-TRAF6-TAK1 and TIFA-TRAF2-cIAP1 complexes, TIFA knockout, proteasome inhibitor experiments","pmids":["34328245"],"confidence":"High","gaps":["Whether the two complexes form in the same or different TIFAsomes unknown","Relative kinetics and stoichiometry of the two pathways not defined","Whether TIFA-TRAF2 function extends to non-infection contexts not tested"]},{"year":2024,"claim":"Structural resolution of the TIFA-TIFAB heterodimer revealed it as a pseudo-TIFA dimer lacking the Thr9 phosphosite and TRAF6-binding motif, providing the molecular basis for TIFAB's dominant-negative inhibition of TIFA signaling.","evidence":"X-ray crystallography of TIFA/TIFAB complex, biochemical binding assays, NF-κB activation assays, SEC","pmids":["38442163"],"confidence":"High","gaps":["In vivo regulation of TIFAB expression and its physiological impact not characterized","Whether TIFAB competes with pThr9-mediated oligomerization under infection unclear","No structural data for TIFAB in the context of full-length oligomeric TIFA"]},{"year":2024,"claim":"Demonstration that pThr9-dependent TIFA oligomers undergo liquid-liquid phase separation to form functional condensates that concentrate TRAF6 and support K63-linked ubiquitin chain synthesis reframed TIFAsomes as biomolecular condensates.","evidence":"Phase separation assays, fluorescence microscopy, chemical LLPS inhibitor (compound 22), K63-ubiquitination assays in condensates","pmids":["38357697"],"confidence":"High","gaps":["Whether LLPS occurs in vivo in physiological infection models not shown","Material properties (viscosity, aging) of TIFAsome condensates not characterized","Selectivity of chemical LLPS inhibitor for TIFA condensates vs. other condensates unknown"]},{"year":2025,"claim":"Identification of a novel TRAF2-binding motif (PxxE) in TIFA and demonstration that both TRAF2 and TRAF6 contribute to TIFA autophagic degradation established that TIFA turnover is an active signaling-coupled process.","evidence":"Mutagenesis of PxxE motif, autophagy flux assays, TRAF2/TRAF6 knockdown","pmids":["40696502"],"confidence":"Medium","gaps":["Structural basis of PxxE-TRAF2 interaction not solved","Whether autophagy of TIFA serves signal termination or signal amplification not distinguished","Relationship to optineurin/TAX1BP1-mediated degradation pathway not integrated"]},{"year":null,"claim":"Key unresolved questions include: the full-length structure of an active TIFAsome oligomer, whether TIFA condensates form in vivo during physiological infection, how different upstream kinases (ALPK1, Aurora A, Akt) are selected in different cellular contexts, and whether TIFA participates in additional innate sensing pathways beyond ADP-heptose.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full-length oligomeric or condensate-state structure available","In vivo condensate formation not confirmed by live imaging","Context-dependent kinase selection mechanism unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,3,8,10]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,14,29]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,8,11,29]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[20]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[20]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[11,16,17,18,19,25]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,3,8,14,24]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[12,15,33]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[13,22,26]}],"complexes":["TIFAsome"],"partners":["TRAF6","TRAF2","ALPK1","IRAK1","TIFAB","NEMO","TAK1","MYO6"],"other_free_text":[]},"mechanistic_narrative":"TIFA is a small adaptor protein that functions as a central signaling hub in innate immunity and NF-κB activation by undergoing phosphorylation-dependent oligomerization and liquid-liquid phase separation to recruit and activate TRAF6 and TRAF2. Phosphorylation of Thr9—by ALPK1 in response to bacterial ADP-heptose/HBP, by Aurora A in leukemia, or by Akt under disturbed flow—enables the FHA domain of one TIFA dimer to engage pThr9 on another, driving higher-order oligomer (TIFAsome) assembly that promotes TRAF6 K63-linked polyubiquitination and downstream TAK1/IKK/NF-κB signaling through both classical and alternative pathways [PMID:26068852, PMID:15492226, PMID:26389808, PMID:34328245, PMID:38357697]. Beyond canonical NF-κB activation, TIFA oligomerization facilitates NLRP3 inflammasome priming and assembly, DNA damage-induced NF-κB signaling via TRAF2-dependent NEMO ubiquitination on damaged chromatin, and pro-apoptotic JNK/p38 signaling in hepatocellular carcinoma [PMID:27965388, PMID:29581234, PMID:29263897]. TIFAB negatively regulates this pathway by forming a pseudo-TIFA heterodimer that lacks the pThr9 site and TRAF6-binding motif, preventing productive oligomerization [PMID:38442163]."},"prefetch_data":{"uniprot":{"accession":"Q96CG3","full_name":"TRAF-interacting protein with FHA domain-containing protein A","aliases":["Putative MAPK-activating protein PM14","Putative NF-kappa-B-activating protein 20","TRAF2-binding protein"],"length_aa":184,"mass_kda":21.4,"function":"Adapter molecule that plays a key role in the activation of pro-inflammatory NF-kappa-B signaling following detection of bacterial pathogen-associated molecular pattern metabolites (PAMPs) (PubMed:12566447, PubMed:15492226, PubMed:26068852, PubMed:28222186, PubMed:28877472, PubMed:30111836). Promotes activation of an innate immune response by inducing the oligomerization and polyubiquitination of TRAF6, which leads to the activation of TAK1 and IKK through a proteasome-independent mechanism (PubMed:15492226, PubMed:26068852). TIFA-dependent innate immune response is triggered by ADP-D-glycero-beta-D-manno-heptose (ADP-Heptose), a potent PAMP present in all Gram-negative and some Gram-positive bacteria: ADP-Heptose is recognized by ALPK1, which phosphorylates TIFA at Thr-9, leading to TIFA homooligomerization and subsequent activation of pro-inflammatory NF-kappa-B signaling (PubMed:30111836)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q96CG3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TIFA","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TIFA","total_profiled":1310},"omim":[{"mim_id":"613692","title":"TERMINAL URIDYLYL TRANSFERASE 4; TUT4","url":"https://www.omim.org/entry/613692"},{"mim_id":"612663","title":"TRAF-INTERACTING PROTEIN WITH FORKHEAD-ASSOCIATED DOMAIN, FAMILY MEMBER B; TIFAB","url":"https://www.omim.org/entry/612663"},{"mim_id":"609028","title":"TRAF-INTERACTING PROTEIN WITH FORKHEAD-ASSOCIATED DOMAIN; TIFA","url":"https://www.omim.org/entry/609028"},{"mim_id":"607347","title":"ALPHA KINASE 1; ALPK1","url":"https://www.omim.org/entry/607347"},{"mim_id":"602355","title":"TNF RECEPTOR-ASSOCIATED FACTOR 6; TRAF6","url":"https://www.omim.org/entry/602355"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Nucleoli","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TIFA"},"hgnc":{"alias_symbol":["MGC20791","T2BP","T6BP","TIFAA"],"prev_symbol":[]},"alphafold":{"accession":"Q96CG3","domains":[{"cath_id":"2.60.200.20","chopping":"14-149","consensus_level":"high","plddt":96.4101,"start":14,"end":149}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96CG3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96CG3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96CG3-F1-predicted_aligned_error_v6.png","plddt_mean":86.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TIFA","jax_strain_url":"https://www.jax.org/strain/search?query=TIFA"},"sequence":{"accession":"Q96CG3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96CG3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96CG3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96CG3"}},"corpus_meta":[{"pmid":"28877472","id":"PMC_28877472","title":"ALPK1- and TIFA-Dependent Innate Immune Response Triggered by the Helicobacter pylori Type IV Secretion System.","date":"2017","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/28877472","citation_count":147,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"26068852","id":"PMC_26068852","title":"INNATE IMMUNITY. Cytosolic detection of the bacterial metabolite HBP activates TIFA-dependent innate immunity.","date":"2015","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/26068852","citation_count":126,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"28811347","id":"PMC_28811347","title":"TIFA Signaling in Gastric Epithelial Cells Initiates the cag Type 4 Secretion System-Dependent Innate Immune Response to Helicobacter pylori Infection.","date":"2017","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/28811347","citation_count":118,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"15492226","id":"PMC_15492226","title":"TIFA activates IkappaB kinase (IKK) by promoting oligomerization and ubiquitination of TRAF6.","date":"2004","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/15492226","citation_count":115,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"28222186","id":"PMC_28222186","title":"ALPK1 controls TIFA/TRAF6-dependent innate immunity against heptose-1,7-bisphosphate of gram-negative bacteria.","date":"2017","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/28222186","citation_count":104,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"33037203","id":"PMC_33037203","title":"The ALPK1/TIFA/NF-κB axis links a bacterial carcinogen to R-loop-induced replication stress.","date":"2020","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/33037203","citation_count":89,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"17635994","id":"PMC_17635994","title":"T6BP and NDP52 are myosin VI binding partners with potential roles in cytokine signalling and cell adhesion.","date":"2007","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/17635994","citation_count":80,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"12566447","id":"PMC_12566447","title":"Identification of TIFA as an adapter protein that links tumor necrosis factor receptor-associated factor 6 (TRAF6) to interleukin-1 (IL-1) receptor-associated kinase-1 (IRAK-1) in IL-1 receptor signaling.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12566447","citation_count":75,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"36036242","id":"PMC_36036242","title":"Akkermansia muciniphila upregulates genes involved in maintaining the intestinal barrier function via ADP-heptose-dependent activation of the ALPK1/TIFA pathway.","date":"2022","source":"Gut microbes","url":"https://pubmed.ncbi.nlm.nih.gov/36036242","citation_count":62,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"10920205","id":"PMC_10920205","title":"T6BP, a TRAF6-interacting protein involved in IL-1 signaling.","date":"2000","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/10920205","citation_count":56,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"11798190","id":"PMC_11798190","title":"T2BP, a novel TRAF2 binding protein, can activate NF-kappaB and AP-1 without TNF stimulation.","date":"2002","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/11798190","citation_count":55,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"28514661","id":"PMC_28514661","title":"Innate Recognition of Intracellular Bacterial Growth Is Driven by the TIFA-Dependent Cytosolic Surveillance Pathway.","date":"2017","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/28514661","citation_count":53,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"28069801","id":"PMC_28069801","title":"Aurora A and NF-κB Survival Pathway Drive Chemoresistance in Acute Myeloid Leukemia via the TRAF-Interacting Protein TIFA.","date":"2016","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/28069801","citation_count":48,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"27965388","id":"PMC_27965388","title":"TIFA as a crucial mediator for NLRP3 inflammasome.","date":"2016","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/27965388","citation_count":44,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"38126163","id":"PMC_38126163","title":"Fusobacterium nucleatum promotes inflammatory and anti-apoptotic responses in colorectal cancer cells via ADP-heptose release and ALPK1/TIFA axis activation.","date":"2023","source":"Gut microbes","url":"https://pubmed.ncbi.nlm.nih.gov/38126163","citation_count":43,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"34328245","id":"PMC_34328245","title":"TIFA has dual functions in Helicobacter pylori-induced classical and alternative NF-κB pathways.","date":"2021","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/34328245","citation_count":41,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"16643855","id":"PMC_16643855","title":"A novel Zinc finger protein, ZCCHC11, interacts with TIFA and modulates TLR signaling.","date":"2006","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/16643855","citation_count":41,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"22566686","id":"PMC_22566686","title":"Intermolecular binding between TIFA-FHA and TIFA-pT mediates tumor necrosis factor alpha stimulation and NF-κB activation.","date":"2012","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/22566686","citation_count":40,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"29263897","id":"PMC_29263897","title":"TIFA