{"gene":"TRAF6","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":1999,"finding":"TRAF6 knockout mice are osteopetrotic with impaired osteoclast function and show defective IL-1, CD40, and LPS signaling, establishing TRAF6 as an essential signaling mediator downstream of these diverse receptor pathways in vivo.","method":"Gene knockout mouse model with in vitro signaling assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with defined cellular phenotypes across multiple pathways, replicated across multiple assay readouts","pmids":["10215628"],"is_preprint":false},{"year":2003,"finding":"TRAF6 is required for dendritic cell maturation; TRAF6-deficient DCs fail to upregulate MHCII and B7.2, produce inflammatory cytokines, or stimulate naive T cells in response to LPS or CD40L, and the CD4+CD8α− splenic DC subset is nearly absent.","method":"TRAF6-deficient mice and bone marrow chimeras; flow cytometry and functional assays","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO and chimera experiments with multiple orthogonal functional readouts","pmids":["14499111"],"is_preprint":false},{"year":2002,"finding":"TRAF6 associates with XEDAR and TROY/TAJ (EDAR-related TNFR superfamily members) and is essential for XEDAR-mediated NF-κB activation, contributing to development of epidermal appendages; TRAF6 does not associate with EDAR.","method":"Co-immunoprecipitation; TRAF6-deficient mice; NF-κB reporter assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus KO phenotype plus functional NF-κB assay, multiple orthogonal methods","pmids":["12060722"],"is_preprint":false},{"year":2008,"finding":"TRAF6 is specifically required for TGF-β-induced, Smad-independent activation of JNK and p38; its C-terminal TRAF homology domain interacts with TGF-β receptors; TGF-β induces K63-linked ubiquitination of TRAF6 and promotes TRAF6–TAK1 association.","method":"Co-immunoprecipitation; siRNA knockdown; ubiquitination assays; kinase activation assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus ubiquitination assay plus kinase assay, single lab with multiple orthogonal methods","pmids":["18922473"],"is_preprint":false},{"year":2004,"finding":"TIFA (TRAF-interacting protein with a forkhead-associated domain) activates IKK by promoting oligomerization and K63-linked polyubiquitination of TRAF6; only oligomeric TIFA activates IKK; activation was reconstituted in vitro with purified TRAF6, TAK1 complex, and Ubc13-Uev1A.","method":"In vitro reconstitution with purified proteins; cell-free IKK activation assay; ubiquitination assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified proteins plus mutagenesis (TRAF6-binding-defective TIFA mutant), single lab","pmids":["15492226"],"is_preprint":false},{"year":2011,"finding":"TRAF6 associates with TβRI and mediates Lys63-linked polyubiquitination of TβRI, promoting PKCζ-dependent cleavage by TACE; the released TβRI intracellular domain translocates to the nucleus and, with p300, activates invasion-promoting genes (Snail, MMP2).","method":"Co-immunoprecipitation; ubiquitination assays; pharmacological inhibition; nuclear fractionation; cell invasion assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, ubiquitination assay, fractionation, functional assays; multiple orthogonal methods in single lab","pmids":["21629263"],"is_preprint":false},{"year":2017,"finding":"TRAF6 E3 ligase activity contributes to but is not solely essential for IL-1-dependent K63-Ub chain formation and TAK1 activation, because Pellino1/2 generate redundant K63-Ub chains; TRAF6/Pellino1/Pellino2 triple-KO abolishes K63-Ub formation on IRAK1, IRAK4, and MyD88. E3 ligase-inactive TRAF6 mutants partially restore IL-1 signaling, demonstrating E3 ligase-independent roles of TRAF6.","method":"CRISPR/Cas9 triple-KO cells; E3 ligase-inactive knockin mice (TRAF6[L74H]); ubiquitination assays; in vitro TAK1 activation assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple KO/knockin genetic models plus in vitro TAK1 assay plus mutagenesis, multiple orthogonal methods","pmids":["28404732"],"is_preprint":false},{"year":2018,"finding":"The TRAF6 RING dimer employs a concerted allosteric mechanism for ubiquitin transfer: both subunits cooperate, with the C-terminal extension of one TRAF6 RING subunit making trans-interactions with donor-Ub to promote