{"gene":"TIFAB","run_date":"2026-04-28T21:42:59","timeline":{"discoveries":[{"year":2004,"finding":"TIFAB binds TIFA and inhibits TIFA-mediated NF-κB activation. TIFAB does not associate with TRAF family members directly, but alters the TIFA-TRAF6 interaction by inducing a conformational change in TIFA, increasing the amount of TRAF6 co-precipitated with TIFA without changing the amount of TIFA co-precipitated with TRAF6.","method":"Co-immunoprecipitation, overexpression in HEK293 cells, NF-κB reporter assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 — single Co-IP/pulldown with functional NF-κB reporter assay, single lab, original discovery paper","pmids":["15047173"],"is_preprint":false},{"year":2009,"finding":"TIFAB is predominantly expressed in B cells, dendritic cells, and macrophages in the spleen. TIFAB expression is downregulated upon TRAF6-mediated stimulation (CD40, sIgM, TLRs). Microinjection of TIFAB into NIH3T3 cells inhibits S-phase entry, indicating TIFAB acts as a negative regulator of TRAF6-induced cellular functions including B cell proliferation and DC/macrophage maturation.","method":"Flow cytometry-based cell sorting, microinjection, cell cycle analysis, immunofluorescence","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 2/3 — direct localization and functional microinjection experiments, single lab, multiple cell types tested","pmids":["19470519"],"is_preprint":false},{"year":2015,"finding":"TIFAB forms a complex with TRAF6 and reduces TRAF6 protein stability by a lysosome-dependent mechanism. Loss of TIFAB increases TRAF6 protein levels and amplifies the dynamic range of TLR4 signaling, leading to hypersensitivity to TLR4 stimulation and ineffective hematopoiesis. Combined deletion of TIFAB and miR-146a cooperatively increases TRAF6 expression and hematopoietic dysfunction.","method":"Co-immunoprecipitation, Tifab knockout mouse model, competitive transplantation assay, gene expression analysis, lysosome inhibitor experiments, TLR4 stimulation assays","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, KO mouse with defined cellular phenotype, multiple orthogonal methods, replicated mechanistic findings","pmids":["26458771"],"is_preprint":false},{"year":2020,"finding":"TIFAB regulates ubiquitin-specific peptidase 15 (USP15) ubiquitin hydrolase activity. TIFAB expression in HSPCs permits USP15 signaling to substrates MDM2 and KEAP1, thereby mitigating p53 expression. Loss of TIFAB compromises USP15 signaling and sensitizes HSPCs to hematopoietic stress through derepression of p53. In MLL-AF9 leukemia, TIFAB deletion increases p53 signaling and decreases leukemic cell function; restoring USP15 partially rescues TIFAB-deficient MLL-AF9 cell function.","method":"Proteomic/mass spectrometry interactome, genetic epistasis (KO + rescue), in vitro ubiquitin hydrolase assay, immunoblotting, leukemia transplantation model","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 — proteomics combined with functional enzymatic assay and genetic epistasis rescue, multiple orthogonal methods in single study","pmids":["32101751"],"is_preprint":false},{"year":2021,"finding":"TIFAB accelerates MLL-AF9-induced AML by upregulating HOXA9. Mechanistically, NF-κB non-canonical component RelB directly suppresses TIFAB transcription; forced TIFAB expression downregulates RelB and upregulates HOXA9, blocking myeloid differentiation and upregulating leukemia stem cell signatures.","method":"Retroviral forced expression, genetic deletion (RelB KO), gene set enrichment analysis, AML transplantation model, ChIP/transcriptional target analysis","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 — KO and forced expression with defined downstream target (HOXA9) in leukemia model, single lab","pmids":["34877491"],"is_preprint":false},{"year":2024,"finding":"TIFAB forms a stable heterodimer specifically with monomeric TIFA (not the TIFA dimer), creating a 'pseudo-TIFA dimer' that lacks the phosphorylation site and TRAF6 binding motif present in TIFAB. This heterodimer inhibits TIFA dimer formation and consequently suppresses TIFA-TRAF6-mediated NF-κB activation. Structural analysis revealed the molecular basis for TIFAB's inhibitory mechanism.","method":"Crystal structure determination, biochemical binding assays, cell-based NF-κB reporter assays, mutagenesis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with biochemical and cell-based