{"gene":"TIFA","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2000,"finding":"TIFA (T6BP) specifically associates with TRAF6 via its coiled-coil region 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; IRAK is not present in TRAF6-T6BP complexes.","method":"Yeast two-hybrid, co-immunoprecipitation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with domain mapping, single lab, two orthogonal methods","pmids":["10920205"],"is_preprint":false},{"year":2002,"finding":"TIFA (T2BP) binds TRAF2 via the TRAF domain of TRAF2 and requires almost the entire TIFA protein for the interaction; overexpression of TIFA activates NF-κB and AP-1 in a dose-dependent manner without TNF stimulation.","method":"Mammalian two-hybrid, co-immunoprecipitation, NF-κB/AP-1 reporter assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP confirmed interaction; functional reporter assays; single lab, two methods","pmids":["11798190"],"is_preprint":false},{"year":2003,"finding":"TIFA bridges IRAK-1 and TRAF6 in IL-1 receptor signaling: TIFA associates with TRAF6 constitutively and with IRAK-1 in an IL-1-stimulation-dependent manner; mutations abolishing TRAF6 binding or FHA domain phosphopeptide binding prevent NF-κB and JNK activation; overexpressed TIFA significantly enhances the IRAK-1/TRAF6 interaction.","method":"Co-immunoprecipitation of endogenous proteins, transient transfection with NF-κB/JNK reporter assays, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — endogenous Co-IP, mutagenesis, functional reporters, replicated across multiple papers","pmids":["12566447"],"is_preprint":false},{"year":2004,"finding":"TIFA activates IKK by promoting oligomerization and K63-linked polyubiquitination of TRAF6, which then activates TAK1 and IKK through a proteasome-independent mechanism. Only high-molecular-weight oligomeric forms of TIFA (not TRAF6-binding-defective mutant) activate IKK. Demonstrated in an in vitro reconstitution system with purified TIFA, TRAF6, TAK1 kinase complex, and Ubc13-Uev1A.","method":"In vitro reconstitution with purified proteins, cell-free IKK activation assay, in vitro ubiquitination assay, mutagenesis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified components, mutagenesis, replicated mechanistic finding across multiple labs","pmids":["15492226"],"is_preprint":false},{"year":2004,"finding":"TIFAB, a TIFA homolog, inhibits TIFA-mediated NF-κB activation by binding TIFA. TIFAB does not associate with TRAF family members but binds TIFA and increases the amount of TRAF6 co-precipitated with TIFA, suggesting TIFAB induces a conformational change in TIFA that inhibits TRAF6 activation.","method":"Co-immunoprecipitation, NF-κB reporter assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and functional reporter, single lab, two orthogonal methods","pmids":["15047173"],"is_preprint":false},{"year":2006,"finding":"ZCCHC11, a zinc finger protein, interacts with TIFA via affinity purification from macrophage phosphoproteins. Upon LPS stimulation, ZCCHC11L translocates from nucleus to cytoplasm and binds TIFA. ZCCHC11 functions as a negative regulator of TLR-mediated NF-κB activation downstream of TIFA-TRAF6.","method":"GST pulldown/affinity purification with mass spectrometry, co-immunoprecipitation, siRNA knockdown, NF-κB reporter assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — affinity purification, Co-IP, and functional knockdown; single lab, multiple methods","pmids":["16643855"],"is_preprint":false},{"year":2007,"finding":"TIFA (T6BP) is a myosin VI binding partner; the interaction was confirmed in vitro and in vivo with binding sites mapped on each protein. T6BP and myosin VI localize to the trans-Golgi complex and perinuclear vesicles. RNAi knockdown of T6BP reduces membrane ruffling, increases stress fibers and focal adhesions, upregulates constitutive secretion, and inhibits NF-κB activation.","method":"Yeast two-hybrid, in vitro binding, co-immunoprecipitation, immunofluorescence, electron microscopy, RNAi knockdown with functional assays","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vitro binding, localization by EM/IF, RNAi with multiple phenotypic readouts; single lab but multiple orthogonal methods","pmids":["17635994"],"is_preprint":false},{"year":2012,"finding":"TIFA undergoes phosphorylation at Thr9, and this phosphorylated Thr9 (pT9) binds intramolecularly with the FHA domain of TIFA from a different dimer, 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.","method":"Biochemical analysis, phosphorylation mapping, TIFA oligomerization assays, siRNA knockdown, NF-κB reporter assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — phosphorylation site identification, oligomerization mechanism, mutagenesis, functional siRNA knockdown; replicated by structural studies","pmids":["22566686"],"is_preprint":false},{"year":2013,"finding":"TIFA upregulation during hypoxia-reoxygenation is TLR4- and MyD88-dependent. TIFA associates with TRAF6 constitutively and with IRAK-1 only after hypoxia-reoxygenation. siRNA-mediated TIFA knockdown reduces NF-κB activation and HMGB1 upregulation/release after hypoxia-reoxygenation.","method":"Co-immunoprecipitation, siRNA knockdown, EMSA, Western blotting, TLR4(-/-) and MyD88(-/-) cells","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout cells, Co-IP, functional siRNA with defined readouts; single lab","pmids":["23722163"],"is_preprint":false},{"year":2015,"finding":"Cytosolic detection of the bacterial metabolite heptose-1,7-bisphosphate (HBP) activates NF-κB through a TIFA-dependent signaling axis mediated by phosphorylation-dependent oligomerization of TIFA. Identified by genome-wide RNAi screen and validated in cell-based assays.","method":"Genome-wide RNAi screen, cell-based NF-κB activation assay, TIFA phosphorylation and oligomerization assays","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide RNAi screen followed by functional validation of phosphorylation-dependent oligomerization; independently replicated across multiple labs","pmids":["26068852"],"is_preprint":false},{"year":2015,"finding":"Crystal structure of TIFA (residues 1-150) and its complex with the N-terminal pThr9 peptide (residues 1-15) reveals: TIFA forms an intrinsic dimer, has an extra β-strand in the FHA structure, and pThr9-FHA domain interaction occurs only between different dimers (not within a dimer), providing the structural mechanism for TIFA oligomerization.","method":"X-ray crystallography, size-exclusion chromatography, mutagenesis, functional NF-κB assays","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation by mutagenesis and NF-κB assays; consistent with prior biochemical data","pmids":["26389808"],"is_preprint":false},{"year":2016,"finding":"Aurora A kinase phosphorylates TIFA at Thr9, triggering NF-κB survival pathway activation in AML cells. TIFA silencing decreases 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":"siRNA knockdown, in vivo xenograft, kinase assay (Aurora A phosphorylation of TIFA Thr9), Western blotting, IC50 assays","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase-substrate identification, siRNA knockdown with defined phenotypes, in vivo xenograft; single lab","pmids":["28069801"],"is_preprint":false},{"year":2016,"finding":"TIFA mediates both signal 1 (priming via NF-κB) and signal 2 (activation) of the NLRP3 inflammasome in vascular endothelial cells. STEROL regulatory element-binding protein 2 transactivates TIFA expression (signal 1). Akt phosphorylates TIFA at Thr9 (signal 2), enabling TIFA-TIFA homophilic oligomerization and interaction with caspase-1 for higher-order NLRP3 assembly.","method":"Transcription factor binding/reporter assay, Akt kinase assay, co-immunoprecipitation (TIFA-caspase-1), siRNA knockdown, in vitro and in vivo atheroprone flow models","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, kinase assay, functional knockdown with dual-signal readouts; single lab, multiple methods","pmids":["27965388"],"is_preprint":false},{"year":2017,"finding":"ALPK1 kinase phosphorylates TIFA at Thr9 in response to H. pylori-delivered βHBP; this triggers TIFAsome formation (large TIFA oligomeric complexes including TRAF2) and NF-κB activation. ALPK1 knockout and CRISPR/Cas9 ablation abrogate TIFA phosphorylation and TIFAsome formation. The pathway is independent of CagA translocation.","method":"CRISPR/Cas9 knockout, RNAi, immunofluorescence microscopy, immunoblotting, mass spectrometry, recombinant protein technology","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR/Cas9 KO, mass spectrometry identification of complex components, immunofluorescence of TIFAsomes, multiple orthogonal methods; replicated across labs","pmids":["28877472"],"is_preprint":false},{"year":2017,"finding":"TIFA threonine 9 phosphorylation and FHA domain are required for TIFA oligomerization in both infected and bystander cells in response to Shigella and Salmonella infection. ALPK1 is the critical kinase responsible for TIFA oligomerization, TRAF6 oligomerization, and IL-8/NF-κB activation. Connexin-dependent cell-cell communication enables bystander cell activation via this pathway.","method":"Genome-wide RNAi screen, mutagenesis (Thr9), siRNA knockdown, NF-κB/IL-8 reporter assays, TIFA oligomerization assays","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide screen followed by mutagenesis and functional validation; replicated independently","pmids":["28222186"],"is_preprint":false},{"year":2017,"finding":"During Shigella infection, TIFA senses HBP released during intracellular bacterial replication (after