Ub transfer to the E2.","method":"Structural modeling; biochemical ubiquitination assays; mutagenesis of RING domain extensions","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — structural modeling combined with biochemical assay and mutagenesis, single lab","pmids":["29432170"],"is_preprint":false},{"year":2018,"finding":"The CBM (CARMA1-BCL10-MALT1) signalosome recruits TRAF6, which cooperatively decorates BCL10 filaments to form higher-order assemblies; cryo-EM structure of BCL10 CARD filament at 4.0-Å resolution redefines CARD-CARD interactions; MALT1 immediately dimerizes on the BCL10 filamentous scaffold before recruiting TRAF6.","method":"Cryo-electron microscopy; time-lapse confocal imaging; biochemical reconstitution","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure plus live imaging plus reconstitution, single study with multiple orthogonal methods","pmids":["29382759"],"is_preprint":false},{"year":2018,"finding":"PRDX1 interacts with the RING finger domain of TRAF6 and inhibits its E3 ubiquitin ligase activity, thereby suppressing K63-linked ubiquitination of ECSIT (required for NF-κB activation) and BECN1 (required for autophagy) in response to TLR4 stimulation.","method":"Co-immunoprecipitation; in vitro ubiquitination assay; siRNA knockdown (PRDX1KD cells); NF-κB reporter assay","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus in vitro ubiquitination assay plus multiple KD cell lines, single lab","pmids":["29929436"],"is_preprint":false},{"year":2013,"finding":"TRAF6 binds HIF-1α and mediates its K63-linked polyubiquitination, increasing HIF-1α protein stability independent of oxygen levels; TRAF6 E3 ligase activity is required for this effect and for promoting tumor angiogenesis.","method":"Co-immunoprecipitation; ubiquitination assay; E3 ligase-deficient mutant; cell-based HIF-1α protein stability assay; in vivo tumor angiogenesis assay","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus E3 ligase mutant, single lab","pmids":["23722539"],"is_preprint":false},{"year":2013,"finding":"PINK1 forms a complex with SARM1 and TRAF6; TRAF6 mediates K63-linked ubiquitination of PINK1 at Lys433, promoted by SARM1; this complex is required for PINK1 stabilization on depolarized mitochondria and subsequent parkin recruitment for mitophagy; some pathogenic PINK1 mutations reduce complex formation and ubiquitination.","method":"Co-immunoprecipitation; ubiquitination assay with Lys433 mutant; siRNA knockdown; mitochondrial fractionation; fluorescence microscopy of parkin recruitment","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, site-specific ubiquitination assay with mutant, siRNA KD phenotype, localization imaging; multiple orthogonal methods, single lab","pmids":["23885119"],"is_preprint":false},{"year":2008,"finding":"TRAF6 is required for RIG-I/MDA5 pathway-dependent NF-κB, JNK, and p38 activation in response to viral infection or poly(I:C); TRAF6-deficient MEFs show impaired IRF3-independent signaling. MEKK1 (not TAK1) is the MAP3K used by TRAF6 for IFN-β induction in this pathway; forced MEKK1 + IRF3 expression induces IFN-β.","method":"TRAF6-deficient MEFs; TAK1-deficient MEFs; siRNA knockdown of MEKK1; overexpression rescue; IFN-β reporter assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic KO cells plus siRNA plus rescue, multiple orthogonal approaches, single lab","pmids":["18984593"],"is_preprint":false},{"year":2019,"finding":"TRAF6 mediates K63-linked ubiquitination of FOXP3 at Lys262, directing its proper nuclear localization; loss of TRAF6 or mutation of K262 causes perinuclear accumulation of FOXP3 and disrupted Treg suppressive function; Treg-specific TRAF6 deletion renders mice resistant to implanted tumors.","method":"Treg-specific TRAF6 conditional KO mice; site-directed mutagenesis (K262R FOXP3); ubiquitination assay; immunofluorescence localization; in vivo tumor models","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — conditional KO plus site-specific ubiquitination mutagenesis plus localization imaging plus in vivo phenotype, multiple orthogonal methods, single lab","pmids":["30886050"],"is_preprint":false},{"year":2008,"finding":"HSV-1 UL37 tegument protein activates NF-κB through direct interaction with TRAF6 via a TRAF6-binding domain in UL37; mutation of this domain reduces NF-κB