suppresses hepatocellular carcinoma progression via MALT1-dependent and -independent signaling pathways.","date":"2016","source":"Signal transduction and targeted therapy","url":"https://pubmed.ncbi.nlm.nih.gov/29263897","citation_count":37,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"26501855","id":"PMC_26501855","title":"TIFA, an inflammatory signaling adaptor, is tumor suppressive for liver cancer.","date":"2015","source":"Oncogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/26501855","citation_count":32,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"23722163","id":"PMC_23722163","title":"TIFA upregulation after hypoxia-reoxygenation is TLR4- and MyD88-dependent and associated with HMGB1 upregulation and release.","date":"2013","source":"Free radical biology & medicine","url":"https://pubmed.ncbi.nlm.nih.gov/23722163","citation_count":31,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"26389808","id":"PMC_26389808","title":"Uncovering the Mechanism of Forkhead-Associated Domain-Mediated TIFA Oligomerization That Plays a Central Role in Immune Responses.","date":"2015","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26389808","citation_count":29,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"32910997","id":"PMC_32910997","title":"TIFA and TIFAB: FHA-domain proteins involved in inflammation, hematopoiesis, and disease.","date":"2020","source":"Experimental hematology","url":"https://pubmed.ncbi.nlm.nih.gov/32910997","citation_count":23,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"15047173","id":"PMC_15047173","title":"TIFAB inhibits TIFA, TRAF-interacting protein with a forkhead-associated domain.","date":"2004","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/15047173","citation_count":20,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"31123889","id":"PMC_31123889","title":"Role of NOD1 and ALPK1/TIFA Signalling in Innate Immunity Against Helicobacter pylori Infection.","date":"2019","source":"Current topics in microbiology and immunology","url":"https://pubmed.ncbi.nlm.nih.gov/31123889","citation_count":19,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"36586274","id":"PMC_36586274","title":"The potential biomarker TIFA regulates pyroptosis in sepsis-induced acute kidney injury.","date":"2022","source":"International immunopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/36586274","citation_count":19,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"29581234","id":"PMC_29581234","title":"TRAF-interacting protein with forkhead-associated domain (TIFA) transduces DNA damage-induced activation of NF-κB.","date":"2018","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29581234","citation_count":15,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"30378729","id":"PMC_30378729","title":"Binding and Enhanced Binding between Key Immunity Proteins TRAF6 and TIFA.","date":"2018","source":"Chembiochem : a European journal of chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/30378729","citation_count":14,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"29975933","id":"PMC_29975933","title":"TIFA Promotes Cell Survival and Migration in Lung Adenocarcinoma.","date":"2018","source":"Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/29975933","citation_count":13,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"35635239","id":"PMC_35635239","title":"TIFA promotes colorectal cancer cell proliferation in an RSK- and PRAS40-dependent manner.","date":"2022","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/35635239","citation_count":12,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"36215666","id":"PMC_36215666","title":"The Autophagy Receptor TAX1BP1 (T6BP) improves antigen presentation by MHC-II molecules.","date":"2022","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/36215666","citation_count":12,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"32198460","id":"PMC_32198460","title":"Structural analysis of TIFA: Insight into TIFA-dependent signal transduction in innate immunity.","date":"2020","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/32198460","citation_count":10,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"39260704","id":"PMC_39260704","title":"Targeting PYK2 with heterobifunctional T6BP helps mitigate MASLD and MASH-HCC progression.","date":"2024","source":"Journal of hepatology","url":"https://pubmed.ncbi.nlm.nih.gov/39260704","citation_count":9,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"32452604","id":"PMC_32452604","title":"Nonhydrolyzable Heptose Bis- and Monophosphate Analogues Modulate Pro-inflammatory TIFA-NF-κB Signaling.","date":"2020","source":"Chembiochem : a European journal of chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/32452604","citation_count":9,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"39924309","id":"PMC_39924309","title":"NSUN3 Aggravates Sepsis-Associated Acute Kidney Injury by Stabilising TIFA mRNA Through m5C.","date":"2025","source":"Clinical and experimental pharmacology & physiology","url":"https://pubmed.ncbi.nlm.nih.gov/39924309","citation_count":8,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"38357697","id":"PMC_38357697","title":"ADP-Hep-Induced Liquid Phase Condensation of TIFA-TRAF6 Activates ALPK1/TIFA-Dependent Innate Immune Responses.","date":"2024","source":"Research (Washington, D.C.)","url":"https://pubmed.ncbi.nlm.nih.gov/38357697","citation_count":7,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"38442163","id":"PMC_38442163","title":"TIFAB regulates the TIFA-TRAF6 signaling pathway involved in innate immunity by forming a heterodimer complex with TIFA.","date":"2024","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/38442163","citation_count":6,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"36965415","id":"PMC_36965415","title":"Helicobacter pylori regulates TIFA turnover in gastric epithelial cells.","date":"2023","source":"European journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/36965415","citation_count":6,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"16023795","id":"PMC_16023795","title":"Identification and characterization of Xenopus laevis homologs of mammalian TRAF6 and its binding protein TIFA.","date":"2005","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/16023795","citation_count":6,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"38099344","id":"PMC_38099344","title":"TIFA contributes to periodontitis in diabetic mice via activating the NF‑κB signaling pathway.","date":"2023","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/38099344","citation_count":4,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"39842611","id":"PMC_39842611","title":"TIFA renders intestinal epithelial cells responsive to microbial ADP-heptose and drives colonic inflammation in mice.","date":"2025","source":"Mucosal immunology","url":"https://pubmed.ncbi.nlm.nih.gov/39842611","citation_count":3,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"37615983","id":"PMC_37615983","title":"Inducing a Proinflammatory Response with Bioengineered Yeast Vacuoles with TLR2-Binding Peptides (VacT2BP) as a Drug Carrier for Daunorubicin Delivery.","date":"2023","source":"ACS applied materials & interfaces","url":"https://pubmed.ncbi.nlm.nih.gov/37615983","citation_count":3,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"39868044","id":"PMC_39868044","title":"IFN-γ licenses normal and pathogenic ALPK1/TIFA pathway in human monocytes.","date":"2024","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/39868044","citation_count":2,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"39481822","id":"PMC_39481822","title":"TIFA enhances glycolysis through E2F1 and promotes the progression of glioma.","date":"2024","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/39481822","citation_count":2,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"40696502","id":"PMC_40696502","title":"TRAF2 binds to TIFA via a novel motif and contributes to its autophagic degradation.","date":"2025","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/40696502","citation_count":0,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"41320097","id":"PMC_41320097","title":"METTL3-mediated m6A modification of TIFA mRNA promotes tubular cell pyroptosis in acute kidney injury.","date":"2025","source":"Free radical biology & medicine","url":"https://pubmed.ncbi.nlm.nih.gov/41320097","citation_count":0,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":null,"id":"bio_10.1101_2025.06.30.662363","title":"Crosstalk Between ALPK1 and STING: A Synergistic Axis in Innate Immune Activation and Human Inflammatory Disease","date":"2025-07-04","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.30.662363","citation_count":0,"is_preprint":true,"source_track":"pubmed_title"},{"pmid":"23128233","id":"PMC_23128233","title":"Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease.","date":"2012","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/23128233","citation_count":3725,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12477932","id":"PMC_12477932","title":"Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences.","date":"2002","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/12477932","citation_count":1479,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"18978771","id":"PMC_18978771","title":"A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity.","date":"2008","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/18978771","citation_count":1061,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"18264110","id":"PMC_18264110","title":"IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia.","date":"2008","source":"Nature medicine","url":"https://pubmed.ncbi.nlm.nih.gov/18264110","citation_count":962,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"32352535","id":"PMC_32352535","title":"COVID-19 and the cardiovascular system: implications for risk assessment, diagnosis, and treatment options.","date":"2020","source":"Cardiovascular research","url":"https://pubmed.ncbi.nlm.nih.gov/32352535","citation_count":943,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"32901017","id":"PMC_32901017","title":"Intestinal microbiota-derived short-chain fatty acids regulation of immune cell IL-22 production and gut immunity.","date":"2020","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/32901017","citation_count":826,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"19920355","id":"PMC_19920355","title":"Th22 cells represent a distinct human T cell subset involved in epidermal immunity and remodeling.","date":"2009","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/19920355","citation_count":807,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"25706098","id":"PMC_25706098","title":"Interleukin-22: immunobiology and pathology.","date":"2015","source":"Annual review of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/25706098","citation_count":730,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21600206","id":"PMC_21600206","title":"Aryl hydrocarbon receptor-induced signals up-regulate IL-22 production and inhibit inflammation in the gastrointestinal tract.","date":"2011","source":"Gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/21600206","citation_count":549,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"19481975","id":"PMC_19481975","title":"Anti-inflammatory and pro-inflammatory roles of TGF-beta, IL-10, and IL-22 in immunity and autoimmunity.","date":"2009","source":"Current opinion in pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/19481975","citation_count":526,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15122762","id":"PMC_15122762","title":"Interleukin 22 (IL-22) plays a protective role in T cell-mediated murine hepatitis: IL-22 is a survival factor for hepatocytes via STAT3 activation.","date":"2004","source":"Hepatology (Baltimore, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/15122762","citation_count":497,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"20032993","id":"PMC_20032993","title":"Circulating Th17, Th22, and Th1 cells are increased in psoriasis.","date":"2009","source":"The Journal of investigative dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/20032993","citation_count":495,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"16537974","id":"PMC_16537974","title":"IL-22 is increased in active Crohn's disease and promotes proinflammatory gene expression and intestinal epithelial cell migration.","date":"2006","source":"American journal of physiology. Gastrointestinal and liver physiology","url":"https://pubmed.ncbi.nlm.nih.gov/16537974","citation_count":472,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"10875937","id":"PMC_10875937","title":"Interleukin (IL)-22, a novel human cytokine that signals through the interferon receptor-related proteins CRF2-4 and IL-22R.