functional validation, multiple orthogonal methods in single study","pmids":["38442163"],"is_preprint":false},{"year":2025,"finding":"TIFAB modulates metabolic pathways in KMT2A::MLLT3-induced AML through hepatocyte nuclear factor 4 alpha (HNF4A). TIFAB inhibits NF-κB component RelB, which suppresses HNF4A; TIFAB expression thus upregulates HNF4A, enhancing glucose uptake, mitochondrial function, and leukemia stem/progenitor cell engraftment. HNF4A rescue restores metabolic defects caused by Tifab deletion, while Hnf4a knockdown attenuates TIFAB-mediated leukemic progenitor enhancement.","method":"Genetic deletion (Tifab KO), forced expression, gene set enrichment analysis, metabolic assays (glucose uptake, mitochondrial function), leukemia transplantation model, shRNA knockdown epistasis","journal":"Blood advances","confidence":"Medium","confidence_rationale":"Tier 2 — KO and overexpression with epistasis rescue using HNF4A, metabolic functional assays, single lab","pmids":["39626355"],"is_preprint":false},{"year":2022,"finding":"miR-626 regulates NF-κB signaling mediated by TIFAB in oral squamous cell carcinoma; miR-626 inhibition reduces TIFAB-mediated NF-κB signaling and enhances radiosensitivity.","method":"miRNA mimic/inhibitor overexpression, dual luciferase reporter assay, Western blot, in vivo xenograft model","journal":"Cancer biotherapy & radiopharmaceuticals","confidence":"Low","confidence_rationale":"Tier 3 — single lab, single pathway reporter, mechanism of miR-626/TIFAB interaction not fully characterized","pmids":["35549438"],"is_preprint":false}],"current_model":"TIFAB is an FHA-domain protein that acts as a negative regulator of innate immune and NF-κB signaling: it forms a stable heterodimer with monomeric TIFA (blocking TIFA oligomerization and TRAF6 activation), reduces TRAF6 protein stability via a lysosome-dependent mechanism, and regulates USP15 ubiquitin hydrolase activity to modulate MDM2/KEAP1 and p53 signaling in hematopoietic stem/progenitor cells; through inhibition of RelB it also controls transcription of downstream targets HOXA9 and HNF4A to influence leukemic progenitor metabolism and function."},"narrative":{"teleology":[{"year":2004,"claim":"The initial discovery that TIFAB binds TIFA and inhibits TIFA-mediated NF-κB activation established TIFAB as a negative regulator of innate immune signaling, though the mechanism of inhibition was unclear.","evidence":"Co-immunoprecipitation and NF-κB reporter assay in HEK293 cells","pmids":["15047173"],"confidence":"Medium","gaps":["Single Co-IP system without reciprocal endogenous validation","Structural basis for TIFAB–TIFA interaction unknown","In vivo relevance not demonstrated"]},{"year":2009,"claim":"Identification of TIFAB's predominant expression in immune cells (B cells, dendritic cells, macrophages) and its downregulation upon TRAF6-mediated stimulation placed TIFAB in a negative feedback loop within the innate immune system.","evidence":"Flow cytometry-based cell sorting, microinjection into NIH3T3 cells with cell cycle analysis","pmids":["19470519"],"confidence":"Medium","gaps":["Mechanism of TIFAB downregulation upon TRAF6 stimulation not defined","No genetic loss-of-function in vivo model yet"]},{"year":2015,"claim":"Demonstration that TIFAB complexes with TRAF6 and promotes its lysosome-dependent degradation revealed a second arm of NF-κB suppression beyond the TIFA interaction, with TIFAB knockout mice showing hematopoietic dysfunction and TLR4 hypersensitivity.","evidence":"Tifab knockout mice, competitive transplantation, lysosome inhibitor rescue, reciprocal Co-IP","pmids":["26458771"],"confidence":"High","gaps":["How TIFAB directs TRAF6 to lysosomal degradation rather than proteasomal is unclear","Whether TIFAB directly contacts TRAF6 or acts through an intermediary not resolved"]},{"year":2020,"claim":"Discovery that TIFAB regulates USP15 deubiquitinase activity toward MDM2 and KEAP1 expanded TIFAB's function beyond NF-κB, linking it to p53 homeostasis and hematopoietic stress responses.","evidence":"Mass spectrometry interactome, in vitro ubiquitin hydrolase assay, genetic epistasis with USP15 rescue in MLL-AF9 leukemia transplantation model","pmids":["32101751"],"confidence":"High","gaps":["Structural basis for TIFAB regulation of USP15 catalytic activity unknown","Whether TIFAB–USP15 interaction is direct or scaffolded not resolved","Relationship