activation during early infection.","method":"Co-immunoprecipitation; NF-κB reporter assay; recombinant virus with mutated TRAF6-binding site","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus reporter assay plus recombinant virus mutagenesis, single lab","pmids":["18682563"],"is_preprint":false},{"year":2000,"finding":"T6BP specifically associates with TRAF6 via the coiled-coil region of T6BP and the N-terminal RING/zinc finger domains of TRAF6; IL-1 (not TNF) induces TRAF6–T6BP complex formation in an IRAK-dependent manner; IRAK is not present in TRAF6–T6BP complexes.","method":"Co-immunoprecipitation; yeast two-hybrid; domain-mapping pulldowns; IL-1 stimulation time course","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP with domain mapping and ligand-dependent complex formation, single lab","pmids":["10920205"],"is_preprint":false},{"year":2010,"finding":"The RING finger motif of TRAF6 is S-glutathionylated under basal conditions; upon IL-1 stimulation, GRX-1 catalyzes deglutathionylation of TRAF6, which is essential for TRAF6 K63-linked auto-polyubiquitination and subsequent NF-κB activation; GRX-1 knockdown severely reduces IL-1R/TLR4-mediated NF-κB induction.","method":"RNAi knockdown; S-glutathionylation detection assay; ubiquitination assay; NF-κB reporter assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — post-translational modification identified biochemically plus functional siRNA knockdown, single lab","pmids":["21078302"],"is_preprint":false},{"year":2013,"finding":"p62/sequestosome-1 interacts with NEMO and is required for TRAF6-mediated K63-linked ubiquitination of NEMO; p62 depletion abolishes TRAF6's ability to ubiquitinate NEMO and severely impairs NF-κB activation following IL-1β stimulation.","method":"Co-immunoprecipitation; siRNA knockdown; ubiquitination assay; NF-κB activation assay","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus siRNA functional validation, single lab","pmids":["24270048"],"is_preprint":false},{"year":2016,"finding":"TRAF6 interacts with Basigin (BSG/CD147) and mediates its K63-linked ubiquitination, promoting BSG recruitment to the plasma membrane and BSG-dependent MMP9 induction; TRAF6 depletion reduces BSG membrane localization and impairs melanoma invasion/metastasis.","method":"Co-immunoprecipitation; shRNA knockdown; ubiquitination assay; cell invasion assays; in vivo metastasis model","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional invasion/metastasis assays, single lab","pmids":["26769849"],"is_preprint":false},{"year":2010,"finding":"NUMBL directly binds TRAF6 in vivo and in vitro, promotes K48-linked (but not K63-linked) polyubiquitination of TRAF6, reduces TRAF6 protein half-life, and thereby inhibits NF-κB signaling; NUMBL knockdown increases endogenous TRAF6 protein in neurons.","method":"Co-immunoprecipitation; in vivo ubiquitination assay with K48 and K63 mutants; siRNA knockdown; pulse-chase protein stability assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus linkage-specific ubiquitination assay plus protein stability assay, single lab","pmids":["20079715"],"is_preprint":false},{"year":2017,"finding":"IL-1β can activate a TAB1-TAK1 heterodimer in the absence of TAB2/TAB3, but this alternative pathway requires TRAF6 expression and E3 ligase activity and depends on Ubc13-directed K63-Ub chain formation; in TAB2/3 DKO cells, IL-1β signaling is transient and JNK1/2 and p38γ activation is greatly reduced.","method":"TAB2/3 double-KO and triple-KO cell lines; TRAF6 siRNA; re-expression rescue with TAB1, TAB2, or ubiquitin-binding-defective TAB2; kinase assays","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with multiple KO combinations plus rescue experiments plus kinase assays, multiple orthogonal methods, single lab","pmids":["28507161"],"is_preprint":false},{"year":2019,"finding":"TRAF6 K63-linked ubiquitinates cGAS, activating its enzymatic activity; TRAF6 knockdown decreases cGAS-induced IFNβ production upon HSV-1 infection.","method":"Ectopic expression and knockdown; ubiquitination assay; IFNβ reporter/expression assay; HSV-1 infection","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single method set (overexpression/KD + ubiquitination), no in vitro reconstitution or mutagenesis of ubiquitination