NOD1-mediated entry), assembling into large signaling complexes; IECs lacking TIFA cannot discriminate between proliferating and stagnant intracellular bacteria, establishing TIFA as a rheostat for intracellular bacterial replication that escalates immune response proportionally to replication rate.","method":"TIFA knockout cells, siRNA knockdown, NF-κB activation assays, bacterial replication quantification, immunofluorescence","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with defined epistatic phenotype, temporal dissection from NOD1 pathway, multiple readouts; replicated independently","pmids":["28514661"],"is_preprint":false},{"year":2017,"finding":"HBP is delivered to host gastric epithelial cells via the cag-T4SS where it activates TIFA-dependent NF-κB signaling independently of NOD1; TIFA activation precedes NOD1 activation during H. pylori infection. CagA contributes to NF-κB downstream of TIFA and NOD1.","method":"siRNA knockdown, bacterial mutant strains (cagPAI-deficient), NF-κB reporter assays, time-course analyses","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic bacterial mutants, siRNA, temporal epistasis with NOD1; independently replicated","pmids":["28811347"],"is_preprint":false},{"year":2018,"finding":"Direct binding between TIFA C-terminal peptide and the TRAF domain of TRAF6 was demonstrated in vitro; crystal structures of TRAF domain complexed with WT and TIFA S174Q/M179D mutant peptides show salt-bridge formation between TIFA residues 177-181 and TRAF6 binding pocket. The double mutant shows enhanced binding to endogenous full-length TRAF6.","method":"X-ray crystallography, in vitro binding assay, co-immunoprecipitation with mutants","journal":"Chembiochem : a European journal of chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with mutagenesis and Co-IP validation; single lab but rigorous structural method","pmids":["30378729"],"is_preprint":false},{"year":2018,"finding":"Upon genotoxic stress, TIFA undergoes nuclear translocation and accumulates on damaged chromatin. DNA damage induces TIFA phosphorylation at Thr9; this pThr9-FHA domain interaction drives enrichment on damaged chromatin and NF-κB activation. TIFA, together with TRAF2, stimulates NEMO ubiquitination to relay DNA damage signals. TRAF2 knockdown suppresses TIFA-enhanced NEMO ubiquitination; unphosphorylatable T9A TIFA mutant has minimal effect on NEMO polyubiquitination.","method":"Subcellular fractionation, immunofluorescence, mutagenesis (Thr9), co-immunoprecipitation, ubiquitination assay, siRNA knockdown","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (localization, mutagenesis, ubiquitination assay), single lab","pmids":["29581234"],"is_preprint":false},{"year":2020,"finding":"Crystal structures of mouse TIFA and Thr9Asp/Thr9Glu phosphomimetic mutants confirm dimer formation similar to human TIFA. Size-exclusion chromatography modeling suggests a higher-order TIFA-TRAF6 signaling complex. Small-angle X-ray scattering confirms the dimer as the solution structure.","method":"X-ray crystallography, size-exclusion chromatography, small-angle X-ray scattering","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure and SAXS with phosphomimetic mutants; single lab but rigorous structural methods","pmids":["32198460"],"is_preprint":false},{"year":2020,"finding":"H. pylori-induced DNA double-strand breaks occur co-transcriptionally in S-phase cells activating NF-κB via β-ADP-heptose/ALPK1/TIFA signaling. R-loops (RNA/DNA hybrids) form during S-phase as a consequence of TIFA/NF-κB signaling, and replication stress/DNA damage depend on these R-loops. DNA damage requires the H. pylori RfaE enzyme and Cag pathogenicity island.","method":"CRISPR/Cas9 knockout, gastric organoid primary cells, R-loop detection (immunofluorescence/S9.6 antibody), DNA fiber assay, bacterial mutant strains","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with multiple readouts, organoids, bacterial mutants; single lab, pathway placed downstream of TIFA/NF-κB","pmids":["33037203"],"is_preprint":false},{"year":2021,"finding":"TIFA interacts with both TRAF6 and TRAF2 in H. pylori-infected gastric epithelial cells, forming TIFAsomes. The TIFA/TRAF6 interaction enables TAK1 binding, leading to classical NF-κB activation. The TIFA/TRAF2 interaction causes transient displacement and proteasomal degradation of cIAP1 from TRAF2, facilitating alternative NF-κB pathway activation.","method":"Co-immunoprecipitation, siRNA knockdown, immunofluorescence, immunoblotting","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP for both TRAF2 and TRAF6 interactions, functional siRNA, single lab with multiple readouts","pmids":["34328245"],"is_preprint":false},{"year":2022,"finding":"TIFA expression is essential for intestinal epithelial cell responsiveness to ADP-heptose from Akkermansia muciniphila; TIFA/ALPK1/TRAF6-dependent NF-κB activation drives MUC2, BIRC3, and TNFAIP3 gene expression for intestinal barrier maintenance. Genetic editing tools confirm the pathway.","method":"CRISPR/Cas9 gene editing, NF-κB reporter assay, siRNA, pharmacological inhibitors, qPCR","journal":"Gut microbes","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with functional gene expression readouts, single lab","pmids":["36036242"],"is_preprint":false},{"year":2022,"finding":"TIFA-T9A (oligomerization site mutation) and TIFA-D6 (TRAF6-binding site deletion) mutants abolish TIFA-mediated CRC cell proliferation enhancement, confirming that both Thr9 phosphorylation-dependent oligomerization and TRAF6 binding are required for TIFA's pro-proliferative function. RSK and PRAS40 activation downstream of TIFA drives CRC progression.","method":"Site-directed mutagenesis, siRNA knockdown, ectopic expression, in vitro/in vivo proliferation assays, Western blotting","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — structure-function mutagenesis with in vivo xenograft; single lab","pmids":["35635239"],"is_preprint":false},{"year":2022,"finding":"TIFA (T6BP) influences MHC-II-restricted endogenous antigen presentation. T6BP silencing causes mislocalization of MHC-II loading compartments and rapid degradation of the invariant chain (CD74) without affecting MHC-II expression or internalization. Calnexin is identified as a T6BP binding partner (via its cytosolic tail), and calnexin silencing replicates T6BP knockdown phenotypes.","method":"siRNA knockdown, immunopeptidome analysis of MHC-II molecules, co-immunoprecipitation (T6BP-calnexin), immunofluorescence, CD4+ T cell activation assay","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP of interaction, functional knockdown with multiple readouts, immunopeptidome; single lab","pmids":["36215666"],"is_preprint":false},{"year":2023,"finding":"H. pylori promotes TIFA degradation via both proteasomal and lysosomal pathways following ALPK1-dependent activation. TIFA interacts with polyubiquitin, optineurin, TAX1BP1, and LAMP1 after H. pylori infection; TRAF2, TRAF6, TAK1, and NEMO are not required for TIFA degradation.","method":"Proteasome/lysosome inhibitor treatment, co-immunoprecipitation, immunoblotting, H. pylori infection time-course","journal":"European journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP of multiple partners, pharmacological inhibitor dissection; single lab, two orthogonal methods","pmids":["36965415"],"is_preprint":false},{"year":2024,"finding":"ADP-heptose (ADP-Hep) recognition by ALPK1 induces liquid-liquid phase separation (LLPS) of TIFA, driven by ALPK1 phosphorylation of pT9, the pT9-FHA domain interaction, and an intrinsically disordered region. TRAF6 is recruited into TIFA condensates, enabling K63-linked polyubiquitin chain synthesis within condensates. A chemical probe (compound 22) confirmed that ALPK1-TIFA-TRAF6 pathway activation requires TIFA phase separation.","method":"Live-cell imaging of phase separation, chemical probe inhibition, in vitro ubiquitination assay, mutagenesis, Co-IP","journal":"Research (Washington, D.C.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live imaging of condensates, chemical probe, in vitro ubiquitination; single lab, multiple methods","pmids":["38357697"],"is_preprint":false},{"year":2024,"finding":"TIFAB forms a stable heterodimer with monomeric TIFA (not with the TIFA dimer), inhibiting TIFA dimer formation and suppressing TIFA-TRAF6 signaling. Crystal structure of the TIFA/TIFAB heterodimer shows it forms a 'pseudo-TIFA dimer' lacking the phosphorylation site and TRAF6-binding motif, preventing formation of the ordered phosphorylated TIFA oligomer required for NF-κB activation.","method":"X-ray crystallography, biochemical assays (SEC, in vitro binding), cell-based NF-κB assays, co-immunoprecipitation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of heterodimer with mutagenesis and functional cell-based validation; multiple orthogonal methods","pmids":["38442163"],"is_preprint":false},{"year":2024,"finding":"TIFA (T6BP) directly targets PYK2 and prevents N-terminal FERM domain-triggered PYK2 dimerization, disrupting PYK2-JNK signaling. TIFA also recruits the E3 ubiquitin ligase CBL to form a complex with PYK2, leading to CBL-mediated PYK2 degradation.","method":"Hepatocyte-specific overexpression/deletion, co-immunoprecipitation (TIFA-PYK2-CBL complex), ubiquitination assay, in vivo dietary rodent models","journal":"Journal of hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP of ternary complex, in vivo genetic models, functional mechanistic readouts; single lab","pmids":["39260704"],"is_preprint":false},{"year":2025,"finding":"TRAF2 interacts with