between TIFAB's NF-κB and USP15 functions not dissected"]},{"year":2021,"claim":"Establishing that TIFAB is transcriptionally repressed by RelB and that TIFAB in turn suppresses RelB to upregulate HOXA9 revealed a reciprocal regulatory circuit driving leukemia stem cell programs in MLL-AF9 AML.","evidence":"RelB KO, retroviral forced expression, gene set enrichment analysis, AML transplantation model","pmids":["34877491"],"confidence":"Medium","gaps":["Single-lab finding; independent replication needed","Mechanism by which TIFAB suppresses RelB protein or activity not defined"]},{"year":2024,"claim":"Crystal structure of the TIFAB–TIFA heterodimer resolved the 20-year-old question of how TIFAB inhibits NF-κB: TIFAB sequesters monomeric TIFA into a pseudo-dimer that lacks key phosphorylation and TRAF6-binding sites, preventing TIFA homodimerization.","evidence":"X-ray crystallography, biochemical binding assays, mutagenesis, cell-based NF-κB reporter validation","pmids":["38442163"],"confidence":"High","gaps":["Whether the pseudo-dimer forms in vivo under physiological stoichiometries not shown","Dynamics of monomer–dimer equilibrium in stimulated immune cells not measured"]},{"year":2025,"claim":"Linking TIFAB to metabolic reprogramming in AML through RelB-dependent upregulation of HNF4A showed that TIFAB's NF-κB inhibitory function directly controls glucose uptake and mitochondrial activity in leukemia stem cells.","evidence":"Tifab KO and forced expression, metabolic assays, HNF4A rescue and shRNA epistasis in leukemia transplantation model","pmids":["39626355"],"confidence":"Medium","gaps":["Single-lab study; independent validation needed","Whether HNF4A regulation is direct or entirely mediated through RelB not fully delineated","Relevance to non-KMT2A-rearranged leukemias unknown"]},{"year":null,"claim":"It remains unknown how TIFAB coordinates its dual functions — TIFA sequestration, TRAF6 lysosomal degradation, and USP15 regulation — within the same cell, and whether these activities are context-dependent or operate concurrently.","evidence":"","pmids":[],"confidence":"Low","gaps":["No integrated model linking all three TIFAB effector arms","Post-translational regulation of TIFAB itself largely uncharacterized","Role in normal steady-state hematopoiesis versus stress hematopoiesis not dissected"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2,3,5]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,5]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,1,2,5]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,2,3,5]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[3,4,6]}],"complexes":[],"partners":["TIFA","TRAF6","USP15","RELA"],"other_free_text":[]},"mechanistic_narrative":"TIFAB is an FHA-domain protein that functions as a negative regulator of innate immune signaling by sequestering monomeric TIFA into a pseudo-dimer that lacks the TRAF6-binding motif, thereby blocking TIFA oligomerization and downstream NF-κB activation [PMID:38442163, PMID:15047173]. TIFAB also forms a complex with TRAF6 and promotes its lysosome-dependent degradation, constraining TLR4 signaling amplitude in hematopoietic cells [PMID:26458771]. Beyond NF-κB, TIFAB regulates USP15 deubiquitinase activity toward substrates MDM2 and KEAP1, thereby attenuating p53 levels in hematopoietic stem/progenitor cells and modulating leukemic cell fitness [PMID:32101751]. In KMT2A-rearranged AML, TIFAB suppresses the non-canonical NF-κB component RelB, leading to transcriptional upregulation of HOXA9 and HNF4A, which enhance leukemia stem cell self-renewal and metabolic reprogramming [PMID:34877491, PMID:39626355]."},"prefetch_data":{"uniprot":{"accession":"Q6ZNK6","full_name":"TRAF-interacting protein with FHA domain-containing protein B","aliases":["TIFA-like protein"],"length_aa":161,"mass_kda":17.9,"function":"Inhibits TIFA-mediated TRAF6 activation possibly by inducing a conformational change in TIFA","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q6ZNK6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TIFAB","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TIFAB","total_profiled":1310},"omim":[{"mim_id":"620469","title":"CRANIAL DYSINNERVATION DISORDER, CONGENITAL, WITH ABSENT CORNEAL REFLEX AND DEVELOPMENTAL DELAY; CCDDRD","url":"https://www.omim.org/entry/620469"},{"mim_id":"612663","title":"TRAF-INTERACTING PROTEIN WITH FORKHEAD-ASSOCIATED DOMAIN, FAMILY