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10875937","citation_count":441,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15489334","id":"PMC_15489334","title":"The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).","date":"2004","source":"Genome research","url":"https://pubmed.ncbi.nlm.nih.gov/15489334","citation_count":438,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"19479869","id":"PMC_19479869","title":"Frequency and phenotype of peripheral blood Th17 cells in ankylosing spondylitis and rheumatoid arthritis.","date":"2009","source":"Arthritis and rheumatism","url":"https://pubmed.ncbi.nlm.nih.gov/19479869","citation_count":437,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12087100","id":"PMC_12087100","title":"Interleukin-22 (IL-22) activates the JAK/STAT, ERK, JNK, and p38 MAP kinase pathways in a rat hepatoma cell line. Pathways that are shared with and distinct from IL-10.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12087100","citation_count":407,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"16143135","id":"PMC_16143135","title":"Interleukin-22, a member of the IL-10 subfamily, induces inflammatory responses in colonic subepithelial myofibroblasts.","date":"2005","source":"Gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/16143135","citation_count":400,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"10657629","id":"PMC_10657629","title":"Cloning and characterization of IL-10-related T cell-derived inducible factor (IL-TIF), a novel cytokine structurally related to IL-10 and inducible by IL-9.","date":"2000","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/10657629","citation_count":399,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"23405899","id":"PMC_23405899","title":"IL-22, not simply a Th17 cytokine.","date":"2013","source":"Immunological reviews","url":"https://pubmed.ncbi.nlm.nih.gov/23405899","citation_count":371,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"19122664","id":"PMC_19122664","title":"Ulcerative colitis-risk loci on chromosomes 1p36 and 12q15 found by genome-wide association study.","date":"2009","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/19122664","citation_count":336,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"25024136","id":"PMC_25024136","title":"CX₃CR1⁺ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22.","date":"2014","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/25024136","citation_count":334,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"11035029","id":"PMC_11035029","title":"Identification of the functional interleukin-22 (IL-22) receptor complex: the IL-10R2 chain (IL-10Rbeta ) is a common chain of both the IL-10 and IL-22 (IL-10-related T cell-derived inducible factor, IL-TIF) receptor complexes.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11035029","citation_count":330,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"24610014","id":"PMC_24610014","title":"Epidermal Th22 and Tc17 cells form a localized disease memory in clinically healed psoriasis.","date":"2014","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/24610014","citation_count":329,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"24816405","id":"PMC_24816405","title":"IL-22(+)CD4(+) T cells promote colorectal cancer stemness via STAT3 transcription factor activation and induction of the methyltransferase DOT1L.","date":"2014","source":"Immunity","url":"https://pubmed.ncbi.nlm.nih.gov/24816405","citation_count":324,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"10954742","id":"PMC_10954742","title":"Human interleukin-10-related T cell-derived inducible factor: molecular cloning and functional characterization as an hepatocyte-stimulating factor.","date":"2000","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/10954742","citation_count":292,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12975309","id":"PMC_12975309","title":"The secreted protein discovery initiative (SPDI), a large-scale effort to identify novel human secreted and transmembrane proteins: a bioinformatics assessment.","date":"2003","source":"Genome research","url":"https://pubmed.ncbi.nlm.nih.gov/12975309","citation_count":285,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21124836","id":"PMC_21124836","title":"Overrepresentation of IL-17A and IL-22 producing CD8 T cells in lesional skin suggests their involvement in the pathogenesis of psoriasis.","date":"2010","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21124836","citation_count":262,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21674558","id":"PMC_21674558","title":"Interleukin-22 promotes human hepatocellular carcinoma by activation of STAT3.","date":"2011","source":"Hepatology (Baltimore, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/21674558","citation_count":259,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15765404","id":"PMC_15765404","title":"Interleukin-21 enhances T-helper cell type I signaling and interferon-gamma production in Crohn's disease.","date":"2005","source":"Gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/15765404","citation_count":243,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":24360,"output_tokens":7254,"usd":0.090945},"stage2":{"model":"claude-opus-4-6","input_tokens":11039,"output_tokens":4197,"usd":0.24018},"total_usd":0.722975,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":39498,"output_tokens":9584,"usd":0.131127},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":13568,"output_tokens":4239,"usd":0.260723}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"TIFA (T6BP) specifically associates with TRAF6 via an interaction between the coiled-coil region of T6BP and the N-terminal ring finger and zinc finger domains of TRAF6; IL-1 (but not TNF) induces TRAF6-T6BP complex formation in a ligand- and IRAK-dependent manner\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, ligand-stimulation assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP with domain mapping, replicated in subsequent studies\",\n      \"pmids\": [\"10920205\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"TIFA (T2BP) binds TRAF2 via the TRAF domain of TRAF2, and overexpression of T2BP activates NF-κB and AP-1 in a dose-dependent manner\",\n      \"method\": \"Mammalian two-hybrid screen, co-immunoprecipitation, reporter assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, Co-IP plus reporter assay but limited mechanistic follow-up\",\n      \"pmids\": [\"11798190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"TIFA acts as an adapter linking IRAK-1 to TRAF6 in IL-1 receptor signaling; TIFA associates with TRAF6 constitutively and with IRAK-1 only upon IL-1 stimulation; mutations abolishing TRAF6 binding or FHA domain phosphopeptide binding prevent NF-κB and JNK activation\",\n      \"method\": \"Co-immunoprecipitation of endogenous proteins, transient transfection, site-directed mutagenesis, NF-κB/JNK reporter assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — endogenous Co-IP plus mutagenesis, replicated across multiple labs\",\n      \"pmids\": [\"12566447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TIFA activates IKK by promoting oligomerization and K63-linked polyubiquitination of TRAF6; only high-molecular-weight oligomeric TIFA is active; activation requires TRAF6 binding and is reconstituted in vitro with purified TIFA, TRAF6, TAK1 complex, and Ubc13-Uev1A\",\n      \"method\": \"In vitro reconstitution with purified proteins, gel-filtration oligomer fractionation, in vitro IKK activation assay, TRAF6-binding-defective mutant analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with purified proteins plus mutagenesis, foundational study replicated by many labs\",\n      \"pmids\": [\"15492226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TIFAB (a TIFA homolog lacking a phosphorylation site and TRAF6-binding motif) binds TIFA and inhibits TIFA-mediated NF-κB activation, potentially by inducing a conformational change in TIFA that alters TRAF6 interaction\",\n      \"method\": \"Co-immunoprecipitation, NF-κB reporter assays, TRAF6 pulldown\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab Co-IP with functional reporter assay but limited mechanistic depth\",\n      \"pmids\": [\"15047173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ZCCHC11, a zinc finger protein, interacts with TIFA (identified by GST-TIFA affinity purification and mass spectrometry from macrophage phosphoproteins) and functions as a negative regulator of TLR-mediated NF-κB activation; ZCCHC11L translocates from nucleus to cytoplasm upon LPS stimulation and binds TIFA\",\n      \"method\": \"GST pulldown/affinity purification, mass spectrometry, siRNA knockdown, NF-κB reporter assays, immunofluorescence\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — MS-identified interaction with functional KD validation in single study\",\n      \"pmids\": [\"16643855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TIFA (T6BP) is a myosin VI binding partner; interaction confirmed in vitro and in vivo with binding sites mapped; T6BP and myosin VI co-localize at the trans-Golgi and perinuclear vesicles; loss of T6BP reduces membrane ruffling, increases stress fibers/focal adhesions, upregulates constitutive secretion, and T6BP inhibits NF-κB activation\",\n      \"method\": \"Yeast two-hybrid, in vitro binding, co-immunoprecipitation, immunofluorescence, electron microscopy, RNAi knockdown with phenotypic readouts\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Y2H, in vitro binding, Co-IP, EM, KD phenotype) in single study\",\n      \"pmids\": [\"17635994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TIFA is phosphorylated at Thr9; phospho-Thr9 binds the FHA domain of a different TIFA dimer (intermolecular FHA-pT9 interaction) driving TIFA oligomerization and NF-κB activation; unphosphorylated TIFA exists as an intrinsic dimer; silencing endogenous TIFA attenuates TNF-α-mediated downstream NF-κB signaling\",\n      \"method\": \"Phosphorylation site identification, in vitro FHA-phosphopeptide binding, size-exclusion chromatography, mutagenesis, siRNA knockdown, NF-κB reporter assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — phosphorylation site mapping, FHA-pT binding assay, mutagenesis, and KD with defined signaling phenotype\",\n      \"pmids\": [\"22566686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TIFA associates constitutively with TRAF6 and associates with IRAK-1 only after hypoxia-reoxygenation; TIFA upregulation under hypoxia-reoxygenation is TLR4- and MyD88-dependent; siRNA knockdown of TIFA reduces NF-κB activation and HMGB1 release\",\n      \"method\": \"Co-immunoprecipitation of endogenous proteins, TLR4/MyD88 knockout cells, siRNA knockdown, EMSA, Western blotting, ELISA\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — endogenous Co-IP plus genetic KO controls and KD phenotype in single study\",\n      \"pmids\": [\"23722163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TIFA oligomerization requires Thr9 phosphorylation; crystal structure of TIFA (residues 1-150) and its complex with pThr9 peptide reveals an intrinsic dimer with an extra β-strand, and pThr9-FHA binding occurs only between different dimers (not within a dimer), providing the structural mechanism for higher-order TIFA oligomerization\",\n      \"method\": \"X-ray crystallography, crystal structure of TIFA and TIFA/pThr9-peptide complex, mutagenesis, biochemical oligomerization assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus functional mutagenesis validation\",\n      \"pmids\": [\"26389808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Cytosolic HBP (heptose-1,7-bisphosphate), a bacterial metabolic intermediate absent from eukaryotes, is detected by host cells and activates NF-κB via a pathway requiring phosphorylation-dependent oligomerization of TIFA; identified by genome-wide RNAi screen\",\n      \"method\": \"Genome-wide RNAi screen, HBP cytosolic delivery assay, NF-κB reporter, in vivo innate/adaptive immune response measurement\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide screen with mechanistic follow-up, replicated by multiple independent labs\",\n      \"pmids\": [\"26068852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Aurora A kinase phosphorylates TIFA at Thr9 in AML cells, triggering NF-κB survival pathway activation; TIFA silencing decreases leukemic cell growth and chemoresistance via downregulation of Bcl-2 and Bcl-XL; in vivo delivery of TIFA-inhibitory fragments potentiates chemotherapy clearance in xenograft mice\",\n      \"method\": \"Kinase assay identifying Aurora A as Thr9 kinase, siRNA/shRNA knockdown, in vivo xenograft, co-immunoprecipitation\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — kinase assay plus in vivo KD phenotype, single lab\",\n      \"pmids\": [\"28069801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TIFA is a crucial mediator of NLRP3 inflammasome priming and activation in vascular endothelial cells; Akt phosphorylates TIFA at Thr9, driving TIFA-TIFA homophilic oligomerization and higher-order NLRP3 assembly; TIFA interacts with caspase-1 in activated endothelial cells; SREBP2 transactivates TIFA for signal 1 priming\",\n      \"method\": \"Co-immunoprecipitation, kinase assay (Akt-TIFA), shRNA knockdown, luciferase reporter, in vitro flow assay, in vivo hyperlipidemia model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — kinase assay, Co-IP of TIFA-caspase-1, in vivo model, single