TIFA via two conserved sequence motifs, one of which (Pro159-Xaa-Xaa-Glu162) is novel. ADP-heptose induces TIFA degradation by autophagy, and both TRAF2 and TRAF6 contribute to this autophagic degradation of TIFA.","method":"Mutagenesis of TIFA binding motifs, co-immunoprecipitation, autophagy inhibitor assays, TRAF2/TRAF6 knockdown","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis defining novel binding motif, functional knockdown, pharmacological dissection; single lab, multiple methods","pmids":["40696502"],"is_preprint":false},{"year":2025,"finding":"METTL3 mediates m6A modification of TIFA mRNA, which is recognized by IGF2BP2 to enhance TIFA mRNA stability, thereby upregulating TIFA protein; elevated TIFA promotes NLRP3 transcription via NF-κB signaling, activating NLRP3 inflammasome and Caspase-1 to drive pyroptosis in tubular epithelial cells during AKI.","method":"MeRIP-seq, RNA sequencing, METTL3 conditional knockout mice, siRNA knockdown, co-immunoprecipitation (IGF2BP2-TIFA mRNA), NLRP3 inflammasome/pyroptosis assays","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP-seq identifies m6A site, conditional KO in vivo with functional pyroptosis readouts, single lab","pmids":["41320097"],"is_preprint":false},{"year":2025,"finding":"NSUN3 stabilizes TIFA mRNA through m5C methylation, increasing TIFA protein expression; NSUN3 knockdown reduces TIFA expression, alleviates LPS-induced HK-2 cell injury, and reduces SA-AKI in mice.","method":"methylation RNA immunoprecipitation-qPCR, actinomycin D mRNA stability assay, siRNA knockdown, in vivo CLP model","journal":"Clinical and experimental pharmacology & physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP-qPCR identifies m5C modification, mRNA stability assay, in vivo model; single lab","pmids":["39924309"],"is_preprint":false},{"year":2025,"finding":"ALPK1 activation enhances STING pathway outputs including LC3B lipidation and NLRP3 inflammasome activation; conversely, STING activation increases ALPK1 protein expression and triggers TIFA Thr9 phosphorylation, demonstrating bidirectional crosstalk between the ALPK1/TIFA and STING pathways. ALPK1 signaling also activates eIF2α (integrated stress response).","method":"Cell-based pathway activation assays, Western blotting (TIFA Thr9 phosphorylation), LC3B lipidation assay, NLRP3 inflammasome assay","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single lab, cell-based assays without deep mechanistic dissection of TIFA-specific contributions","pmids":["bio_10.1101_2025.06.30.662363"],"is_preprint":true}],"current_model":"TIFA is an FHA domain-containing adaptor protein that functions as a central hub in innate immune NF-κB signaling: upon sensing of bacterial ADP-heptose metabolites (HBP/ADP-Hep) by the upstream kinase ALPK1, TIFA is phosphorylated at Thr9, triggering phospho-Thr9/FHA-domain-mediated homo-oligomerization and liquid-liquid phase separation into TIFAsomes, which recruit and activate TRAF6 (via K63-linked polyubiquitination) and TRAF2 to activate classical and alternative NF-κB pathways respectively, while TIFAB negatively regulates this process by heterodimerizing with monomeric TIFA to block dimer/oligomer assembly, and TIFA is subsequently degraded via both proteasomal and autophagic pathways; additionally, TIFA bridges IRAK-1 to TRAF6 in IL-1R signaling, translocates to damaged chromatin upon genotoxic stress to drive NEMO ubiquitination, and is post-transcriptionally regulated by METTL3/m6A and NSUN3/m5C modifications that stabilize its mRNA."},"narrative":{"mechanistic_narrative":"TIFA is an FHA-domain adaptor protein that serves as a central signaling hub coupling innate immune sensing of bacterial metabolites to NF-κB activation [PMID:26068852, PMID:28877472]. In its resting state TIFA exists as an intrinsic dimer; phosphorylation at Thr9 generates a pThr9 motif that engages the FHA domain of a neighboring dimer, driving higher-order oligomerization that is the trigger for downstream signaling [PMID:22566686, PMID:26389808]. Cytosolic detection of bacterial ADP-heptose/HBP metabolites by the upstream kinase ALPK1 catalyzes this Thr9 phosphorylation during infection by H. pylori, Shigella, Salmonella, and other organisms, nucleating large TIFA oligomeric complexes (TIFAsomes) that behave as liquid-liquid phase-separated condensates [PMID:28877472, PMID:28222186, PMID:38357697]. Within these assemblies TIFA recruits TRAF6 to drive K63-linked polyubiquitination and TAK1/IKK-dependent classical NF-κB signaling, and engages TRAF2 to displace cIAP1 and activate the alternative NF-κB pathway [PMID:15492226, PMID:34328245, PMID:30378729]. TIFA additionally bridges IRAK-1 to TRAF6 in IL-1 receptor signaling [PMID:10920205, PMID:12566447], and translocates to damaged chromatin upon genotoxic stress where its pThr9/FHA interaction and TRAF2 cooperation stimulate NEMO ubiquitination [PMID:29581234]. Signaling is restrained by TIFAB, which heterodimerizes with monomeric TIFA to form a pseudo-dimer lacking the phosphorylation site and TRAF6-binding motif, blocking productive oligomer assembly [PMID:38442163, PMID:15047173], and activated TIFA is cleared via proteasomal, lysosomal, and TRAF2/TRAF6-dependent autophagic degradation [PMID:36965415, PMID:40696502]. Through this pathway TIFA controls intestinal barrier gene expression and inflammasome priming, and its dysregulation promotes leukemic and colorectal cancer cell survival and proliferation [PMID:36036242, PMID:27965388, PMID:28069801, PMID:35635239].","teleology":[{"year":2000,"claim":"Established TIFA as a TRAF6-associated adaptor selectively engaged downstream of IL-1 but not TNF, placing it in receptor-proximal innate immune signaling.","evidence":"Yeast two-hybrid and co-immunoprecipitation mapping the coiled-coil/TRAF6 ring-finger interaction","pmids":["10920205"],"confidence":"Medium","gaps":["Did not define the activation trigger upstream of TIFA","IRAK absent from the complex left the IRAK-TRAF6 bridging unexplained"]},{"year":2002,"claim":"Showed TIFA also binds TRAF2 and is sufficient to activate NF-κB and AP-1 when overexpressed, broadening its connectivity to multiple TRAF effectors.","evidence":"Mammalian two-hybrid, Co-IP, and NF-κB/AP-1 reporter assays","pmids":["11798190"],"confidence":"Medium","gaps":["Overexpression-driven activation did not establish the physiological stimulus","Distinct roles of TRAF2 vs TRAF6 binding not resolved"]},{"year":2003,"claim":"Defined TIFA as the bridge that recruits IRAK-1 to TRAF6 upon IL-1 stimulation, resolving how an upstream kinase links to the TRAF6 effector.","evidence":"Endogenous Co-IP, FHA/TRAF6-binding mutagenesis, and NF-κB/JNK reporters","pmids":["12566447"],"confidence":"High","gaps":["The kinase phosphorylating TIFA itself was not identified","Mechanism converting binding into TRAF6 activation unknown"]},{"year":2004,"claim":"Demonstrated the biochemical mechanism by which TIFA activates IKK: oligomeric TIFA promotes K63-linked TRAF6 polyubiquitination and TAK1/IKK activation proteasome-independently.","evidence":"In vitro reconstitution with purified TIFA, TRAF6, TAK1, Ubc13-Uev1A and cell-free IKK assays","pmids":["15492226"],"confidence":"High","gaps":["What drives TIFA into the active oligomeric form in cells was undefined","Structural basis of oligomerization unresolved"]},{"year":2004,"claim":"Identified TIFAB as a homolog that inhibits TIFA-mediated NF-κB activation, establishing negative regulation of the pathway.","evidence":"Co-IP and NF-κB reporter assays","pmids":["15047173"],"confidence":"Medium","gaps":["Structural basis of inhibition inferred but not directly shown","Whether TIFAB acts on monomer vs oligomer not resolved at this stage"]},{"year":2007,"claim":"Connected TIFA to membrane trafficking via myosin VI binding and trans-Golgi localization, indicating roles beyond cytosolic NF-κB signaling.","evidence":"Yeast two-hybrid, in vitro binding, IF/EM localization, and RNAi phenotyping","pmids":["17635994"],"confidence":"High","gaps":["Mechanistic link between trafficking role and NF-κB signaling unclear","How myosin VI binding relates to oligomerization not addressed"]},{"year":2012,"claim":"Pinpointed Thr9 phosphorylation and the pThr9-FHA inter-dimer interaction as the molecular switch driving TIFA oligomerization and NF-κB activation.","evidence":"Phosphorylation mapping, oligomerization assays, siRNA knockdown, and NF-κB reporters","pmids":["22566686"],"confidence":"High","gaps":["The physiological kinase and stimulus were not identified","Higher-order complex architecture not visualized"]},{"year":2015,"claim":"Identified the bacterial metabolite HBP as the cytosolic ligand triggering TIFA-dependent NF-κB activation, defining the upstream stimulus.","evidence":"Genome-wide RNAi screen and cell-based NF-κB/oligomerization assays","pmids":["26068852"],"confidence":"High","gaps":["The sensor/kinase coupling HBP to TIFA phosphorylation was not yet identified"]},{"year":2015,"claim":"Provided the structural mechanism for oligomerization, showing TIFA is an intrinsic dimer and pThr9-FHA contacts occur only between distinct dimers.","evidence":"X-ray crystallography of TIFA 1-150 and the pThr9 peptide complex with mutagenesis","pmids":["26389808"],"confidence":"High","gaps":["Full higher-order oligomer/condensate structure not resolved","TRAF6 incorporation geometry not determined"]},{"year":2016,"claim":"Showed multiple host kinases (Aurora A, Akt) can phosphorylate TIFA Thr9 to drive NF-κB survival signaling and inflammasome priming, expanding TIFA into oncogenic and vascular