MEMBER B; TIFAB","url":"https://www.omim.org/entry/612663"},{"mim_id":"153550","title":"CHROMOSOME 5q DELETION SYNDROME","url":"https://www.omim.org/entry/153550"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"lymphoid tissue","ntpm":5.3}],"url":"https://www.proteinatlas.org/search/TIFAB"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q6ZNK6","domains":[{"cath_id":"2.60.200.20","chopping":"6-132","consensus_level":"high","plddt":95.998,"start":6,"end":132}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6ZNK6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6ZNK6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6ZNK6-F1-predicted_aligned_error_v6.png","plddt_mean":89.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TIFAB","jax_strain_url":"https://www.jax.org/strain/search?query=TIFAB"},"sequence":{"accession":"Q6ZNK6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6ZNK6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6ZNK6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6ZNK6"}},"corpus_meta":[{"pmid":"26458771","id":"PMC_26458771","title":"Loss of Tifab, a del(5q) MDS gene, alters hematopoiesis through derepression of Toll-like receptor-TRAF6 signaling.","date":"2015","source":"The Journal of experimental medicine","url":"https://pubmed.ncbi.nlm.nih.gov/26458771","citation_count":96,"is_preprint":false},{"pmid":"29860346","id":"PMC_29860346","title":"DNA methylation as a marker for prenatal smoke exposure in adults.","date":"2018","source":"International journal of epidemiology","url":"https://pubmed.ncbi.nlm.nih.gov/29860346","citation_count":89,"is_preprint":false},{"pmid":"32101751","id":"PMC_32101751","title":"TIFAB Regulates USP15-Mediated p53 Signaling during Stressed and Malignant Hematopoiesis.","date":"2020","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/32101751","citation_count":34,"is_preprint":false},{"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},{"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},{"pmid":"19470519","id":"PMC_19470519","title":"TRAF-interacting protein with a forkhead-associated domain B (TIFAB) is a negative regulator of the TRAF6-induced cellular functions.","date":"2009","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19470519","citation_count":18,"is_preprint":false},{"pmid":"30267110","id":"PMC_30267110","title":"Identification of the TIFAB Gene as a Susceptibility Locus for Coronary Artery Aneurysm in Patients with Kawasaki Disease.","date":"2018","source":"Pediatric cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/30267110","citation_count":16,"is_preprint":false},{"pmid":"27733012","id":"PMC_27733012","title":"Constitutive Activation of NIK Impairs the Self-Renewal of Hematopoietic Stem/Progenitor Cells and Induces Bone Marrow Failure.","date":"2016","source":"Stem cells (Dayton, Ohio)","url":"https://pubmed.ncbi.nlm.nih.gov/27733012","citation_count":14,"is_preprint":false},{"pmid":"23419067","id":"PMC_23419067","title":"A boy with homozygous microdeletion of NEUROG1 presents with a congenital cranial dysinnervation disorder [Moebius syndrome variant].","date":"2013","source":"Behavioral and brain functions : BBF","url":"https://pubmed.ncbi.nlm.nih.gov/23419067","citation_count":13,"is_preprint":false},{"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},{"pmid":"34877491","id":"PMC_34877491","title":"TIFAB accelerates MLL-AF9-Induced acute myeloid leukemia through upregulation of HOXA9.","date":"2021","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/34877491","citation_count":3,"is_preprint":false},{"pmid":"39626355","id":"PMC_39626355","title":"TIFAB modulates metabolic pathways in KMT2A::MLLT3-induced AML through HNF4A.","date":"2025","source":"Blood advances","url":"https://pubmed.ncbi.nlm.nih.gov/39626355","citation_count":1,"is_preprint":false},{"pmid":"35549438","id":"PMC_35549438","title":"miR-626 Inhibition Enhanced the Radiosensitivity to Oral Squamous Cell Carcinoma via the Downregulation of Nuclear Factor Kappa-B Signaling.","date":"2022","source":"Cancer biotherapy & radiopharmaceuticals","url":"https://pubmed.ncbi.nlm.nih.gov/35549438","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8139,"output_tokens":2244,"usd":0.029038},"stage2":{"model":"claude-opus-4-6","input_tokens":5545,"output_tokens":2157,"usd":0.122475},"total_usd":0.151513,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2004,\n      \"finding\": \"TIFAB binds TIFA and inhibits TIFA-mediated NF-κB activation. TIFAB does not associate with TRAF family members directly, but alters the TIFA-TRAF6 interaction by inducing a conformational change in TIFA, increasing the amount of TRAF6 co-precipitated with TIFA without changing the amount of TIFA co-precipitated with TRAF6.