lab\",\n      \"pmids\": [\"27965388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ALPK1 kinase phosphorylates TIFA in response to the bacterial metabolite HBP/β-ADP-heptose (delivered via H. pylori cag T4SS); phosphorylation drives TIFAsome (large TIFA oligomeric complex) formation including TRAF2; TIFAsome formation and NF-κB activation are ALPK1-dependent and CagA-independent\",\n      \"method\": \"CRISPR/Cas9 knockout, recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry, mutant H. pylori strains\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO, MS-confirmed complex, multiple orthogonal methods, replicated across labs\",\n      \"pmids\": [\"28877472\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TIFA-dependent cytosolic surveillance of HBP is activated by the H. pylori cag-T4SS in gastric epithelial cells, preceding NOD1 activation; TIFA pathway is NOD1-independent and drives rapid NF-κB-dependent inflammation within hours of infection\",\n      \"method\": \"CRISPR/Cas9 knockout (TIFA, NOD1), infection time-course assays, NF-κB reporter, IL-8 ELISA, siRNA knockdown\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO with epistasis analysis, replicated findings consistent with other studies\",\n      \"pmids\": [\"28811347\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TIFA senses HBP released during intracellular bacterial replication (Shigella) and assembles into large signaling complexes to drive dynamic NF-κB inflammatory response proportional to bacterial replication rate; IECs lacking TIFA cannot discriminate between proliferating and stagnant intracellular bacteria despite intact NOD1/2 pathways; TIFA acts downstream of NOD1\",\n      \"method\": \"TIFA-knockout IECs, CRISPR/Cas9, intracellular bacterial growth assays, NF-κB reporter, epistasis with NOD1/2\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO with epistasis and quantitative bacterial replication phenotype\",\n      \"pmids\": [\"28514661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ALPK1 is the critical kinase responsible for TIFA Thr9 phosphorylation and oligomerization during Shigella, Salmonella, and Neisseria infections; TIFA oligomerization depends on Thr9 and the FHA domain; TIFA oligomerization triggers TRAF6 oligomerization and NF-κB-dependent IL-8 expression; HBP is the bacterial trigger\",\n      \"method\": \"Genome-wide RNAi screen, TIFA oligomerization assays, siRNA, mutagenesis (T9A, FHA mutants), IL-8 ELISA\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide screen plus mechanistic mutagenesis, multiple bacterial species tested, consistent with independent labs\",\n      \"pmids\": [\"28222186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TIFA undergoes nuclear translocation upon genotoxic stress and accumulates on damaged chromatin in a pThr9-dependent manner; TIFA works with TRAF2 to stimulate NEMO ubiquitination (K63-linked), relaying DNA damage signals to NF-κB; TRAF2 knockdown suppresses TIFA-enhanced NEMO ubiquitination under genotoxic stress\",\n      \"method\": \"Immunofluorescence (nuclear localization), chromatin fractionation, ubiquitination assays, siRNA knockdown (TRAF2), T9A mutagenesis, overexpression studies\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods in single lab including mutagenesis and ubiquitination assays\",\n      \"pmids\": [\"29581234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Direct binding between TIFA and the TRAF domain of TRAF6 was demonstrated; crystal structures of the TRAF domain complexed with C-terminal TIFA peptides (wild-type and S174Q/M179D mutant) show salt-bridge formation between TIFA residues 177-181 and the TRAF6 binding pocket; rational mutagenesis (S174Q/M179D) enhances TIFA-TRAF6 binding\",\n      \"method\": \"X-ray crystallography of TRAF domain-TIFA peptide complexes, in vitro binding assays, rational mutagenesis, co-immunoprecipitation of endogenous TRAF6\",\n      \"journal\": \"Chembiochem : a European journal of chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with mutagenesis validation and endogenous Co-IP confirmation\",\n      \"pmids\": [\"30378729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"H. pylori-induced DNA double-strand breaks and replication fork stalling occur via ALPK1/TIFA/NF-κB signaling; TIFA-dependent NF-κB activation leads to R-loop formation in S-phase cells, which underlies DNA damage; R-loop formation is downstream of β-ADP-heptose/ALPK1/TIFA/NF-κB signaling\",\n      \"method\": \"Genetic KO/KD of ALPK1/TIFA, γH2AX/53BP1 foci quantification, R-loop detection (S9.6 antibody), replication fork stalling (DNA fiber assay), gastric organoid primary cells\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, KO epistasis, primary organoid cells, single lab but rigorous\",\n      \"pmids\": [\"33037203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structures of mouse TIFA and phosphomimetic mutants (T9D, T9E) confirm dimeric architecture; size-exclusion chromatography suggests model for TIFA-TRAF6 signaling complex oligomeric assembly upon phosphorylation\",\n      \"method\": \"X-ray crystallography, SAXS, size-exclusion chromatography, mutagenesis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structures with SAXS solution validation\",\n      \"pmids\": [\"32198460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TIFA interacts with both TRAF6 and TRAF2 forming TIFAsomes upon H. pylori infection; TIFA/TRAF6 interaction enables TAK1 binding leading to classical NF-κB activation; TIFA/TRAF2 interaction causes transient displacement and proteasomal degradation of cIAP1 from TRAF2, activating the alternative NF-κB pathway\",\n      \"method\": \"Co-immunoprecipitation, CRISPR/Cas9 knockout, immunofluorescence, proteasome inhibitor experiments, proximity ligation assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO with reciprocal Co-IP, dual pathway epistasis, multiple orthogonal methods\",\n      \"pmids\": [\"34328245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"H. pylori infection leads to ALPK1-dependent TIFA activation followed by proteasomal and lysosomal degradation of TIFA; TIFA interacts with free polyubiquitin, optineurin, TAX1BP1, and LAMP1 (adaptors for lysosomal trafficking); TRAF2, TRAF6, TAK1, or NEMO are not required for TIFA degradation\",\n      \"method\": \"Co-immunoprecipitation, proteasome/lysosome inhibitor treatment, immunofluorescence, Western blotting time-course\",\n      \"journal\": \"European journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with multiple partners, pharmacological inhibition, single lab\",\n      \"pmids\": [\"36965415\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TIFAB forms a stable heterodimer with monomeric TIFA (not with the TIFA dimer); crystal structure of TIFA/TIFAB complex shows TIFAB creates a 'pseudo-TIFA dimer' lacking phosphorylation site and TRAF6-binding motif, thereby inhibiting TIFA dimer formation and suppressing TIFA-TRAF6 signaling and NF-κB activation\",\n      \"method\": \"X-ray crystallography of TIFA/TIFAB complex, co-immunoprecipitation, biochemical and cell-based NF-κB activation assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with biochemical and functional validation\",\n      \"pmids\": [\"38442163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TIFA undergoes liquid-liquid phase separation (LLPS) driven by ALPK1 phosphorylation (pT9), the FHA domain, and an intrinsically disordered region; TRAF6 is recruited into TIFA condensates, where K63-linked polyubiquitin chain synthesis occurs; phase separation of TIFA is required for downstream ALPK1-TIFA-TRAF6 inflammatory signaling\",\n      \"method\": \"Live-cell imaging of LLPS, chemical probe inhibition of condensate formation, NF-κB reporter assays, ubiquitination assays in condensates\",\n      \"journal\": \"Research (Washington, D.C.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — LLPS imaging with functional small-molecule probe validation, single lab\",\n      \"pmids\": [\"38357697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"T6BP (TIFA) directly targets PYK2 and prevents its N-terminal FERM domain-triggered dimerization, disrupting downstream PYK2-JNK signaling; T6BP recruits E3 ubiquitin ligase CBL to form a complex with PYK2, leading to PYK2 degradation\",\n      \"method\": \"Hepatocyte-specific overexpression/deletion, co-immunoprecipitation, in vivo dietary MASH models, heterobifunctional crosslinking\",\n      \"journal\": \"Journal of hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with in vivo genetic models, single lab\",\n      \"pmids\": [\"39260704\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TIFA contains two conserved sequence motifs essential for TRAF2 interaction: a canonical TRAF-binding motif and a novel Pro159-Xaa-Xaa-Glu162 motif; ADP-heptose induces TIFA degradation by autophagy; both TRAF2 and TRAF6 contribute to autophagic TIFA degradation\",\n      \"method\": \"Mutagenesis of TIFA interaction motifs, co-immunoprecipitation, autophagy flux assays, ADP-heptose stimulation\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis plus functional autophagy assays, single lab, no prior replication\",\n      \"pmids\": [\"40696502\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"METTL3 mediates m6A modification of TIFA mRNA, which is recognized by IGF2BP2 to enhance TIFA mRNA stability; increased TIFA drives NLRP3 transcription via NF-κB signaling, activating the NLRP3 inflammasome/Caspase-1/GSDMD pyroptosis axis in tubular cells during AKI\",\n      \"method\": \"MeRIP-seq, RNA-seq, METTL3 conditional KO mice, RIP assay (IGF2BP2-TIFA mRNA), mRNA stability assay, siRNA knockdown, pyroptosis functional assays\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — MeRIP-seq with conditional KO and functional pyroptosis readout, single lab\",\n      \"pmids\": [\"41320097\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NSUN3 increases TIFA mRNA stability via m5C modification, upregulating TIFA expression; NSUN3 knockdown alleviates LPS-induced tubular cell injury by reducing TIFA levels\",\n      \"method\": \"m5C methylated RNA immunoprecipitation-qPCR, actinomycin D mRNA stability assay, siRNA knockdown, AKI mouse model\",\n      \"journal\": \"Clinical and experimental pharmacology & physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — m5C-RIP with functional KD validation, single lab\",\n      \"pmids\": [\"39924309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"STING activation triggers TIFA Thr9 phosphorylation and increases ALPK1 protein expression; conversely, ALPK1 activation (via TIFA) enhances STING pathway outputs including NLRP3 inflammasome activation and LC3B lipidation; bidirectional crosstalk between ALPK1-TIFA and STING pathways amplifies innate immune signaling\",\n      \"method\": \"Pharmacological activation/inhibition of ALPK1 and STING, phospho-TIFA (pT9) immunoblotting, NLRP3/LC3B functional assays, patient samples with gain-of-function ALPK1 mutations\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple pathway assays with clinical correlation, but preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.06.30.662363\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"TIFA is a phosphorylation-dependent oligomeric adaptor protein that is phosphorylated at Thr9 by upstream kinases (ALPK1 in response to bacterial ADP-heptose/HBP; Aurora A in AML; Akt in endothelial cells), whereupon its FHA domain binds pThr9 in trans between dimers to form higher-order oligomers (TIFAsomes) that recruit and activate TRAF6 (via K63-linked polyubiquitination) and TRAF2 (causing cIAP1 displacement), driving both classical and alternative NF-κB pathways, NLRP3 inflammasome assembly, and DNA damage-linked NF-κB activation, with negative regulation by TIFAB (which sequesters monomeric TIFA as a non-functional heterodimer) and termination via proteasomal/autophagic degradation.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries only from KEEP papers.\n\n**Classification:**\n- Papers [1]-[47] in main corpus: Most are about TIFA (TRAF-interacting protein with FHA domain) — KEEP\n- Paper [31] (T6BP/TAX1BP1 autophagy receptor for MHC-II): T6BP here refers to TAX1BP1/CALCOCO3, a different protein that shares the nickname \"T6BP\" — EXCLUDE (alias collision)\n- Paper [33] (T6BP targeting PYK2 in MASH): Also refers to TAX1BP1/T6BP autophagy receptor — EXCLUDE (alias collision)\n- Paper [42] (VacT2BP yeast vacuoles): About TLR2-binding peptide, not TIFA — EXCLUDE (alias collision)\n- Additional corpus papers [1]-[30]: All appear to be about IL-22 or general genomics, not TIFA — EXCLUDE (alias collision / off-target)\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"T6BP (later called TIFA) was identified as a protein that specifically associates with TRAF6 via the coiled-coil region of T6BP interacting with the N-terminal ring finger and zinc finger domains of TRAF6. IL-1, but not TNF, induces TRAF6-T6BP complex formation in a ligand-dependent manner requiring IRAK.