contexts.","evidence":"Kinase assays, siRNA knockdown, Co-IP (TIFA-caspase-1), reporter assays, and xenograft/flow models","pmids":["28069801","27965388"],"confidence":"Medium","gaps":["Relative contribution of each kinase in physiological settings unclear","Direct caspase-1 engagement mechanism not structurally defined"]},{"year":2017,"claim":"Identified ALPK1 as the ADP-heptose/HBP sensor kinase that phosphorylates TIFA Thr9 to nucleate TIFAsomes during enteric and gastric bacterial infection, completing the ligand-kinase-adaptor axis.","evidence":"CRISPR/Cas9 knockout, RNAi, mass spectrometry of TIFAsome components, IF, and bacterial mutants across H. pylori, Shigella, and Salmonella models","pmids":["28877472","28222186","28514661","28811347"],"confidence":"High","gaps":["Quantitative biophysics of TIFAsome assembly not yet defined","How TIFA discriminates replication rate mechanistically partly inferred"]},{"year":2018,"claim":"Extended TIFA function to the DNA damage response, showing genotoxic stress drives TIFA pThr9-dependent chromatin accumulation and TRAF2-dependent NEMO ubiquitination.","evidence":"Subcellular fractionation, IF, Thr9 mutagenesis, ubiquitination assays, and siRNA knockdown","pmids":["29581234"],"confidence":"Medium","gaps":["The kinase phosphorylating TIFA after DNA damage not identified","How TIFA targets damaged chromatin mechanistically unresolved"]},{"year":2018,"claim":"Defined the direct TIFA C-terminus/TRAF6 TRAF-domain interface at atomic resolution, mapping the salt-bridge contacts required for engagement.","evidence":"X-ray crystallography of TRAF6 with WT and mutant TIFA peptides plus Co-IP","pmids":["30378729"],"confidence":"High","gaps":["Stoichiometry of TIFA-TRAF6 in the active condensate not defined"]},{"year":2020,"claim":"Confirmed conserved TIFA dimer architecture in mouse and modeled a higher-order TIFA-TRAF6 signaling complex, reinforcing the dimer-to-oligomer mechanism.","evidence":"Crystallography of mouse TIFA and phosphomimetic mutants, SEC, and SAXS","pmids":["32198460"],"confidence":"High","gaps":["Solution oligomer beyond the dimer modeled but not directly visualized"]},{"year":2020,"claim":"Linked TIFA/NF-κB signaling to genome instability, showing the ALPK1/TIFA axis drives co-transcriptional R-loop formation and DNA double-strand breaks during H. pylori infection.","evidence":"CRISPR knockout, gastric organoids, R-loop/S9.6 detection, DNA fiber assays, and bacterial mutants","pmids":["33037203"],"confidence":"Medium","gaps":["Direct molecular link from NF-κB output to R-loop induction not fully defined","Causal role of TIFA in carcinogenesis not established"]},{"year":2021,"claim":"Dissected how TIFAsomes branch into classical and alternative NF-κB outputs: TRAF6/TAK1 for classical and TRAF2-mediated cIAP1 displacement/degradation for alternative signaling.","evidence":"Reciprocal Co-IP, siRNA knockdown, IF, and immunoblotting in H. pylori-infected epithelial cells","pmids":["34328245"],"confidence":"Medium","gaps":["Temporal coordination of the two branches within one condensate not resolved","Single-lab observation of the cIAP1 displacement step"]},{"year":2022,"claim":"Placed TIFA in intestinal homeostasis and cancer, showing the ALPK1/TIFA/TRAF6 axis drives barrier gene programs and pro-proliferative RSK/PRAS40 signaling requiring both Thr9 oligomerization and TRAF6 binding.","evidence":"CRISPR editing, structure-function mutagenesis (T9A, TRAF6-binding deletion), reporters, qPCR, and in vivo proliferation models","pmids":["36036242","35635239"],"confidence":"Medium","gaps":["Whether commensal-driven and pathogen-driven TIFA signaling differ mechanistically unclear","In vivo causal contribution to tumorigenesis not fully established"]},{"year":2022,"claim":"Revealed a trafficking/antigen-presentation function in which TIFA binds calnexin and controls MHC-II compartment localization and invariant chain stability.","evidence":"siRNA knockdown, immunopeptidome analysis, Co-IP (TIFA-calnexin), IF, and CD4+ T cell assays","pmids":["36215666"],"confidence":"Medium","gaps":["Relationship between this trafficking role and NF-κB signaling unresolved","Whether oligomerization is required not tested"]},{"year":2023,"claim":"Established that activated TIFA is turned over through both proteasomal and lysosomal routes via autophagy receptors, defining signal termination.","evidence":"Proteasome/lysosome inhibitors, Co-IP with polyubiquitin, optineurin, TAX1BP1, LAMP1, and infection time-course","pmids":["36965415"],"confidence":"Medium","gaps":["The E3 ligase ubiquitinating TIFA not identified","Which degradation route dominates physiologically unclear"]},{"year":2024,"claim":"Showed TIFAsome formation is a liquid-liquid phase separation event requiring pThr9-FHA interaction and an intrinsically disordered region, with TRAF6 ubiquitination occurring inside condensates.","evidence":"Live-cell phase-separation imaging, chemical probe inhibition, in vitro ubiquitination, mutagenesis, and Co-IP","pmids":["38357697"],"confidence":"Medium","gaps":["Material properties and regulation of condensate dissolution not defined","Single-lab characterization of the LLPS requirement"]},{"year":2024,"claim":"Provided the structural basis for TIFAB inhibition, showing it forms a pseudo-TIFA heterodimer with monomeric TIFA that lacks the phosphorylation site and TRAF6-binding motif.","evidence":"Crystallography of the TIFA/TIFAB heterodimer with SEC, in vitro binding, and NF-κB cell-based assays","pmids":["38442163"],"confidence":"High","gaps":["How TIFAB levels are regulated to set the signaling threshold not addressed"]},{"year":2024,"claim":"Identified a non-NF-κB TIFA function in hepatocytes, where TIFA blocks PYK2 dimerization and recruits CBL for PYK2 degradation, dampening PYK2-JNK signaling.","evidence":"Hepatocyte-specific overexpression/deletion, Co-IP of the TIFA-PYK2-CBL complex, ubiquitination assays, and dietary rodent models","pmids":["39260704"],"confidence":"Medium","gaps":["Whether this role depends on Thr9/oligomerization not tested","Tissue specificity versus the immune signaling role unclear"]},{"year":2025,"claim":"Refined TRAF2 engagement and degradation, defining a novel TIFA TRAF2-binding motif and showing ADP-heptose triggers TRAF2/TRAF6-dependent autophagic clearance of TIFA.","evidence":"Binding-motif mutagenesis, Co-IP, autophagy inhibitors, and TRAF2/TRAF6 knockdown","pmids":["40696502"],"confidence":"Medium","gaps":["How TRAFs route TIFA to autophagy mechanistically unclear","Single-lab finding"]},{"year":2025,"claim":"Identified post-transcriptional control of TIFA, with METTL3/m6A-IGF2BP2 and NSUN3/m5C modifications stabilizing TIFA mRNA to amplify NF-κB/NLRP3-driven pyroptosis in kidney injury.","evidence":"MeRIP-seq/qPCR, RNA stability assays, conditional/knockdown models, Co-IP (IGF2BP2-TIFA mRNA), and in vivo AKI models","pmids":["41320097","39924309"],"confidence":"Medium","gaps":["Whether these RNA modifications regulate TIFA in other tissues unknown","Crosstalk between mRNA stabilization and protein degradation not integrated"]},{"year":null,"claim":"How the ALPK1/TIFA axis is integrated with other innate immune pathways such as STING and the integrated stress response, and how TIFAsome assembly/dissolution is quantitatively tuned in vivo, remains open.","evidence":"","pmids":[],"confidence":"Low","gaps":["Bidirectional ALPK1/TIFA-STING crosstalk reported only in a preprint without deep TIFA-specific dissection","No unified model linking condensate dynamics, degradation, and mRNA stabilization in setting signaling output"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2,3,18]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[7,10]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[13,26]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[6]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[18]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[18]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[9,13,14]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,7,21]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[11,20,23]}],"complexes":["TIFAsome","TIFA/TIFAB heterodimer","TIFA-TRAF6 complex","TIFA-TRAF2 complex"],"partners":["TRAF6","TRAF2","TIFAB","IRAK1","ALPK1","NEMO","PYK2","CALNEXIN"],"other_free_text":[]}},"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":148,"is_preprint":false},{"pmid":"26068852","id":"PMC_26068852","title":"INNATE IMMUNITY. 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Disease","date":"2025-07-04","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.30.662363","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":25129,"output_tokens":8587,"usd":0.102096,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":18309,"output_tokens":6949,"usd":0.132635,"stage2_stop_reason":"end_turn"},"total_usd":0.234731,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"TIFA (T6BP) specifically associates with TRAF6 via its coiled-coil region 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; IRAK is not present in TRAF6-T6BP complexes.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with domain mapping, single lab, two orthogonal methods\",\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 requires almost the entire TIFA protein for the interaction; overexpression of TIFA activates NF-κB and AP-1 in a dose-dependent manner without TNF stimulation.