site","pmids":["31078259"],"is_preprint":false},{"year":2019,"finding":"TRAF6 binds and K63-ubiquitinates hDNA2, increasing its stability and promoting its nuclear localization; TRAF6-mediated polyubiquitination of hDNA2 is required for DNA end resection and homology-directed repair; inhibiting this ubiquitination abolishes nuclear hDNA2 localization.","method":"Co-immunoprecipitation; ubiquitination assay; nuclear fractionation; DNA end resection assay; HDR assay","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus localization and functional DNA repair assays, single lab","pmids":["31216032"],"is_preprint":false},{"year":2016,"finding":"K63-linked ubiquitin chains and the E3 ligase TRAF6 regulate mammalian P-body formation and mRNA decapping; TRAF6 binds DCP1a and indirectly regulates its phosphorylation; mutation of six C-terminal lysines of DCP1a suppresses decapping activity and impairs interaction with DCP2, EDC4, and XRN1.","method":"Co-immunoprecipitation; DCP1a lysine-mutant analysis; P-body formation imaging; mRNA decay assays; ubiquitination assays","journal":"Molecular cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus mutagenesis plus functional mRNA decay assays, single lab with multiple methods","pmids":["27315556"],"is_preprint":false},{"year":2009,"finding":"IL-1β stimulates a TRAF6–MLK3 interaction; TRAF6 RNAi dramatically impairs MLK3 kinase activation by IL-1β; TNF/IL-1β also stimulates K63-linked ubiquitination of MLK3 that is important for its kinase activity, while K48-linked ubiquitination directs MLK3 for proteasomal degradation.","method":"Co-immunoprecipitation; RNAi; in vitro kinase assay; linkage-specific ubiquitination analysis","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus RNAi plus kinase assay plus ubiquitination assay, single lab","pmids":["19586614"],"is_preprint":false},{"year":2019,"finding":"GCA (grancalcin) activates TRAF6 ubiquitin ligase activity to induce K63-linked ubiquitination of ULK1, resulting in ULK1 stabilization and activation and consequent autophagy induction; this GCA-TRAF6-ULK1 axis mediates imatinib resistance in CML.","method":"Co-immunoprecipitation; ubiquitination assay; siRNA/overexpression; autophagy flux assays; drug resistance assays","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional autophagy and resistance assays, single lab","pmids":["30929559"],"is_preprint":false},{"year":2021,"finding":"TRAF6 acts as both a positive regulator (scaffolding for NF-κB activation) and a negative regulator (suppressor of basal MALT1 protease activity) in T cells; loss of TRAF6-mediated homeostatic suppression of MALT1 protease leads to severe autoimmune inflammation fully reverted by genetic or pharmacological MALT1 protease inactivation.","method":"Genetically engineered mouse models; biochemical analyses; pharmacological MALT1 inhibition; epistasis experiments","journal":"Science immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic mouse models plus biochemical analysis plus pharmacological rescue, multiple orthogonal methods","pmids":["34767456"],"is_preprint":false},{"year":2016,"finding":"Cullin-5 (Cul-5) directly binds TRAF6 (C-terminal domain of Cul-5 interacts with TRAF-C domain of TRAF6) and promotes TRAF6 K63-linked polyubiquitination in response to LPS; hemizygous Cul-5 KO delays NF-κB and MAPK activation in LPS-stimulated macrophages.","method":"Co-immunoprecipitation; domain-mapping pulldown; ubiquitination assay; hemizygous KO mice; macrophage signaling assays","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-specific Co-IP plus ubiquitination assay plus KO phenotype, single lab","pmids":["27233966"],"is_preprint":false},{"year":2018,"finding":"RSK2 directly phosphorylates TRAF6 at Ser46, Ser47, and Ser48 (in vitro kinase assay); RSK2 positively regulates TRAF6 K63-linked ubiquitination; TRAF6 is also required for RSK2 ubiquitination; RSK2 KO mice show reduced TRAF6 K63-ubiquitination and attenuated colon inflammation.","method":"In vitro kinase assay; Co-immunoprecipitation; ubiquitination assay; RSK2 KO mice; colon inflammation model","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay identifies specific phosphorylation sites, plus Co-IP, ubiquitination assay, and KO mouse phenotype, single lab","pmids":["29563609"],"is_preprint":false},{"year":2019,"finding":"TRAF6 directly interacts with