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, ligand-stimulation assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP with domain mapping, single lab\",\n      \"pmids\": [\"10920205\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"T2BP (TIFA) was identified as a TRAF2-binding protein via a mammalian two-hybrid screen. The TRAF domain of TRAF2 is required for binding to T2BP, and almost the entire T2BP protein is needed for TRAF2 interaction. Overexpression of T2BP activates NF-κB and AP-1 in a dose-dependent manner.\",\n      \"method\": \"Mammalian two-hybrid screening, co-immunoprecipitation, NF-κB/AP-1 reporter assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, co-IP with functional reporter assay\",\n      \"pmids\": [\"11798190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"TIFA was identified as an adaptor protein linking TRAF6 to IRAK-1 in IL-1 receptor signaling. TIFA binds TRAF6 constitutively and associates with IRAK-1 in an IL-1 stimulation-dependent manner. Mutations abolishing TRAF6 binding or FHA domain phosphopeptide binding prevented NF-κB and JNK activation. TIFA overexpression significantly enhanced the IRAK-1/TRAF6 interaction.\",\n      \"method\": \"Co-immunoprecipitation, transient transfection, NF-κB/JNK reporter assays, FHA domain mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, mutagenesis, IL-1 stimulation-dependent interaction confirmed with endogenous proteins\",\n      \"pmids\": [\"12566447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TIFA activates IKK by promoting oligomerization and K63-linked polyubiquitination of TRAF6, leading to TAK1 and IKK activation. Only high-molecular-weight oligomeric forms of TIFA (not monomer/dimer) activate IKK. TIFA requires a functional TRAF6-binding site for this activity. This was reconstituted in vitro with purified TIFA, TRAF6, TAK1 complex, and Ubc13-Uev1A, independently of the proteasome.\",\n      \"method\": \"In vitro IKK reconstitution with purified proteins, crude cytosolic extract assays, gel filtration to separate oligomeric forms, ubiquitination assays, TRAF6-binding-defective mutant\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with purified proteins plus mutagenesis, mechanistically rigorous\",\n      \"pmids\": [\"15492226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TIFAB, a TIFA-related protein lacking TRAF family binding, was identified as a negative regulator of TIFA-mediated NF-κB activation. TIFAB binds TIFA but not TRAF proteins, and increases the amount of TRAF6 co-precipitated with TIFA, suggesting TIFAB inhibits TIFA-mediated TRAF6 activation possibly by inducing a conformational change in TIFA.\",\n      \"method\": \"Co-immunoprecipitation, transient transfection, NF-κB reporter assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, co-IP plus reporter assay, mechanism partially inferred\",\n      \"pmids\": [\"15047173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Xenopus laevis homologs of TRAF6 (XTRAF6) and TIFA (XTIFA) were identified. Overexpression of XTIFA activated NF-κB by binding XTRAF6, and a mutation abolishing XTRAF6 binding abolished NF-κB activation, confirming conservation of the TIFA-TRAF6 signaling mechanism in vertebrate development.\",\n      \"method\": \"Xenopus overexpression, NF-κB reporter assay, binding-defective mutant\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — ortholog functional validation, single lab, mutagenesis confirms mechanism\",\n      \"pmids\": [\"16023795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ZCCHC11, a novel zinc finger protein, was identified as a TIFA-interacting partner. ZCCHC11L translocates from nucleus to cytoplasm in response to LPS and binds TIFA. ZCCHC11 functions as a negative regulator of TLR-mediated NF-κB activation in a TRAF6-dependent manner, as shown by overexpression and siRNA knockdown experiments.\",\n      \"method\": \"GST-TIFA affinity purification, mass spectrometry, co-immunoprecipitation, siRNA knockdown, NF-κB reporter assay, immunofluorescence\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — affinity purification/MS identification, co-IP confirmation, functional siRNA knockdown\",\n      \"pmids\": [\"16643855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TIFA (T6BP) was identified as a myosin VI binding partner by yeast two-hybrid screen, confirmed in vitro and in vivo. T6BP and myosin VI localize to the trans-Golgi complex and perinuclear vesicles. RNAi knockdown of T6BP reduced membrane ruffling, increased stress fibres and focal adhesions, upregulated constitutive secretion, and T6BP was found to inhibit NF-κB activation.\",\n      \"method\": \"Yeast two-hybrid screen, in vitro binding, co-immunoprecipitation, immunofluorescence, electron microscopy, RNAi knockdown, secretion assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, localization with functional consequence via RNAi\",\n      \"pmids\": [\"17635994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TIFA contains a novel phosphorylation site at Thr9, and phosphorylated Thr9 (pThr9) binds the FHA domain of a different TIFA molecule, driving intermolecular oligomerization. Unphosphorylated TIFA exists as an intrinsic dimer; FHA-pThr9 binding occurs between different dimers. TIFA silencing attenuates TNF-α-mediated NF-κB downstream signaling, establishing a link between TNF-α stimulation and NF-κB activation.\",\n      \"method\": \"Phosphorylation site identification, FHA-pT binding assay, size-exclusion chromatography, NMR/biochemical analysis, siRNA silencing, NF-κB reporter assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — novel PTM identified, intermolecular FHA-pT mechanism demonstrated biochemically, functional silencing confirmed\",\n      \"pmids\": [\"22566686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TIFA expression is upregulated after hypoxia-reoxygenation in a TLR4- and MyD88-dependent manner. Under these conditions, TIFA associates with TRAF6 constitutively and with IRAK-1 only after hypoxia-reoxygenation. siRNA knockdown of TIFA reduced NF-κB activation and HMGB1 upregulation/release, placing TIFA in a TLR4/MyD88-dependent feed-forward signaling loop.\",\n      \"method\": \"qRT-PCR, western blotting, co-immunoprecipitation, siRNA knockdown, EMSA, TLR4−/− and MyD88−/− cells\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic knockouts plus siRNA with defined signaling readouts, single lab\",\n      \"pmids\": [\"23722163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Crystal structures of TIFA revealed that the FHA domain forms an intrinsic dimer in solution, and the pThr9 peptide binds at a site that can only interact with a different dimer (not within the same dimer), providing the structural mechanism for phosphorylation-dependent TIFA oligomerization. This oligomerization is essential for NF-κB activation in immune responses.\",\n      \"method\": \"X-ray crystallography of TIFA and TIFA-pThr9 peptide complex, small angle X-ray scattering, functional mutagenesis\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional validation, explains oligomerization mechanism at atomic level\",\n      \"pmids\": [\"26389808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Cytosolic detection of the bacterial metabolite heptose-1,7-bisphosphate (HBP), a conserved Gram-negative LPS biosynthetic intermediate absent from eukaryotes, triggers TIFA-dependent NF-κB activation. A genome-wide RNAi screen identified TIFA as essential for this response. HBP induces phosphorylation-dependent TIFA oligomerization, activating innate and adaptive immune responses in vivo.\",\n      \"method\": \"Genome-wide RNAi screen, HBP cytosolic delivery assay, NF-κB reporter, in vivo mouse model, TIFA phosphorylation analysis\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — genome-wide screen plus mechanistic follow-up, in vitro and in vivo, replicated by multiple labs\",\n      \"pmids\": [\"26068852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TIFA reconstitution in hepatocellular carcinoma cells promotes two independent apoptosis pathways (p53-dependent cell cycle arrest and caspase-8/caspase-3 activation). A non-oligomerizing TIFA mutant had minimal effect, demonstrating that TIFA oligomerization is required for its tumor-suppressive apoptotic function.\",\n      \"method\": \"TIFA reconstitution in HCC cell lines, non-oligomerizing mutant expression, caspase assays, p53 pathway analysis, xenograft mouse model, TUNEL staining\",\n      \"journal\": \"Oncogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — gain-of-function reconstitution plus mutant controls, in vivo xenograft, single lab\",\n      \"pmids\": [\"26501855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TIFA is phosphorylated at Thr9 by Aurora A kinase, triggering NF-κB survival pathway activation in acute myeloid leukemia. Silencing TIFA decreased leukemic cell growth and chemoresistance by downregulating Bcl-2 and Bcl-XL. In vivo delivery of TIFA-inhibitory fragments potentiated myeloblast clearance in xenograft mice.\",\n      \"method\": \"Aurora A kinase assay for Thr9 phosphorylation, siRNA silencing in AML lines and primary patient cells, Bcl-2/Bcl-XL western blotting, xenograft mouse model\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — kinase-substrate relationship established, functional silencing in patient cells and xenograft, multiple readouts\",\n      \"pmids\": [\"28069801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TIFA acts as a crucial mediator of NLRP3 inflammasome in vascular endothelial cells, regulating both priming (signal 1) and activation (signal 2). For signal 1, TIFA is transactivated by SREBP2 and induces NF-κB to upregulate NLRP3 components. For signal 2, Akt phosphorylates TIFA at Thr9, enabling TIFA-TIFA homophilic oligomerization that facilitates higher-order NLRP3 assembly and TIFA-caspase-1 interaction.\",\n      \"method\": \"SREBP2 transactivation assay, Akt kinase assay, TIFA Thr9 phosphorylation, co-immunoprecipitation (TIFA-caspase-1), oligomerization assays, atheroprone flow model, hyperlipidemia mouse model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple mechanisms defined with orthogonal methods, in vitro and in vivo, Akt as upstream kinase identified\",\n      \"pmids\": [\"27965388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TIFA suppresses hepatocellular carcinoma via MALT1-dependent and MALT1-independent signaling. MALT1 competes with TIFA and its silencing enhances TIFA-induced apoptosis. Long-duration TIFA reconstitution activates JNK and p38 via TRAF6 binding: JNK activation drives apoptosis while p38 governs cell cycle arrest via p53-p21 signaling.\",\n      \"method\": \"MALT1 shRNA silencing, JNK/p38 activation assays, TRAF6 co-immunoprecipitation, caspase assays, in vitro and xenograft in vivo models\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple signaling pathway readouts, in vivo confirmation, single lab\",\n      \"pmids\": [\"29263897\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In H. pylori-infected gastric epithelial cells, ALPK1 kinase phosphorylates TIFA in response to the T4SS-delivered bacterial metabolite β-HBP (D-glycero-β-D-manno-heptose 1,7-bisphosphate), leading to formation of large TIFA complexes (TIFAsomes) that include TRAF2. NF-κB activation, TIFA phosphorylation, and TIFAsome formation all depend on functional ALPK1, establishing ALPK1 as the upstream kinase in this innate immune axis.\",\n      \"method\": \"Genome-wide RNAi screen, CRISPR/Cas9 knockout, recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry, mutant H. pylori strains\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods including CRISPR KO, MS, and recombinant proteins; replicated across labs\",\n      \"pmids\": [\"28877472\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HBP delivered to the host cytosol via the H. pylori cag-T4SS activates the TIFA-dependent cytosolic surveillance pathway independently of NOD1, driving NF-κB-dependent inflammation within hours of infection and preceding NOD1 activation. CagA toxin contributes to NF-κB responses subsequent to TIFA and NOD1 activation, establishing sequential activation: TIFA → NOD1 → CagA.\",\n      \"method\": \"Genetic epistasis with TIFA and NOD1 knockdown/knockout, timed NF-κB activation assays, cag-T4SS mutant bacteria, HBP delivery assays\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis established with genetic tools, pathway ordering defined, replicated across labs\",\n      \"pmids\": [\"28811347\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TIFA threonine 9 and the FHA domain are required for TIFA oligomerization in both infected and bystander cells during Shigella and Salmonella infection. TIFA oligomerization triggers TRAF6 oligomerization and NF-κB activation. ALPK1 is the critical kinase responsible for TIFA oligomerization and IL-8 expression in response to invasive and extracellular Gram-negative bacteria.\",\n      \"method\": \"Genome-wide RNAi screen, T9A and FHA domain mutants, NF-κB/IL-8 reporter assays, bacterial infection models, ALPK1 siRNA\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide screen, domain mutagenesis, multiple bacterial pathogens tested; replicated across labs\",\n      \"pmids\": [\"28222186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TIFA senses HBP released during intracellular bacterial replication in the host cytosol, assembling into large signaling complexes to drive dynamic NF-κB activation proportional to bacterial proliferation rate. IECs lacking TIFA could not discriminate between proliferating and stagnant intracellular bacteria despite intact NOD1/2 pathways, defining TIFA as a rheostat for intracellular bacterial replication.