\",\n      \"method\": \"Mammalian two-hybrid, co-immunoprecipitation, NF-κB/AP-1 reporter assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP confirmed interaction; functional reporter assays; single lab, two methods\",\n      \"pmids\": [\"11798190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"TIFA bridges IRAK-1 and TRAF6 in IL-1 receptor signaling: TIFA associates with TRAF6 constitutively and with IRAK-1 in an IL-1-stimulation-dependent manner; mutations abolishing TRAF6 binding or FHA domain phosphopeptide binding prevent NF-κB and JNK activation; overexpressed TIFA significantly enhances the IRAK-1/TRAF6 interaction.\",\n      \"method\": \"Co-immunoprecipitation of endogenous proteins, transient transfection with NF-κB/JNK reporter assays, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — endogenous Co-IP, mutagenesis, functional reporters, replicated across multiple papers\",\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, which then activates TAK1 and IKK through a proteasome-independent mechanism. Only high-molecular-weight oligomeric forms of TIFA (not TRAF6-binding-defective mutant) activate IKK. Demonstrated in an in vitro reconstitution system with purified TIFA, TRAF6, TAK1 kinase complex, and Ubc13-Uev1A.\",\n      \"method\": \"In vitro reconstitution with purified proteins, cell-free IKK activation assay, in vitro ubiquitination assay, mutagenesis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified components, mutagenesis, replicated mechanistic finding across multiple labs\",\n      \"pmids\": [\"15492226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TIFAB, a TIFA homolog, inhibits TIFA-mediated NF-κB activation by binding TIFA. TIFAB does not associate with TRAF family members but binds TIFA and increases the amount of TRAF6 co-precipitated with TIFA, suggesting TIFAB induces a conformational change in TIFA that inhibits TRAF6 activation.\",\n      \"method\": \"Co-immunoprecipitation, NF-κB reporter assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and functional reporter, single lab, two orthogonal methods\",\n      \"pmids\": [\"15047173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ZCCHC11, a zinc finger protein, interacts with TIFA via affinity purification from macrophage phosphoproteins. Upon LPS stimulation, ZCCHC11L translocates from nucleus to cytoplasm and binds TIFA. ZCCHC11 functions as a negative regulator of TLR-mediated NF-κB activation downstream of TIFA-TRAF6.\",\n      \"method\": \"GST pulldown/affinity purification with mass spectrometry, co-immunoprecipitation, siRNA knockdown, NF-κB reporter assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — affinity purification, Co-IP, and functional knockdown; single lab, multiple methods\",\n      \"pmids\": [\"16643855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TIFA (T6BP) is a myosin VI binding partner; the interaction was confirmed in vitro and in vivo with binding sites mapped on each protein. T6BP and myosin VI localize to the trans-Golgi complex and perinuclear vesicles. RNAi knockdown of T6BP reduces membrane ruffling, increases stress fibers and focal adhesions, upregulates constitutive secretion, and inhibits NF-κB activation.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding, co-immunoprecipitation, immunofluorescence, electron microscopy, RNAi knockdown with functional assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in vitro binding, localization by EM/IF, RNAi with multiple phenotypic readouts; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"17635994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TIFA undergoes phosphorylation at Thr9, and this phosphorylated Thr9 (pT9) binds intramolecularly with the FHA domain of TIFA from a different dimer, 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\": \"Biochemical analysis, phosphorylation mapping, TIFA oligomerization assays, siRNA knockdown, NF-κB reporter assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — phosphorylation site identification, oligomerization mechanism, mutagenesis, functional siRNA knockdown; replicated by structural studies\",\n      \"pmids\": [\"22566686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TIFA upregulation during hypoxia-reoxygenation is TLR4- and MyD88-dependent. TIFA associates with TRAF6 constitutively and with IRAK-1 only after hypoxia-reoxygenation. siRNA-mediated TIFA knockdown reduces NF-κB activation and HMGB1 upregulation/release after hypoxia-reoxygenation.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, EMSA, Western blotting, TLR4(-/-) and MyD88(-/-) cells\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout cells, Co-IP, functional siRNA with defined readouts; single lab\",\n      \"pmids\": [\"23722163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Cytosolic detection of the bacterial metabolite heptose-1,7-bisphosphate (HBP) activates NF-κB through a TIFA-dependent signaling axis mediated by phosphorylation-dependent oligomerization of TIFA. Identified by genome-wide RNAi screen and validated in cell-based assays.\",\n      \"method\": \"Genome-wide RNAi screen, cell-based NF-κB activation assay, TIFA phosphorylation and oligomerization assays\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide RNAi screen followed by functional validation of phosphorylation-dependent oligomerization; independently replicated across multiple labs\",\n      \"pmids\": [\"26068852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Crystal structure of TIFA (residues 1-150) and its complex with the N-terminal pThr9 peptide (residues 1-15) reveals: TIFA forms an intrinsic dimer, has an extra β-strand in the FHA structure, and pThr9-FHA domain interaction occurs only between different dimers (not within a dimer), providing the structural mechanism for TIFA oligomerization.\",\n      \"method\": \"X-ray crystallography, size-exclusion chromatography, mutagenesis, functional NF-κB assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation by mutagenesis and NF-κB assays; consistent with prior biochemical data\",\n      \"pmids\": [\"26389808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Aurora A kinase phosphorylates TIFA at Thr9, triggering NF-κB survival pathway activation in AML cells. TIFA silencing decreases 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\": \"siRNA knockdown, in vivo xenograft, kinase assay (Aurora A phosphorylation of TIFA Thr9), Western blotting, IC50 assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase-substrate identification, siRNA knockdown with defined phenotypes, in vivo xenograft; single lab\",\n      \"pmids\": [\"28069801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TIFA mediates both signal 1 (priming via NF-κB) and signal 2 (activation) of the NLRP3 inflammasome in vascular endothelial cells. STEROL regulatory element-binding protein 2 transactivates TIFA expression (signal 1). Akt phosphorylates TIFA at Thr9 (signal 2), enabling TIFA-TIFA homophilic oligomerization and interaction with caspase-1 for higher-order NLRP3 assembly.\",\n      \"method\": \"Transcription factor binding/reporter assay, Akt kinase assay, co-immunoprecipitation (TIFA-caspase-1), siRNA knockdown, in vitro and in vivo atheroprone flow models\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, kinase assay, functional knockdown with dual-signal readouts; single lab, multiple methods\",\n      \"pmids\": [\"27965388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ALPK1 kinase phosphorylates TIFA at Thr9 in response to H. pylori-delivered βHBP; this triggers TIFAsome formation (large TIFA oligomeric complexes including TRAF2) and NF-κB activation. ALPK1 knockout and CRISPR/Cas9 ablation abrogate TIFA phosphorylation and TIFAsome formation. The pathway is independent of CagA translocation.\",\n      \"method\": \"CRISPR/Cas9 knockout, RNAi, immunofluorescence microscopy, immunoblotting, mass spectrometry, recombinant protein technology\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR/Cas9 KO, mass spectrometry identification of complex components, immunofluorescence of TIFAsomes, multiple orthogonal methods; replicated across labs\",\n      \"pmids\": [\"28877472\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TIFA threonine 9 phosphorylation and FHA domain are required for TIFA oligomerization in both infected and bystander cells in response to Shigella and Salmonella infection. ALPK1 is the critical kinase responsible for TIFA oligomerization, TRAF6 oligomerization, and IL-8/NF-κB activation. Connexin-dependent cell-cell communication enables bystander cell activation via this pathway.\",\n      \"method\": \"Genome-wide RNAi screen, mutagenesis (Thr9), siRNA knockdown, NF-κB/IL-8 reporter assays, TIFA oligomerization assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide screen followed by mutagenesis and functional validation; replicated independently\",\n      \"pmids\": [\"28222186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"During Shigella infection, TIFA senses HBP released during intracellular bacterial replication (after NOD1-mediated entry), assembling into large signaling complexes; IECs lacking TIFA cannot discriminate between proliferating and stagnant intracellular bacteria, establishing TIFA as a rheostat for intracellular bacterial replication that escalates immune response proportionally to replication rate.\",\n      \"method\": \"TIFA knockout cells, siRNA knockdown, NF-κB activation assays, bacterial replication quantification, immunofluorescence\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with defined epistatic phenotype, temporal dissection from NOD1 pathway, multiple readouts; replicated independently\",\n      \"pmids\": [\"28514661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HBP is delivered to host gastric epithelial cells via the cag-T4SS where it activates TIFA-dependent NF-κB signaling independently of NOD1; TIFA activation precedes NOD1 activation during H. pylori infection. CagA contributes to NF-κB downstream of TIFA and NOD1.