\",\n      \"method\": \"Co-immunoprecipitation, overexpression in HEK293 cells, NF-κB reporter assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP/pulldown with functional NF-κB reporter assay, single lab, original discovery paper\",\n      \"pmids\": [\"15047173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TIFAB is predominantly expressed in B cells, dendritic cells, and macrophages in the spleen. TIFAB expression is downregulated upon TRAF6-mediated stimulation (CD40, sIgM, TLRs). Microinjection of TIFAB into NIH3T3 cells inhibits S-phase entry, indicating TIFAB acts as a negative regulator of TRAF6-induced cellular functions including B cell proliferation and DC/macrophage maturation.\",\n      \"method\": \"Flow cytometry-based cell sorting, microinjection, cell cycle analysis, immunofluorescence\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — direct localization and functional microinjection experiments, single lab, multiple cell types tested\",\n      \"pmids\": [\"19470519\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TIFAB forms a complex with TRAF6 and reduces TRAF6 protein stability by a lysosome-dependent mechanism. Loss of TIFAB increases TRAF6 protein levels and amplifies the dynamic range of TLR4 signaling, leading to hypersensitivity to TLR4 stimulation and ineffective hematopoiesis. Combined deletion of TIFAB and miR-146a cooperatively increases TRAF6 expression and hematopoietic dysfunction.\",\n      \"method\": \"Co-immunoprecipitation, Tifab knockout mouse model, competitive transplantation assay, gene expression analysis, lysosome inhibitor experiments, TLR4 stimulation assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, KO mouse with defined cellular phenotype, multiple orthogonal methods, replicated mechanistic findings\",\n      \"pmids\": [\"26458771\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TIFAB regulates ubiquitin-specific peptidase 15 (USP15) ubiquitin hydrolase activity. TIFAB expression in HSPCs permits USP15 signaling to substrates MDM2 and KEAP1, thereby mitigating p53 expression. Loss of TIFAB compromises USP15 signaling and sensitizes HSPCs to hematopoietic stress through derepression of p53. In MLL-AF9 leukemia, TIFAB deletion increases p53 signaling and decreases leukemic cell function; restoring USP15 partially rescues TIFAB-deficient MLL-AF9 cell function.\",\n      \"method\": \"Proteomic/mass spectrometry interactome, genetic epistasis (KO + rescue), in vitro ubiquitin hydrolase assay, immunoblotting, leukemia transplantation model\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — proteomics combined with functional enzymatic assay and genetic epistasis rescue, multiple orthogonal methods in single study\",\n      \"pmids\": [\"32101751\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TIFAB accelerates MLL-AF9-induced AML by upregulating HOXA9. Mechanistically, NF-κB non-canonical component RelB directly suppresses TIFAB transcription; forced TIFAB expression downregulates RelB and upregulates HOXA9, blocking myeloid differentiation and upregulating leukemia stem cell signatures.\",\n      \"method\": \"Retroviral forced expression, genetic deletion (RelB KO), gene set enrichment analysis, AML transplantation model, ChIP/transcriptional target analysis\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO and forced expression with defined downstream target (HOXA9) in leukemia model, single lab\",\n      \"pmids\": [\"34877491\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TIFAB forms a stable heterodimer specifically with monomeric TIFA (not the TIFA dimer), creating a 'pseudo-TIFA dimer' that lacks the phosphorylation site and TRAF6 binding motif present in TIFAB. This heterodimer inhibits TIFA dimer formation and consequently suppresses TIFA-TRAF6-mediated NF-κB activation. Structural analysis revealed the molecular basis for TIFAB's inhibitory mechanism.