\",\n      \"method\": \"TIFA-knockout IECs, Shigella intracellular replication assay, NF-κB activation kinetics, HBP detection assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — TIFA KO with specific phenotypic readout, epistasis with NOD1/2, defined bacterial sensing function\",\n      \"pmids\": [\"28514661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TIFA undergoes nuclear translocation and accumulates on damaged chromatin following genotoxic stress. DNA damage induces TIFA phosphorylation at Thr9, and this pThr9-FHA interaction is required for chromatin enrichment. TIFA then partners with TRAF2 to stimulate ubiquitination of NEMO (whose sumoylation, phosphorylation, and ubiquitination relay DNA damage to NF-κB). TRAF2 knockdown suppressed TIFA-enhanced NEMO ubiquitination under genotoxic stress.\",\n      \"method\": \"Nuclear fractionation, chromatin immunoprecipitation, TIFA overexpression and T9A mutant, TRAF2 knockdown, NEMO ubiquitination assay, NF-κB target gene expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — localization linked to function, multiple orthogonal methods including ChIP, mutagenesis, and TRAF2 KD\",\n      \"pmids\": [\"29581234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Direct binding between TIFA and the TRAF domain of TRAF6 was demonstrated biochemically, and the crystal structures of TRAF6-TRAF domain complexed with wild-type and S174Q/M179D mutant TIFA C-terminal peptides were solved. The structures revealed salt-bridge formation between TIFA residues 177-181 and TRAF6 binding-pocket residues. A rationally designed TIFA double mutant (S174Q/M179D) showed enhanced binding to endogenous full-length TRAF6.\",\n      \"method\": \"In vitro binding assays, X-ray crystallography of TRAF domain-TIFA peptide complexes, co-immunoprecipitation with full-length TRAF6\",\n      \"journal\": \"Chembiochem\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with mutagenesis and biochemical validation of direct TIFA-TRAF6 interaction\",\n      \"pmids\": [\"30378729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"H. pylori-induced DNA double-strand breaks occur co-transcriptionally in S-phase cells and depend on ALPK1/TIFA/NF-κB signaling triggered by β-ADP-heptose. DNA damage requires co-transcriptional RNA/DNA hybrids (R-loops) that form as a consequence of β-ADP-heptose/ALPK1/TIFA/NF-κB signaling, linking bacterial innate immune activation to replication stress.\",\n      \"method\": \"DNA damage markers (γH2AX), R-loop immunofluorescence (S9.6 antibody), ALPK1/TIFA pathway inhibition, gastric organoid primary cells, replication fork stalling assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, primary organoid cells, mechanistic pathway linking TIFA to R-loop-dependent DNA damage\",\n      \"pmids\": [\"33037203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structures of mouse TIFA and phosphomimetic mutants (T9D and T9E) confirmed the dimeric structure similar to human TIFA. Size-exclusion chromatography analysis of TIFA and TIFA-TRAF6 complexes suggested a model for the TIFA-TRAF6 signaling complex assembly wherein TIFA oligomers recruit TRAF6.\",\n      \"method\": \"X-ray crystallography, small angle X-ray scattering, size-exclusion chromatography, phosphomimetic mutagenesis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structures with multiple mutants, SAXS, biochemical complex analysis\",\n      \"pmids\": [\"32198460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TIFA has dual functions in H. pylori-infected gastric epithelial cells: (1) TIFA-TRAF6 interaction enables TAK1 binding, activating classical NF-κB signaling; (2) TIFA-TRAF2 interaction causes transient displacement of cIAP1 from TRAF2 and proteasomal degradation of cIAP1, facilitating alternative NF-κB pathway activation. Both interactions contribute to TIFAsome formation.\",\n      \"method\": \"Co-immunoprecipitation (TIFA-TRAF6-TAK1 and TIFA-TRAF2-cIAP1 complexes), TIFA knockout, proteasome inhibitor experiments, classical and alternative NF-κB pathway reporters\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple co-IP complexes, TIFA KO, two distinct NF-κB pathway mechanisms defined, single lab with strong mechanistic evidence\",\n      \"pmids\": [\"34328245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TIFA expression in intestinal epithelial cells is required for NF-κB activation by ADP-heptose released from the commensal bacterium Akkermansia muciniphila, via the ALPK1/TIFA/TRAF6 axis. This signaling induces MUC2, BIRC3, and TNFAIP3 expression involved in intestinal barrier maintenance.\",\n      \"method\": \"Drug inhibitors, CRISPR gene editing (ALPK1, TIFA, TRAF6 KO), ADP-heptose characterization, NF-κB reporter, gene expression assays\",\n      \"journal\": \"Gut microbes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO, multiple pathway components validated, functional gene expression readouts\",\n      \"pmids\": [\"36036242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TIFA promotes colorectal cancer cell proliferation via TRAF6 binding and oligomerization: mutants at the oligomerization site (T9A) or TRAF6-binding site (D6 deletion) abolished TIFA-mediated proliferation enhancement. The mechanism involves RSK and PRAS40 kinase activation downstream of TIFA.\",\n      \"method\": \"TIFA knockdown, T9A and D6 mutant overexpression, cell proliferation assays in vitro and xenograft in vivo, RSK/PRAS40 phosphorylation assays\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis of key functional residues, in vivo xenograft, downstream kinase pathway identified\",\n      \"pmids\": [\"35639239\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"H. pylori promotes TIFA turnover in gastric epithelial cells via both proteasomal and lysosomal degradation pathways following ALPK1-dependent TIFA activation. H. pylori infection promotes TIFA interaction with free polyubiquitin and with autophagy/trafficking adaptors optineurin, TAX1BP1, and LAMP1, implicating selective autophagy in TIFA degradation. TRAF2, TRAF6, TAK1, and NEMO are not required for TIFA degradation.\",\n      \"method\": \"Proteasome and lysosome inhibitors, co-immunoprecipitation of TIFA with optineurin/TAX1BP1/LAMP1, H. pylori infection assays, western blotting for TIFA levels\",\n      \"journal\": \"European journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple inhibitors plus co-IP of degradation complex, genetic dissection of required factors\",\n      \"pmids\": [\"36965415\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TIFAB forms a stable heterodimer with monomeric TIFA (not the intrinsic TIFA dimer), inhibiting TIFA dimer formation and suppressing TIFA-TRAF6 signaling. Structural analysis showed the TIFA/TIFAB complex is a 'pseudo-TIFA dimer' lacking the phosphorylation site and TRAF6-binding motif contributed by TIFAB, preventing formation of the orderly phosphorylated TIFA oligomer required for NF-κB activation.\",\n      \"method\": \"X-ray crystallography of TIFA/TIFAB complex, biochemical binding assays, cell-based NF-κB activation assays, size-exclusion chromatography\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure of inhibitory complex with functional validation, mechanistic basis for TIFAB negative regulation established\",\n      \"pmids\": [\"38442163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ALPK1-induced TIFA phosphorylation at Thr9 drives liquid-liquid phase separation (LLPS) of TIFA, mediated by the pT9-FHA domain interaction and an intrinsically disordered region. TRAF6 is subsequently recruited into TIFA condensates, where K63-linked polyubiquitin chain synthesis occurs and downstream effectors are enriched and activated. Chemical inhibition of LLPS (compound 22) blocked the ALPK1-TIFA-TRAF6 pathway.\",\n      \"method\": \"Phase separation assays, fluorescence microscopy of condensates, chemical probe inhibitor, K63-ubiquitination assays in condensates, intrinsically disordered region analysis\",\n      \"journal\": \"Research (Washington, D.C.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct demonstration of LLPS with functional validation, chemical inhibitor confirmation, mechanistic link to TRAF6 ubiquitination\",\n      \"pmids\": [\"38357697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"IFN-γ licenses the ALPK1/TIFA pathway in human monocytes by upregulating TIFA expression; TIFA induction alone is sufficient to recapitulate the licensing effect of IFN-γ. This licensing was blocked by JAK inhibitors. B cells did not require IFN-γ for ALPK1/TIFA responsiveness.\",\n      \"method\": \"ADP-heptose stimulation of human mononuclear cell subsets, IFN-γ treatment, TIFA overexpression, JAK inhibitor blockade, NF-κB activation assays\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific pathway licensing, TIFA overexpression rescue, pharmacological inhibition, single lab\",\n      \"pmids\": [\"39868044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TRAF2 interacts with TIFA via two conserved sequence motifs, one of which (Pro159-Xaa-Xaa-Glu162) is a novel TRAF2-binding motif. ADP-heptose induces TIFA degradation by autophagy, and both TRAF2 and TRAF6 contribute to this autophagic degradation process.\",\n      \"method\": \"Mutagenesis of TIFA motifs, co-immunoprecipitation, autophagy flux assays, TRAF2/TRAF6 knockdown\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — novel binding motif identified by mutagenesis, autophagy degradation mechanism established with genetic tools\",\n      \"pmids\": [\"40696502\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NSUN3 stabilizes TIFA mRNA through m5C modification recognized by IGF2BP2, increasing TIFA expression. Knockdown of NSUN3 reduced TIFA expression and alleviated LPS-induced kidney injury, placing NSUN3 as an upstream regulator of TIFA at the post-transcriptional level.\",\n      \"method\": \"MeRIP-seq, methylation RNA immunoprecipitation-qPCR, actinomycin D mRNA stability assay, NSUN3 knockdown, TIFA western blotting\",\n      \"journal\": \"Clinical and experimental pharmacology & physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — m5C modification mapped by MeRIP-seq, mRNA stability assay, functional knockdown, single lab\",\n      \"pmids\": [\"39924309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"METTL3-mediated m6A modification of TIFA mRNA is recognized by IGF2BP2 to enhance TIFA mRNA stability. Increased TIFA promotes NLRP3 transcription via NF-κB signaling, activating the NLRP3 inflammasome and Caspase-1, driving pyroptosis in tubular epithelial cells during ischemic AKI.\",\n      \"method\": \"MeRIP-seq, RNA-seq, m6A-RIP, METTL3 conditional KO mice, TIFA knockdown, NLRP3/Caspase-1/GSDMD western blot, pyroptosis assays\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — MeRIP-seq identifies modification site, conditional KO mouse, multiple mechanistic readouts along METTL3→TIFA→NF-κB→NLRP3→pyroptosis axis\",\n      \"pmids\": [\"41320097\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ALPK1 activation enhances STING pathway outputs including canonical NF-κB/interferon responses and noncanonical STING proton channel-dependent LC3B lipidation and NLRP3 inflammasome activation. Conversely, STING activation increases ALPK1 protein expression and triggers TIFA Thr9 phosphorylation, establishing bidirectional crosstalk between the ALPK1-TIFA and STING innate immune pathways.\",\n      \"method\": \"ALPK1 gain-of-function mutations, STING pathway reporters, LC3B lipidation assay, NLRP3 inflammasome activation, TIFA pThr9 immunoblot, eIF2α phosphorylation assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple pathway readouts, bidirectional signaling demonstrated, preprint not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"TIFA is a small adaptor protein with an FHA domain that, upon phosphorylation at Thr9 by ALPK1 (in response to bacterial ADP-heptose/HBP) or by Aurora A/Akt (in response to cellular stresses), undergoes FHA-pThr9-mediated intermolecular oligomerization and liquid-liquid phase separation to form TIFAsomes; these condensates recruit and activate TRAF6 (via a C-terminal binding motif) and TRAF2 (via a novel Pro-X-X-Glu motif), promoting K63-linked TRAF6 polyubiquitination and downstream TAK1/IKK/NF-κB activation (both classical and alternative pathways), NLRP3 inflammasome priming and assembly, and DNA damage-induced NF-κB signaling via NEMO ubiquitination, while TIFAB negatively regulates this pathway by forming an inhibitory heterodimer with monomeric TIFA.