\",\n      \"method\": \"siRNA knockdown, bacterial mutant strains (cagPAI-deficient), NF-κB reporter assays, time-course analyses\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic bacterial mutants, siRNA, temporal epistasis with NOD1; independently replicated\",\n      \"pmids\": [\"28811347\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Direct binding between TIFA C-terminal peptide and the TRAF domain of TRAF6 was demonstrated in vitro; crystal structures of TRAF domain complexed with WT and TIFA S174Q/M179D mutant peptides show salt-bridge formation between TIFA residues 177-181 and TRAF6 binding pocket. The double mutant shows enhanced binding to endogenous full-length TRAF6.\",\n      \"method\": \"X-ray crystallography, in vitro binding assay, co-immunoprecipitation with mutants\",\n      \"journal\": \"Chembiochem : a European journal of chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with mutagenesis and Co-IP validation; single lab but rigorous structural method\",\n      \"pmids\": [\"30378729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Upon genotoxic stress, TIFA undergoes nuclear translocation and accumulates on damaged chromatin. DNA damage induces TIFA phosphorylation at Thr9; this pThr9-FHA domain interaction drives enrichment on damaged chromatin and NF-κB activation. TIFA, together with TRAF2, stimulates NEMO ubiquitination to relay DNA damage signals. TRAF2 knockdown suppresses TIFA-enhanced NEMO ubiquitination; unphosphorylatable T9A TIFA mutant has minimal effect on NEMO polyubiquitination.\",\n      \"method\": \"Subcellular fractionation, immunofluorescence, mutagenesis (Thr9), co-immunoprecipitation, ubiquitination assay, siRNA knockdown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (localization, mutagenesis, ubiquitination assay), single lab\",\n      \"pmids\": [\"29581234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structures of mouse TIFA and Thr9Asp/Thr9Glu phosphomimetic mutants confirm dimer formation similar to human TIFA. Size-exclusion chromatography modeling suggests a higher-order TIFA-TRAF6 signaling complex. Small-angle X-ray scattering confirms the dimer as the solution structure.\",\n      \"method\": \"X-ray crystallography, size-exclusion chromatography, small-angle X-ray scattering\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure and SAXS with phosphomimetic mutants; single lab but rigorous structural methods\",\n      \"pmids\": [\"32198460\"],\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 activating NF-κB via β-ADP-heptose/ALPK1/TIFA signaling. R-loops (RNA/DNA hybrids) form during S-phase as a consequence of TIFA/NF-κB signaling, and replication stress/DNA damage depend on these R-loops. DNA damage requires the H. pylori RfaE enzyme and Cag pathogenicity island.\",\n      \"method\": \"CRISPR/Cas9 knockout, gastric organoid primary cells, R-loop detection (immunofluorescence/S9.6 antibody), DNA fiber assay, bacterial mutant strains\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with multiple readouts, organoids, bacterial mutants; single lab, pathway placed downstream of TIFA/NF-κB\",\n      \"pmids\": [\"33037203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TIFA interacts with both TRAF6 and TRAF2 in H. pylori-infected gastric epithelial cells, forming TIFAsomes. The TIFA/TRAF6 interaction enables TAK1 binding, leading to classical NF-κB activation. The TIFA/TRAF2 interaction causes transient displacement and proteasomal degradation of cIAP1 from TRAF2, facilitating alternative NF-κB pathway activation.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, immunofluorescence, immunoblotting\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP for both TRAF2 and TRAF6 interactions, functional siRNA, single lab with multiple readouts\",\n      \"pmids\": [\"34328245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TIFA expression is essential for intestinal epithelial cell responsiveness to ADP-heptose from Akkermansia muciniphila; TIFA/ALPK1/TRAF6-dependent NF-κB activation drives MUC2, BIRC3, and TNFAIP3 gene expression for intestinal barrier maintenance. Genetic editing tools confirm the pathway.\",\n      \"method\": \"CRISPR/Cas9 gene editing, NF-κB reporter assay, siRNA, pharmacological inhibitors, qPCR\",\n      \"journal\": \"Gut microbes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with functional gene expression readouts, single lab\",\n      \"pmids\": [\"36036242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TIFA-T9A (oligomerization site mutation) and TIFA-D6 (TRAF6-binding site deletion) mutants abolish TIFA-mediated CRC cell proliferation enhancement, confirming that both Thr9 phosphorylation-dependent oligomerization and TRAF6 binding are required for TIFA's pro-proliferative function. RSK and PRAS40 activation downstream of TIFA drives CRC progression.\",\n      \"method\": \"Site-directed mutagenesis, siRNA knockdown, ectopic expression, in vitro/in vivo proliferation assays, Western blotting\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — structure-function mutagenesis with in vivo xenograft; single lab\",\n      \"pmids\": [\"35635239\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TIFA (T6BP) influences MHC-II-restricted endogenous antigen presentation. T6BP silencing causes mislocalization of MHC-II loading compartments and rapid degradation of the invariant chain (CD74) without affecting MHC-II expression or internalization. Calnexin is identified as a T6BP binding partner (via its cytosolic tail), and calnexin silencing replicates T6BP knockdown phenotypes.\",\n      \"method\": \"siRNA knockdown, immunopeptidome analysis of MHC-II molecules, co-immunoprecipitation (T6BP-calnexin), immunofluorescence, CD4+ T cell activation assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP of interaction, functional knockdown with multiple readouts, immunopeptidome; single lab\",\n      \"pmids\": [\"36215666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"H. pylori promotes TIFA degradation via both proteasomal and lysosomal pathways following ALPK1-dependent activation. TIFA interacts with polyubiquitin, optineurin, TAX1BP1, and LAMP1 after H. pylori infection; TRAF2, TRAF6, TAK1, and NEMO are not required for TIFA degradation.\",\n      \"method\": \"Proteasome/lysosome inhibitor treatment, co-immunoprecipitation, immunoblotting, H. pylori infection time-course\",\n      \"journal\": \"European journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP of multiple partners, pharmacological inhibitor dissection; single lab, two orthogonal methods\",\n      \"pmids\": [\"36965415\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ADP-heptose (ADP-Hep) recognition by ALPK1 induces liquid-liquid phase separation (LLPS) of TIFA, driven by ALPK1 phosphorylation of pT9, the pT9-FHA domain interaction, and an intrinsically disordered region. TRAF6 is recruited into TIFA condensates, enabling K63-linked polyubiquitin chain synthesis within condensates. A chemical probe (compound 22) confirmed that ALPK1-TIFA-TRAF6 pathway activation requires TIFA phase separation.\",\n      \"method\": \"Live-cell imaging of phase separation, chemical probe inhibition, in vitro ubiquitination assay, mutagenesis, Co-IP\",\n      \"journal\": \"Research (Washington, D.C.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging of condensates, chemical probe, in vitro ubiquitination; single lab, multiple methods\",\n      \"pmids\": [\"38357697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TIFAB forms a stable heterodimer with monomeric TIFA (not with the TIFA dimer), inhibiting TIFA dimer formation and suppressing TIFA-TRAF6 signaling. Crystal structure of the TIFA/TIFAB heterodimer shows it forms a 'pseudo-TIFA dimer' lacking the phosphorylation site and TRAF6-binding motif, preventing formation of the ordered phosphorylated TIFA oligomer required for NF-κB activation.\",\n      \"method\": \"X-ray crystallography, biochemical assays (SEC, in vitro binding), cell-based NF-κB assays, co-immunoprecipitation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of heterodimer with mutagenesis and functional cell-based validation; multiple orthogonal methods\",\n      \"pmids\": [\"38442163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TIFA (T6BP) directly targets PYK2 and prevents N-terminal FERM domain-triggered PYK2 dimerization, disrupting PYK2-JNK signaling. TIFA also recruits the E3 ubiquitin ligase CBL to form a complex with PYK2, leading to CBL-mediated PYK2 degradation.\",\n      \"method\": \"Hepatocyte-specific overexpression/deletion, co-immunoprecipitation (TIFA-PYK2-CBL complex), ubiquitination assay, in vivo dietary rodent models\",\n      \"journal\": \"Journal of hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP of ternary complex, in vivo genetic models, functional mechanistic readouts; single lab\",\n      \"pmids\": [\"39260704\"],\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 novel. ADP-heptose induces TIFA degradation by autophagy, and both TRAF2 and TRAF6 contribute to this autophagic degradation of TIFA.