\",\n      \"method\": \"Crystal structure determination, biochemical binding assays, cell-based NF-κB reporter assays, mutagenesis\",\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 cell-based functional validation, multiple orthogonal methods in single study\",\n      \"pmids\": [\"38442163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TIFAB modulates metabolic pathways in KMT2A::MLLT3-induced AML through hepatocyte nuclear factor 4 alpha (HNF4A). TIFAB inhibits NF-κB component RelB, which suppresses HNF4A; TIFAB expression thus upregulates HNF4A, enhancing glucose uptake, mitochondrial function, and leukemia stem/progenitor cell engraftment. HNF4A rescue restores metabolic defects caused by Tifab deletion, while Hnf4a knockdown attenuates TIFAB-mediated leukemic progenitor enhancement.\",\n      \"method\": \"Genetic deletion (Tifab KO), forced expression, gene set enrichment analysis, metabolic assays (glucose uptake, mitochondrial function), leukemia transplantation model, shRNA knockdown epistasis\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO and overexpression with epistasis rescue using HNF4A, metabolic functional assays, single lab\",\n      \"pmids\": [\"39626355\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"miR-626 regulates NF-κB signaling mediated by TIFAB in oral squamous cell carcinoma; miR-626 inhibition reduces TIFAB-mediated NF-κB signaling and enhances radiosensitivity.\",\n      \"method\": \"miRNA mimic/inhibitor overexpression, dual luciferase reporter assay, Western blot, in vivo xenograft model\",\n      \"journal\": \"Cancer biotherapy & radiopharmaceuticals\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, single pathway reporter, mechanism of miR-626/TIFAB interaction not fully characterized\",\n      \"pmids\": [\"35549438\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TIFAB is an FHA-domain protein that acts as a negative regulator of innate immune and NF-κB signaling: it forms a stable heterodimer with monomeric TIFA (blocking TIFA oligomerization and TRAF6 activation), reduces TRAF6 protein stability via a lysosome-dependent mechanism, and regulates USP15 ubiquitin hydrolase activity to modulate MDM2/KEAP1 and p53 signaling in hematopoietic stem/progenitor cells; through inhibition of RelB it also controls transcription of downstream targets HOXA9 and HNF4A to influence leukemic progenitor metabolism and function.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TIFAB is an FHA-domain protein that functions as a negative regulator of innate immune signaling by sequestering monomeric TIFA into a pseudo-dimer that lacks the TRAF6-binding motif, thereby blocking TIFA oligomerization and downstream NF-κB activation [PMID:38442163, PMID:15047173]. TIFAB also forms a complex with TRAF6 and promotes its lysosome-dependent degradation, constraining TLR4 signaling amplitude in hematopoietic cells [PMID:26458771]. Beyond NF-κB, TIFAB regulates USP15 deubiquitinase activity toward substrates MDM2 and KEAP1, thereby attenuating p53 levels in hematopoietic stem/progenitor cells and modulating leukemic cell fitness [PMID:32101751]. In KMT2A-rearranged AML, TIFAB suppresses the non-canonical NF-κB component RelB, leading to transcriptional upregulation of HOXA9 and HNF4A, which enhance leukemia stem cell self-renewal and metabolic reprogramming [PMID:34877491, PMID:39626355].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"The initial discovery that TIFAB binds TIFA and inhibits TIFA-mediated NF-κB activation established TIFAB as a negative regulator of innate immune signaling, though the mechanism of inhibition was unclear.\",\n      \"evidence\": \"Co-immunoprecipitation and NF-κB reporter assay in HEK293 cells\",\n      \"pmids\": [\"15047173\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single Co-IP system without reciprocal endogenous validation\",\n        \"Structural basis for TIFAB–TIFA interaction unknown\",\n        \"In vivo relevance not demonstrated\"\n      ]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identification of TIFAB's predominant expression in immune cells (B cells, dendritic cells, macrophages) and its downregulation upon TRAF6-mediated stimulation placed TIFAB in a negative feedback loop within the innate immune system.