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TIFA is a phosphorylation-dependent oligomeric adaptor protein that functions as a central innate immune sensor linking bacterial metabolite detection to NF-κB activation and inflammasome assembly. TIFA exists as an intrinsic dimer; phosphorylation of Thr9 by ALPK1 (in response to bacterial ADP-heptose/HBP), Aurora A, or Akt enables its FHA domain to bind pThr9 on adjacent dimers in trans, driving assembly of higher-order oligomeric signaling platforms (TIFAsomes) that recruit TRAF6 to promote K63-linked polyubiquitination and classical NF-κB activation, and recruit TRAF2 to displace cIAP1 and activate the alternative NF-κB pathway [PMID:15492226, PMID:22566686, PMID:26389808, PMID:34328245, PMID:28877472]. TIFA signaling is negatively regulated by TIFAB, which sequesters TIFA monomers into non-functional pseudo-dimers lacking phosphorylation and TRAF6-binding capacity, and is terminated by proteasomal and autophagic degradation of activated TIFA [PMID:38442163, PMID:36965415, PMID:40696502]. Beyond innate immunity, TIFA translocates to damaged chromatin upon genotoxic stress to relay DNA damage signals to NF-κB via TRAF2-dependent NEMO ubiquitination, and in hepatocytes targets PYK2 for CBL-mediated degradation to suppress JNK signaling [PMID:29581234, PMID:39260704].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Identifying TIFA as a TRAF6-associated adaptor downstream of IL-1R/IRAK established that an intermediate protein links receptor-proximal kinases to TRAF6-mediated NF-κB activation.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation with domain mapping in IL-1-stimulated cells\",\n      \"pmids\": [\"10920205\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The phosphorylation event controlling TIFA activation was unknown\", \"Whether TIFA functioned with other TRAFs was untested\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Demonstrating that TIFA also binds TRAF2 and activates both NF-κB and AP-1 broadened its role beyond a TRAF6-specific adaptor.\",\n      \"evidence\": \"Mammalian two-hybrid screen and co-immunoprecipitation with reporter assays\",\n      \"pmids\": [\"11798190\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TRAF2 binding sites on TIFA were not mapped\", \"Physiological context for TIFA-TRAF2 signaling was unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showing that TIFA bridges IRAK-1 to TRAF6 in IL-1 signaling, and that both the FHA domain and TRAF6-binding motif are required, established TIFA as a bona fide signal relay adaptor requiring phosphopeptide recognition.\",\n      \"evidence\": \"Endogenous co-immunoprecipitation, site-directed mutagenesis, NF-κB/JNK reporter assays\",\n      \"pmids\": [\"12566447\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the phosphorylation site and upstream kinase were unknown\", \"Whether TIFA oligomerizes was not addressed\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"In vitro reconstitution revealed that only high-molecular-weight oligomeric TIFA activates IKK by promoting TRAF6 oligomerization and K63-linked polyubiquitination, establishing the oligomerization-dependent activation mechanism.\",\n      \"evidence\": \"Reconstitution with purified TIFA, TRAF6, TAK1, Ubc13-Uev1A; gel-filtration fractionation\",\n      \"pmids\": [\"15492226\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The trigger for TIFA oligomerization was not identified\", \"No structural information on oligomeric TIFA\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Discovery of TIFAB as a TIFA homolog that inhibits TIFA-mediated NF-κB activation introduced a dedicated negative regulatory mechanism for this pathway.\",\n      \"evidence\": \"Co-immunoprecipitation and NF-κB reporter assays\",\n      \"pmids\": [\"15047173\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis for TIFAB inhibition was unknown\", \"Whether TIFAB disrupts oligomerization or TRAF6 binding was not resolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identification of Thr9 as the phosphorylation site whose modification drives intermolecular FHA-pT9 binding between TIFA dimers solved how TIFA transitions from inactive dimer to active oligomer.\",\n      \"evidence\": \"Phosphorylation site identification, FHA-phosphopeptide binding assays, size-exclusion chromatography, mutagenesis\",\n      \"pmids\": [\"22566686\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The upstream kinase phosphorylating Thr9 was unknown\", \"Structural details of the pT9-FHA interaction were lacking\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Crystal structures of TIFA and its pThr9 peptide complex revealed the dimeric architecture and confirmed that pThr9-FHA binding occurs exclusively between dimers, providing the atomic-level mechanism for oligomeric chain assembly.\",\n      \"evidence\": \"X-ray crystallography of TIFA (residues 1–150) and TIFA/pThr9-peptide complex with mutagenesis validation\",\n      \"pmids\": [\"26389808\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of full higher-order oligomer not resolved\", \"How TRAF6 binds in the context of the oligomer was unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"A genome-wide RNAi screen identified bacterial HBP as the pathogen-associated molecular pattern sensed via TIFA-dependent oligomerization and NF-κB activation, establishing TIFA as a cytosolic innate immune sensor for bacterial metabolism.\",\n      \"evidence\": \"Genome-wide RNAi screen with HBP cytosolic delivery, NF-κB reporter, in vivo immune responses\",\n      \"pmids\": [\"26068852\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The kinase linking HBP detection to TIFA phosphorylation was unknown\", \"Whether other bacterial metabolites activate TIFA was untested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Multiple independent studies converged on ALPK1 as the kinase that phosphorylates TIFA Thr9 in response to HBP/β-ADP-heptose delivered by diverse Gram-negative pathogens, completing the ALPK1→TIFA→TRAF6→NF-κB innate immune signaling axis.\",\n      \"evidence\": \"CRISPR/Cas9 knockout of ALPK1, genome-wide RNAi screen, TIFA oligomerization assays with multiple bacterial species (H. pylori, Shigella, Salmonella, Neisseria)\",\n      \"pmids\": [\"28877472\", \"28222186\", \"28811347\", \"28514661\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ALPK1 directly phosphorylates TIFA or acts through an intermediate was debated\", \"Structural basis of ALPK1-TIFA recognition was unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showing that TIFA enables intestinal epithelial cells to discriminate proliferating from quiescent intracellular bacteria via proportional NF-κB responses revealed TIFA as a replication sensor, not merely a pattern-recognition receptor.\",\n      \"evidence\": \"TIFA-knockout IECs infected with Shigella, quantitative NF-κB output correlated with bacterial growth rate\",\n      \"pmids\": [\"28514661\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TIFA signaling kinetics are tuned by HBP concentration thresholds was not quantified\", \"Contribution of TIFA versus NOD1 in vivo during chronic infection was unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Crystal structures of the TRAF6 TRAF domain bound to TIFA C-terminal peptides defined the molecular interface (residues 177–181) for TRAF6 recruitment, and TIFA's nuclear translocation upon genotoxic stress extended its role to DNA damage-induced NF-κB signaling via TRAF2-dependent NEMO ubiquitination.\",\n      \"evidence\": \"X-ray crystallography of TRAF6–TIFA peptide complexes; chromatin fractionation and ubiquitination assays under genotoxic stress\",\n      \"pmids\": [\"30378729\", \"29581234\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The kinase phosphorylating TIFA Thr9 under genotoxic stress was not identified\", \"Full-length TIFA-TRAF6 structural complex was not solved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identification of Aurora A (in AML cells) and Akt (in endothelial cells) as alternative Thr9 kinases demonstrated that TIFA integrates diverse upstream signals beyond innate immunity, including oncogenic survival signaling and NLRP3 inflammasome priming.\",\n      \"evidence\": \"In vitro kinase assays, siRNA/shRNA knockdown with xenograft models (Aurora A); kinase assay and NLRP3 assembly assays (Akt)\",\n      \"pmids\": [\"28069801\", \"27965388\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Aurora A and Akt phosphorylate TIFA directly on Thr9 in physiological contexts needs independent confirmation\", \"Relative contributions of different kinases in specific tissues were not compared\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Dissecting TIFAsome composition revealed that TIFA/TRAF6 activates classical NF-κB via TAK1 while TIFA/TRAF2 activates alternative NF-κB by displacing and degrading cIAP1, establishing TIFA as a bifunctional platform for dual NF-κB pathway activation.\",\n      \"evidence\": \"CRISPR/Cas9 knockout, reciprocal co-immunoprecipitation, proximity ligation assay, proteasome inhibitor experiments during H. pylori infection\",\n      \"pmids\": [\"34328245\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of TRAF2 versus TRAF6 within individual TIFAsomes was not determined\", \"Whether both pathways are co-activated in all cell types was unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrating that activated TIFA is degraded via both proteasomal and lysosomal pathways involving optineurin, TAX1BP1, and polyubiquitin, independently of its signaling partners TRAF2/TRAF6/TAK1/NEMO, revealed a dedicated termination mechanism for TIFAsome signaling.\",\n      \"evidence\": \"Co-immunoprecipitation with autophagy adaptors, proteasome/lysosome inhibitor treatment, Western blot time-course\",\n      \"pmids\": [\"36965415\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The E3 ligase(s) ubiquitinating TIFA for degradation were not identified\", \"Whether degradation occurs within condensates was not addressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The crystal structure of the TIFA/TIFAB heterodimer showed that TIFAB captures TIFA monomers into pseudo-dimers lacking both Thr9 phosphorylation capacity and TRAF6-binding motif, providing the structural basis for negative regulation by preventing functional TIFA dimer formation.\",\n      \"evidence\": \"X-ray crystallography of TIFA/TIFAB complex with biochemical and NF-κB activation assays\",\n      \"pmids\": [\"38442163\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Regulation of TIFAB expression levels and its stoichiometry relative to TIFA in tissues were not characterized\", \"Whether TIFAB can disassemble pre-formed TIFAsomes was not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showing that pThr9-driven TIFA oligomerization proceeds via liquid-liquid phase separation to form functional condensates that concentrate TRAF6 and K63-polyubiquitin chain synthesis introduced a biophysical framework for understanding TIFAsome signal amplification.\",\n      \"evidence\": \"Live-cell imaging of LLPS, chemical probe inhibition of condensate formation, ubiquitination assays within condensates\",\n      \"pmids\": [\"38357697\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether LLPS is required in vivo or is an emergent property of overexpression was not resolved\", \"Relationship between condensates and the crystallographically defined oligomeric chains was not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Mapping two conserved TRAF2-binding motifs on TIFA and showing that ADP-heptose triggers autophagic TIFA degradation dependent on both TRAF2 and TRAF6 refined the termination mechanism and the molecular interface with TRAF2.\",\n      \"evidence\": \"Mutagenesis of TIFA interaction motifs, autophagy flux assays with ADP-heptose stimulation\",\n      \"pmids\": [\"40696502\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether autophagic and proteasomal degradation are sequential or parallel was not resolved\", \"Structural basis of TIFA-TRAF2 interaction at atomic resolution is lacking\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the full structural architecture of higher-order TIFAsomes, the in vivo relevance of TIFA phase separation versus defined oligomeric assembly, the identity of E3 ligases targeting TIFA for degradation, and whether TIFA serves non-redundant roles in tissue-specific innate immunity beyond epithelial and endothelial contexts.