\",\n      \"method\": \"Mutagenesis of TIFA binding motifs, co-immunoprecipitation, autophagy inhibitor assays, TRAF2/TRAF6 knockdown\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis defining novel binding motif, functional knockdown, pharmacological dissection; single lab, multiple methods\",\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, thereby upregulating TIFA protein; elevated TIFA promotes NLRP3 transcription via NF-κB signaling, activating NLRP3 inflammasome and Caspase-1 to drive pyroptosis in tubular epithelial cells during AKI.\",\n      \"method\": \"MeRIP-seq, RNA sequencing, METTL3 conditional knockout mice, siRNA knockdown, co-immunoprecipitation (IGF2BP2-TIFA mRNA), NLRP3 inflammasome/pyroptosis assays\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP-seq identifies m6A site, conditional KO in vivo with functional pyroptosis readouts, single lab\",\n      \"pmids\": [\"41320097\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NSUN3 stabilizes TIFA mRNA through m5C methylation, increasing TIFA protein expression; NSUN3 knockdown reduces TIFA expression, alleviates LPS-induced HK-2 cell injury, and reduces SA-AKI in mice.\",\n      \"method\": \"methylation RNA immunoprecipitation-qPCR, actinomycin D mRNA stability assay, siRNA knockdown, in vivo CLP model\",\n      \"journal\": \"Clinical and experimental pharmacology & physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP-qPCR identifies m5C modification, mRNA stability assay, in vivo model; single lab\",\n      \"pmids\": [\"39924309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ALPK1 activation enhances STING pathway outputs including LC3B lipidation and NLRP3 inflammasome activation; conversely, STING activation increases ALPK1 protein expression and triggers TIFA Thr9 phosphorylation, demonstrating bidirectional crosstalk between the ALPK1/TIFA and STING pathways. ALPK1 signaling also activates eIF2α (integrated stress response).\",\n      \"method\": \"Cell-based pathway activation assays, Western blotting (TIFA Thr9 phosphorylation), LC3B lipidation assay, NLRP3 inflammasome assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single lab, cell-based assays without deep mechanistic dissection of TIFA-specific contributions\",\n      \"pmids\": [\"bio_10.1101_2025.06.30.662363\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"TIFA is an FHA domain-containing adaptor protein that functions as a central hub in innate immune NF-κB signaling: upon sensing of bacterial ADP-heptose metabolites (HBP/ADP-Hep) by the upstream kinase ALPK1, TIFA is phosphorylated at Thr9, triggering phospho-Thr9/FHA-domain-mediated homo-oligomerization and liquid-liquid phase separation into TIFAsomes, which recruit and activate TRAF6 (via K63-linked polyubiquitination) and TRAF2 to activate classical and alternative NF-κB pathways respectively, while TIFAB negatively regulates this process by heterodimerizing with monomeric TIFA to block dimer/oligomer assembly, and TIFA is subsequently degraded via both proteasomal and autophagic pathways; additionally, TIFA bridges IRAK-1 to TRAF6 in IL-1R signaling, translocates to damaged chromatin upon genotoxic stress to drive NEMO ubiquitination, and is post-transcriptionally regulated by METTL3/m6A and NSUN3/m5C modifications that stabilize its mRNA.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TIFA is an FHA-domain adaptor protein that serves as a central signaling hub coupling innate immune sensing of bacterial metabolites to NF-\\u03baB activation [#9, #13]. In its resting state TIFA exists as an intrinsic dimer; phosphorylation at Thr9 generates a pThr9 motif that engages the FHA domain of a neighboring dimer, driving higher-order oligomerization that is the trigger for downstream signaling [#7, #10]. Cytosolic detection of bacterial ADP-heptose/HBP metabolites by the upstream kinase ALPK1 catalyzes this Thr9 phosphorylation during infection by H. pylori, Shigella, Salmonella, and other organisms, nucleating large TIFA oligomeric complexes (TIFAsomes) that behave as liquid-liquid phase-separated condensates [#13, #14, #26]. Within these assemblies TIFA recruits TRAF6 to drive K63-linked polyubiquitination and TAK1/IKK-dependent classical NF-\\u03baB signaling, and engages TRAF2 to displace cIAP1 and activate the alternative NF-\\u03baB pathway [#3, #21, #17]. TIFA additionally bridges IRAK-1 to TRAF6 in IL-1 receptor signaling [#0, #2], and translocates to damaged chromatin upon genotoxic stress where its pThr9/FHA interaction and TRAF2 cooperation stimulate NEMO ubiquitination [#18]. Signaling is restrained by TIFAB, which heterodimerizes with monomeric TIFA to form a pseudo-dimer lacking the phosphorylation site and TRAF6-binding motif, blocking productive oligomer assembly [#27, #4], and activated TIFA is cleared via proteasomal, lysosomal, and TRAF2/TRAF6-dependent autophagic degradation [#25, #29]. Through this pathway TIFA controls intestinal barrier gene expression and inflammasome priming, and its dysregulation promotes leukemic and colorectal cancer cell survival and proliferation [#22, #12, #11, #23].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established TIFA as a TRAF6-associated adaptor selectively engaged downstream of IL-1 but not TNF, placing it in receptor-proximal innate immune signaling.\",\n      \"evidence\": \"Yeast two-hybrid and co-immunoprecipitation mapping the coiled-coil/TRAF6 ring-finger interaction\",\n      \"pmids\": [\"10920205\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not define the activation trigger upstream of TIFA\", \"IRAK absent from the complex left the IRAK-TRAF6 bridging unexplained\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Showed TIFA also binds TRAF2 and is sufficient to activate NF-\\u03baB and AP-1 when overexpressed, broadening its connectivity to multiple TRAF effectors.\",\n      \"evidence\": \"Mammalian two-hybrid, Co-IP, and NF-\\u03baB/AP-1 reporter assays\",\n      \"pmids\": [\"11798190\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Overexpression-driven activation did not establish the physiological stimulus\", \"Distinct roles of TRAF2 vs TRAF6 binding not resolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined TIFA as the bridge that recruits IRAK-1 to TRAF6 upon IL-1 stimulation, resolving how an upstream kinase links to the TRAF6 effector.\",\n      \"evidence\": \"Endogenous Co-IP, FHA/TRAF6-binding mutagenesis, and NF-\\u03baB/JNK reporters\",\n      \"pmids\": [\"12566447\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The kinase phosphorylating TIFA itself was not identified\", \"Mechanism converting binding into TRAF6 activation unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstrated the biochemical mechanism by which TIFA activates IKK: oligomeric TIFA promotes K63-linked TRAF6 polyubiquitination and TAK1/IKK activation proteasome-independently.\",\n      \"evidence\": \"In vitro reconstitution with purified TIFA, TRAF6, TAK1, Ubc13-Uev1A and cell-free IKK assays\",\n      \"pmids\": [\"15492226\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"What drives TIFA into the active oligomeric form in cells was undefined\", \"Structural basis of oligomerization unresolved\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified TIFAB as a homolog that inhibits TIFA-mediated NF-\\u03baB activation, establishing negative regulation of the pathway.\",\n      \"evidence\": \"Co-IP and NF-\\u03baB reporter assays\",\n      \"pmids\": [\"15047173\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of inhibition inferred but not directly shown\", \"Whether TIFAB acts on monomer vs oligomer not resolved at this stage\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Connected TIFA to membrane trafficking via myosin VI binding and trans-Golgi localization, indicating roles beyond cytosolic NF-\\u03baB signaling.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro binding, IF/EM localization, and RNAi phenotyping\",\n      \"pmids\": [\"17635994\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic link between trafficking role and NF-\\u03baB signaling unclear\", \"How myosin VI binding relates to oligomerization not addressed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Pinpointed Thr9 phosphorylation and the pThr9-FHA inter-dimer interaction as the molecular switch driving TIFA oligomerization and NF-\\u03baB activation.\",\n      \"evidence\": \"Phosphorylation mapping, oligomerization assays, siRNA knockdown, and NF-\\u03baB reporters\",\n      \"pmids\": [\"22566686\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The physiological kinase and stimulus were not identified\", \"Higher-order complex architecture not visualized\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified the bacterial metabolite HBP as the cytosolic ligand triggering TIFA-dependent NF-\\u03baB activation, defining the upstream stimulus.\",\n      \"evidence\": \"Genome-wide RNAi screen and cell-based NF-\\u03baB/oligomerization assays\",\n      \"pmids\": [\"26068852\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The sensor/kinase coupling HBP to TIFA phosphorylation was not yet identified\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Provided the structural mechanism for oligomerization, showing TIFA is an intrinsic dimer and pThr9-FHA contacts occur only between distinct dimers.\",\n      \"evidence\": \"X-ray crystallography of TIFA 1-150 and the pThr9 peptide complex with mutagenesis\",\n      \"pmids\": [\"26389808\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full higher-order oligomer/condensate structure not resolved\", \"TRAF6 incorporation geometry not determined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed multiple host kinases (Aurora A, Akt) can phosphorylate TIFA Thr9 to drive NF-\\u03baB survival signaling and inflammasome priming, expanding TIFA into oncogenic and vascular contexts.