\",\n      \"evidence\": \"Flow cytometry-based cell sorting, microinjection into NIH3T3 cells with cell cycle analysis\",\n      \"pmids\": [\"19470519\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism of TIFAB downregulation upon TRAF6 stimulation not defined\",\n        \"No genetic loss-of-function in vivo model yet\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstration that TIFAB complexes with TRAF6 and promotes its lysosome-dependent degradation revealed a second arm of NF-κB suppression beyond the TIFA interaction, with TIFAB knockout mice showing hematopoietic dysfunction and TLR4 hypersensitivity.\",\n      \"evidence\": \"Tifab knockout mice, competitive transplantation, lysosome inhibitor rescue, reciprocal Co-IP\",\n      \"pmids\": [\"26458771\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How TIFAB directs TRAF6 to lysosomal degradation rather than proteasomal is unclear\",\n        \"Whether TIFAB directly contacts TRAF6 or acts through an intermediary not resolved\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Discovery that TIFAB regulates USP15 deubiquitinase activity toward MDM2 and KEAP1 expanded TIFAB's function beyond NF-κB, linking it to p53 homeostasis and hematopoietic stress responses.\",\n      \"evidence\": \"Mass spectrometry interactome, in vitro ubiquitin hydrolase assay, genetic epistasis with USP15 rescue in MLL-AF9 leukemia transplantation model\",\n      \"pmids\": [\"32101751\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for TIFAB regulation of USP15 catalytic activity unknown\",\n        \"Whether TIFAB–USP15 interaction is direct or scaffolded not resolved\",\n        \"Relationship between TIFAB's NF-κB and USP15 functions not dissected\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Establishing that TIFAB is transcriptionally repressed by RelB and that TIFAB in turn suppresses RelB to upregulate HOXA9 revealed a reciprocal regulatory circuit driving leukemia stem cell programs in MLL-AF9 AML.\",\n      \"evidence\": \"RelB KO, retroviral forced expression, gene set enrichment analysis, AML transplantation model\",\n      \"pmids\": [\"34877491\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab finding; independent replication needed\",\n        \"Mechanism by which TIFAB suppresses RelB protein or activity not defined\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Crystal structure of the TIFAB–TIFA heterodimer resolved the 20-year-old question of how TIFAB inhibits NF-κB: TIFAB sequesters monomeric TIFA into a pseudo-dimer that lacks key phosphorylation and TRAF6-binding sites, preventing TIFA homodimerization.\",\n      \"evidence\": \"X-ray crystallography, biochemical binding assays, mutagenesis, cell-based NF-κB reporter validation\",\n      \"pmids\": [\"38442163\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether the pseudo-dimer forms in vivo under physiological stoichiometries not shown\",\n        \"Dynamics of monomer–dimer equilibrium in stimulated immune cells not measured\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linking TIFAB to metabolic reprogramming in AML through RelB-dependent upregulation of HNF4A showed that TIFAB's NF-κB inhibitory function directly controls glucose uptake and mitochondrial activity in leukemia stem cells.\",\n      \"evidence\": \"Tifab KO and forced expression, metabolic assays, HNF4A rescue and shRNA epistasis in leukemia transplantation model\",\n      \"pmids\": [\"39626355\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single-lab study; independent validation needed\",\n        \"Whether HNF4A regulation is direct or entirely mediated through RelB not fully delineated\",\n        \"Relevance to non-KMT2A-rearranged leukemias unknown\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how TIFAB coordinates its dual functions — TIFA sequestration, TRAF6 lysosomal degradation, and USP15 regulation — within the same cell, and whether these activities are context-dependent or operate concurrently.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No integrated model linking all three TIFAB effector arms\",\n        \"Post-translational regulation of TIFAB itself largely uncharacterized\",\n        \"Role in normal steady-state hematopoiesis versus stress hematopoiesis not dissected\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2, 3, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 1, 2, 5]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2, 3, 5]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3, 4, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"TIFA\",\n      \"TRAF6\",\n      \"USP15\",\n      \"RELA\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}