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full-length TIFAsome structure at near-atomic resolution\", \"Tissue-specific kinase usage for Thr9 phosphorylation not systematically compared\", \"In vivo genetic models (TIFA-knockout mice) for host defense phenotyping are limited\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2, 3, 7]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 21, 25]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [10, 15, 24]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [17]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [10, 13, 14, 15, 16]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2, 3, 7, 21]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [12, 27]}\n    ],\n    \"complexes\": [\n      \"TIFAsome\"\n    ],\n    \"partners\": [\n      \"TRAF6\",\n      \"TRAF2\",\n      \"ALPK1\",\n      \"TIFAB\",\n      \"IRAK1\",\n      \"NEMO\",\n      \"PYK2\",\n      \"MYO6\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"TIFA is a small adaptor protein that functions as a central signaling hub in innate immunity and NF-κB activation by undergoing phosphorylation-dependent oligomerization and liquid-liquid phase separation to recruit and activate TRAF6 and TRAF2. Phosphorylation of Thr9—by ALPK1 in response to bacterial ADP-heptose/HBP, by Aurora A in leukemia, or by Akt under disturbed flow—enables the FHA domain of one TIFA dimer to engage pThr9 on another, driving higher-order oligomer (TIFAsome) assembly that promotes TRAF6 K63-linked polyubiquitination and downstream TAK1/IKK/NF-κB signaling through both classical and alternative pathways [PMID:26068852, PMID:15492226, PMID:26389808, PMID:34328245, PMID:38357697]. Beyond canonical NF-κB activation, TIFA oligomerization facilitates NLRP3 inflammasome priming and assembly, DNA damage-induced NF-κB signaling via TRAF2-dependent NEMO ubiquitination on damaged chromatin, and pro-apoptotic JNK/p38 signaling in hepatocellular carcinoma [PMID:27965388, PMID:29581234, PMID:29263897]. TIFAB negatively regulates this pathway by forming a pseudo-TIFA heterodimer that lacks the pThr9 site and TRAF6-binding motif, preventing productive oligomerization [PMID:38442163].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Identification of TIFA (T6BP) as a TRAF6-interacting adaptor established the first molecular link between this protein and IL-1/IRAK-dependent NF-κB signaling, answering how TRAF6 receives upstream signals.\",\n      \"evidence\": \"Yeast two-hybrid screen and co-immunoprecipitation with domain mapping in IL-1-stimulated cells\",\n      \"pmids\": [\"10920205\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; endogenous interaction under physiological conditions not fully characterized\", \"Upstream kinase unknown\", \"TIFA's binding to other TRAF family members not tested\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Discovery that TIFA also binds TRAF2 and activates both NF-κB and AP-1 expanded the adaptor's functional scope beyond the TRAF6 axis.\",\n      \"evidence\": \"Mammalian two-hybrid screen and NF-κB/AP-1 reporter assays with co-immunoprecipitation\",\n      \"pmids\": [\"11798190\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TRAF2-binding site on TIFA not mapped\", \"Physiological stimulus driving TIFA-TRAF2 interaction unknown\", \"Relationship between TRAF2 and TRAF6 binding not tested\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstration that TIFA bridges IRAK-1 and TRAF6, and that both the FHA domain and TRAF6-binding motif are required for NF-κB/JNK activation, defined TIFA as a scaffolding adaptor rather than a passive binder.\",\n      \"evidence\": \"Reciprocal co-IP with endogenous proteins, IL-1 stimulation, FHA and TRAF6-binding mutants in reporter assays\",\n      \"pmids\": [\"12566447\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"FHA domain ligand (phosphopeptide source) not identified\", \"In vivo relevance not tested\", \"Stoichiometry of the IRAK-1/TIFA/TRAF6 complex undefined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"In vitro reconstitution proved that only oligomeric TIFA activates TRAF6 K63-linked polyubiquitination and downstream TAK1/IKK, establishing oligomerization as the activating switch and ruling out proteasome dependence.\",\n      \"evidence\": \"Purified protein reconstitution with TIFA, TRAF6, TAK1 complex, and Ubc13-Uev1A; gel filtration separation of oligomeric forms\",\n      \"pmids\": [\"15492226\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"What triggers TIFA oligomerization in cells was unknown\", \"Structural basis of oligomerization not resolved\", \"TIFAB's inhibitory mechanism only partially characterized\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identification of TIFAB as a TIFA-binding negative regulator that lacks TRAF-binding capacity revealed the first endogenous brake on TIFA-mediated signaling.\",\n      \"evidence\": \"Co-immunoprecipitation and NF-κB reporter assays with TIFAB overexpression\",\n      \"pmids\": [\"15047173\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TIFAB inhibitory mechanism (conformational vs. competitive) unclear\", \"Structural basis of TIFA-TIFAB interaction not solved\", \"Physiological contexts of TIFAB regulation unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Discovery that Thr9 phosphorylation drives FHA-mediated intermolecular oligomerization between TIFA dimers solved the long-standing question of how TIFA oligomerization is triggered.\",\n      \"evidence\": \"Phosphosite identification, NMR/biochemical analysis, size-exclusion chromatography, FHA-pThr9 binding assays, siRNA silencing\",\n      \"pmids\": [\"22566686\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the Thr9 kinase(s) in different signaling contexts unknown\", \"Structural visualization of the oligomeric assembly not achieved\", \"Contribution of pThr9-independent oligomerization mechanisms not excluded\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Crystal structures of TIFA and the FHA-pThr9 peptide complex provided the atomic-level explanation for why pThr9 binding can only occur between different dimers, rationalizing phosphorylation-dependent polymerization.\",\n      \"evidence\": \"X-ray crystallography, SAXS, functional mutagenesis\",\n      \"pmids\": [\"26389808\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length oligomeric structure not solved\", \"In vivo stoichiometry of TIFAsomes unknown\", \"Dynamics of assembly and disassembly not captured\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"A genome-wide RNAi screen identified TIFA as essential for the cytosolic innate immune response to bacterial HBP, establishing TIFA as a pattern recognition adaptor for a conserved Gram-negative metabolite.\",\n      \"evidence\": \"Genome-wide RNAi screen, HBP cytosolic delivery, NF-κB reporter, in vivo mouse model\",\n      \"pmids\": [\"26068852\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The direct sensor/kinase upstream of TIFA for HBP not yet identified\", \"Whether other bacterial metabolites activate TIFA unknown\", \"Contribution of TIFA to adaptive immunity only partially explored\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identification of Akt as a Thr9 kinase linking disturbed hemodynamic flow to TIFA oligomerization and NLRP3 inflammasome assembly in endothelial cells demonstrated that TIFA integrates metabolic and mechanical stress signals beyond pathogen detection.\",\n      \"evidence\": \"Akt kinase assay, TIFA oligomerization assays, TIFA-caspase-1 co-IP, atheroprone flow model, hyperlipidemia mouse model\",\n      \"pmids\": [\"27965388\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How TIFA physically bridges NLRP3 ASC specks not resolved\", \"Whether TIFA directly interacts with NLRP3 components structurally unknown\", \"Relative contribution of Akt vs. ALPK1 in different tissues not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Convergent studies identified ALPK1 as the kinase that phosphorylates TIFA at Thr9 in response to bacterial β-HBP/ADP-heptose delivered by T4SS and other secretion systems, completing the ALPK1→TIFA→TRAF6→NF-κB innate immune axis.\",\n      \"evidence\": \"Genome-wide RNAi screens, CRISPR knockout of ALPK1, recombinant protein technology, multiple Gram-negative bacterial pathogens (H. pylori, Shigella, Salmonella)\",\n      \"pmids\": [\"28877472\", \"28222186\", \"28514661\", \"28811347\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct in vitro kinase assay of ALPK1 on TIFA not reported in all studies\", \"How ADP-heptose activates ALPK1 enzymatically not resolved\", \"Whether additional kinases act on Thr9 in infection contexts not excluded\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Discovery that TIFA translocates to damaged chromatin and promotes TRAF2-dependent NEMO ubiquitination extended TIFA function to DNA damage-induced NF-κB signaling, a context independent of pathogen detection.\",\n      \"evidence\": \"Nuclear fractionation, chromatin enrichment, TIFA T9A mutant, TRAF2 knockdown, NEMO ubiquitination assay\",\n      \"pmids\": [\"29581234\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible for Thr9 phosphorylation in genotoxic stress not identified\", \"Whether TIFA forms TIFAsome-like condensates on chromatin untested\", \"Relationship to ATM/ATR signaling not explored\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Crystal structures of the TRAF6-TIFA C-terminal peptide complex defined the salt-bridge network mediating TIFA-TRAF6 binding and enabled rational design of enhanced-affinity mutants.\",\n      \"evidence\": \"X-ray crystallography of TRAF6 TRAF domain with WT and S174Q/M179D TIFA peptides, binding assays\",\n      \"pmids\": [\"30378729\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length TIFA-TRAF6 complex structure not achieved\", \"Whether enhanced-affinity mutants alter signaling output in cells not fully tested\", \"Structural basis for TIFA-TRAF2 interaction not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Dissection of TIFA's dual engagement of TRAF6 and TRAF2 revealed that TIFA-TRAF6 activates classical NF-κB via TAK1, while TIFA-TRAF2 triggers cIAP1 displacement and degradation to activate the alternative NF-κB pathway.\",\n      \"evidence\": \"Co-IP of TIFA-TRAF6-TAK1 and TIFA-TRAF2-cIAP1 complexes, TIFA knockout, proteasome inhibitor experiments\",\n      \"pmids\": [\"34328245\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the two complexes form in the same or different TIFAsomes unknown\", \"Relative kinetics and stoichiometry of the two pathways not defined\", \"Whether TIFA-TRAF2 function extends to non-infection contexts not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Structural resolution of the TIFA-TIFAB heterodimer revealed it as a pseudo-TIFA dimer lacking the Thr9 phosphosite and TRAF6-binding motif, providing the molecular basis for TIFAB's dominant-negative inhibition of TIFA signaling.\",\n      \"evidence\": \"X-ray crystallography of TIFA/TIFAB complex, biochemical binding assays, NF-κB activation assays, SEC\",\n      \"pmids\": [\"38442163\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo regulation of TIFAB expression and its physiological impact not characterized\", \"Whether TIFAB competes with pThr9-mediated oligomerization under infection unclear\", \"No structural data for TIFAB in the context of full-length oligomeric TIFA\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstration that pThr9-dependent TIFA oligomers undergo liquid-liquid phase separation to form functional condensates that concentrate TRAF6 and support K63-linked ubiquitin chain synthesis reframed TIFAsomes as biomolecular condensates.\",\n      \"evidence\": \"Phase separation assays, fluorescence microscopy, chemical LLPS inhibitor (compound 22), K63-ubiquitination assays in condensates\",\n      \"pmids\": [\"38357697\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether LLPS occurs in vivo in physiological infection models not shown\", \"Material properties (viscosity, aging) of TIFAsome condensates not characterized\", \"Selectivity of chemical LLPS inhibitor for TIFA condensates vs. other condensates unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identification of a novel TRAF2-binding motif (PxxE) in TIFA and demonstration that both TRAF2 and TRAF6 contribute to TIFA autophagic degradation established that TIFA turnover is an active signaling-coupled process.\",\n      \"evidence\": \"Mutagenesis of PxxE motif, autophagy flux assays, TRAF2/TRAF6 knockdown\",\n      \"pmids\": [\"40696502\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of PxxE-TRAF2 interaction not solved\", \"Whether autophagy of TIFA serves signal termination or signal amplification not distinguished\", \"Relationship to optineurin/TAX1BP1-mediated degradation pathway not integrated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: the full-length structure of an active TIFAsome oligomer, whether TIFA condensates form in vivo during physiological infection, how different upstream kinases (ALPK1, Aurora A, Akt) are selected in different cellular contexts, and whether TIFA participates in additional innate sensing pathways beyond ADP-heptose.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full-length oligomeric or condensate-state structure available\", \"In vivo condensate formation not confirmed by live imaging\", \"Context-dependent kinase selection mechanism unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 3, 8, 10]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 14, 29]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 8, 11, 29]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [20]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [20]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [11, 16, 17, 18, 19, 25]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 3, 8, 14, 24]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [12, 15, 33]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [13, 22, 26]}\n    ],\n    \"complexes\": [\n      \"TIFAsome\"\n    ],\n    \"partners\": [\n      \"TRAF6\",\n      \"TRAF2\",\n      \"ALPK1\",\n      \"IRAK1\",\n      \"TIFAB\",\n      \"NEMO\",\n      \"TAK1\",\n      \"MYO6\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}