\",\n      \"evidence\": \"Kinase assays, siRNA knockdown, Co-IP (TIFA-caspase-1), reporter assays, and xenograft/flow models\",\n      \"pmids\": [\"28069801\", \"27965388\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contribution of each kinase in physiological settings unclear\", \"Direct caspase-1 engagement mechanism not structurally defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified ALPK1 as the ADP-heptose/HBP sensor kinase that phosphorylates TIFA Thr9 to nucleate TIFAsomes during enteric and gastric bacterial infection, completing the ligand-kinase-adaptor axis.\",\n      \"evidence\": \"CRISPR/Cas9 knockout, RNAi, mass spectrometry of TIFAsome components, IF, and bacterial mutants across H. pylori, Shigella, and Salmonella models\",\n      \"pmids\": [\"28877472\", \"28222186\", \"28514661\", \"28811347\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative biophysics of TIFAsome assembly not yet defined\", \"How TIFA discriminates replication rate mechanistically partly inferred\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended TIFA function to the DNA damage response, showing genotoxic stress drives TIFA pThr9-dependent chromatin accumulation and TRAF2-dependent NEMO ubiquitination.\",\n      \"evidence\": \"Subcellular fractionation, IF, Thr9 mutagenesis, ubiquitination assays, and siRNA knockdown\",\n      \"pmids\": [\"29581234\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The kinase phosphorylating TIFA after DNA damage not identified\", \"How TIFA targets damaged chromatin mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined the direct TIFA C-terminus/TRAF6 TRAF-domain interface at atomic resolution, mapping the salt-bridge contacts required for engagement.\",\n      \"evidence\": \"X-ray crystallography of TRAF6 with WT and mutant TIFA peptides plus Co-IP\",\n      \"pmids\": [\"30378729\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of TIFA-TRAF6 in the active condensate not defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Confirmed conserved TIFA dimer architecture in mouse and modeled a higher-order TIFA-TRAF6 signaling complex, reinforcing the dimer-to-oligomer mechanism.\",\n      \"evidence\": \"Crystallography of mouse TIFA and phosphomimetic mutants, SEC, and SAXS\",\n      \"pmids\": [\"32198460\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Solution oligomer beyond the dimer modeled but not directly visualized\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linked TIFA/NF-\\u03baB signaling to genome instability, showing the ALPK1/TIFA axis drives co-transcriptional R-loop formation and DNA double-strand breaks during H. pylori infection.\",\n      \"evidence\": \"CRISPR knockout, gastric organoids, R-loop/S9.6 detection, DNA fiber assays, and bacterial mutants\",\n      \"pmids\": [\"33037203\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular link from NF-\\u03baB output to R-loop induction not fully defined\", \"Causal role of TIFA in carcinogenesis not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Dissected how TIFAsomes branch into classical and alternative NF-\\u03baB outputs: TRAF6/TAK1 for classical and TRAF2-mediated cIAP1 displacement/degradation for alternative signaling.\",\n      \"evidence\": \"Reciprocal Co-IP, siRNA knockdown, IF, and immunoblotting in H. pylori-infected epithelial cells\",\n      \"pmids\": [\"34328245\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Temporal coordination of the two branches within one condensate not resolved\", \"Single-lab observation of the cIAP1 displacement step\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed TIFA in intestinal homeostasis and cancer, showing the ALPK1/TIFA/TRAF6 axis drives barrier gene programs and pro-proliferative RSK/PRAS40 signaling requiring both Thr9 oligomerization and TRAF6 binding.\",\n      \"evidence\": \"CRISPR editing, structure-function mutagenesis (T9A, TRAF6-binding deletion), reporters, qPCR, and in vivo proliferation models\",\n      \"pmids\": [\"36036242\", \"35635239\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether commensal-driven and pathogen-driven TIFA signaling differ mechanistically unclear\", \"In vivo causal contribution to tumorigenesis not fully established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed a trafficking/antigen-presentation function in which TIFA binds calnexin and controls MHC-II compartment localization and invariant chain stability.\",\n      \"evidence\": \"siRNA knockdown, immunopeptidome analysis, Co-IP (TIFA-calnexin), IF, and CD4+ T cell assays\",\n      \"pmids\": [\"36215666\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relationship between this trafficking role and NF-\\u03baB signaling unresolved\", \"Whether oligomerization is required not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established that activated TIFA is turned over through both proteasomal and lysosomal routes via autophagy receptors, defining signal termination.\",\n      \"evidence\": \"Proteasome/lysosome inhibitors, Co-IP with polyubiquitin, optineurin, TAX1BP1, LAMP1, and infection time-course\",\n      \"pmids\": [\"36965415\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The E3 ligase ubiquitinating TIFA not identified\", \"Which degradation route dominates physiologically unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed TIFAsome formation is a liquid-liquid phase separation event requiring pThr9-FHA interaction and an intrinsically disordered region, with TRAF6 ubiquitination occurring inside condensates.\",\n      \"evidence\": \"Live-cell phase-separation imaging, chemical probe inhibition, in vitro ubiquitination, mutagenesis, and Co-IP\",\n      \"pmids\": [\"38357697\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Material properties and regulation of condensate dissolution not defined\", \"Single-lab characterization of the LLPS requirement\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided the structural basis for TIFAB inhibition, showing it forms a pseudo-TIFA heterodimer with monomeric TIFA that lacks the phosphorylation site and TRAF6-binding motif.\",\n      \"evidence\": \"Crystallography of the TIFA/TIFAB heterodimer with SEC, in vitro binding, and NF-\\u03baB cell-based assays\",\n      \"pmids\": [\"38442163\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How TIFAB levels are regulated to set the signaling threshold not addressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified a non-NF-\\u03baB TIFA function in hepatocytes, where TIFA blocks PYK2 dimerization and recruits CBL for PYK2 degradation, dampening PYK2-JNK signaling.\",\n      \"evidence\": \"Hepatocyte-specific overexpression/deletion, Co-IP of the TIFA-PYK2-CBL complex, ubiquitination assays, and dietary rodent models\",\n      \"pmids\": [\"39260704\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether this role depends on Thr9/oligomerization not tested\", \"Tissue specificity versus the immune signaling role unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Refined TRAF2 engagement and degradation, defining a novel TIFA TRAF2-binding motif and showing ADP-heptose triggers TRAF2/TRAF6-dependent autophagic clearance of TIFA.\",\n      \"evidence\": \"Binding-motif mutagenesis, Co-IP, autophagy inhibitors, and TRAF2/TRAF6 knockdown\",\n      \"pmids\": [\"40696502\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How TRAFs route TIFA to autophagy mechanistically unclear\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified post-transcriptional control of TIFA, with METTL3/m6A-IGF2BP2 and NSUN3/m5C modifications stabilizing TIFA mRNA to amplify NF-\\u03baB/NLRP3-driven pyroptosis in kidney injury.\",\n      \"evidence\": \"MeRIP-seq/qPCR, RNA stability assays, conditional/knockdown models, Co-IP (IGF2BP2-TIFA mRNA), and in vivo AKI models\",\n      \"pmids\": [\"41320097\", \"39924309\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether these RNA modifications regulate TIFA in other tissues unknown\", \"Crosstalk between mRNA stabilization and protein degradation not integrated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the ALPK1/TIFA axis is integrated with other innate immune pathways such as STING and the integrated stress response, and how TIFAsome assembly/dissolution is quantitatively tuned in vivo, remains open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Bidirectional ALPK1/TIFA-STING crosstalk reported only in a preprint without deep TIFA-specific dissection\", \"No unified model linking condensate dynamics, degradation, and mRNA stabilization in setting signaling output\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2, 3, 18]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [7, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [13, 26]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [18]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [9, 13, 14]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 7, 21]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [11, 20, 23]}\n    ],\n    \"complexes\": [\n      \"TIFAsome\",\n      \"TIFA/TIFAB heterodimer\",\n      \"TIFA-TRAF6 complex\",\n      \"TIFA-TRAF2 complex\"\n    ],\n    \"partners\": [\n      \"TRAF6\",\n      \"TRAF2\",\n      \"TIFAB\",\n      \"IRAK1\",\n      \"ALPK1\",\n      \"NEMO\",\n      \"PYK2\",\n      \"CALNEXIN\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}