PIK3CA (p110α catalytic subunit of PI3K) and promotes its non-proteolytic K63-linked polyubiquitination under serum stimulation; TRAF6 overexpression enhances PI3K activation and downstream AKT phosphorylation and cell growth.","method":"Co-immunoprecipitation; ubiquitination assay; overexpression/knockdown; AKT phosphorylation assay; cell growth assay","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional signaling readout, single lab","pmids":["29729098"],"is_preprint":false},{"year":2019,"finding":"HIV-1 Tat directly interacts with TRAF6, increases TRAF6 oligomerization and auto-ubiquitination, and promotes K63-linked polyubiquitin chain synthesis, thereby activating NF-κB and HIV-1 transcription; TRAF6 knockdown inhibits HIV-1 transcription.","method":"Co-immunoprecipitation; ubiquitination assay; TRAF6 KD/overexpression; NF-κB reporter and HIV-1 transcription assays","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional transcription assays, single lab","pmids":["38241362"],"is_preprint":false},{"year":2010,"finding":"TRAF6 binds the IL-2Rβ chain at a site overlapping the Jak1-binding site and negatively regulates IL-2-induced Jak1-Erk pathway activation; a β-chain mutation that inactivates TRAF6 binding but retains Jak1 binding abrogates TRAF6-dependent suppression of IL-2 signaling; Traf6−/− CD4+ T cells show hyperactivated IL-2 signaling.","method":"Traf6−/− mouse T cells and MEFs; β-chain binding-site mutants; IL-2 stimulation assays; Jak1 and Erk activation assays","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO plus binding-site mutagenesis plus functional signaling assays, single lab","pmids":["21155952"],"is_preprint":false},{"year":2015,"finding":"Satellite cell-specific deletion of Traf6 causes profound muscle regeneration defects with reduced PAX7 and late myogenesis markers; TRAF6 activates ERK1/2 and JNK1/2 → c-JUN → Pax7 promoter binding, augmenting Pax7 expression; TRAF6/c-JUN signaling also represses miR-1 and miR-206 to maintain PAX7 levels.","method":"Conditional (satellite cell-specific) KO mice; ChIP (c-JUN at Pax7 promoter); miRNA expression analysis; kinase activation assays; mdx double-mutant model","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with specific phenotype plus ChIP plus miRNA analysis plus disease model, multiple orthogonal methods","pmids":["26619121"],"is_preprint":false},{"year":2021,"finding":"OTUB1 directly binds TRAF6 and suppresses its K63-linked polyubiquitination, thereby inhibiting TRAF6-mediated activation of ASK1 and downstream pathways; OTUB1 KO aggravates NASH, and hepatocyte-specific OTUB1 overexpression alleviates it.","method":"Immunoprecipitation-mass spectrometry; Co-immunoprecipitation; ubiquitination assay; OTUB1 KO and overexpression mouse models; NASH diet models","journal":"Hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — IP-MS plus Co-IP plus ubiquitination assay plus in vivo KO/OE, single lab","pmids":["34591986"],"is_preprint":false},{"year":2023,"finding":"TRAF6 undergoes phase separation (liquid-liquid condensate/droplet formation) during LPS-induced inflammation, which is essential for its ubiquitination-mediated NF-κB activation; Sufu directly interacts with TRAF6, prevents its oligomerization and autoubiquitination, and inhibits TRAF6 phase-separated droplet formation; Sufu deletion augments LPS/CLP-induced lung injury and is rescued by TRAF6 depletion.","method":"Co-immunoprecipitation; FRAP; Native-PAGE; in vitro phase separation assay with purified proteins; GFP-TRAF6 stable cell line; CLP sepsis mouse model","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro phase separation assay with purified proteins plus FRAP plus multiple in vivo genetic models, multiple orthogonal methods","pmids":["37441604"],"is_preprint":false},{"year":2020,"finding":"TRAF6 mediates K63-linked ubiquitination of STAT6, promoting STAT6 protein stability by reducing its K48-linked ubiquitination (degradation); TRAF6's E3 ligase activity is dispensable for STAT6 stabilization despite promoting K63-ubiquitination; TRAF6 deficiency impairs IL-4-induced M2 macrophage gene expression.","method":"Co-immunoprecipitation; overexpression/knockdown/KO; ubiquitination assays (K48/K63 specific); STAT6 stability assay","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus linkage-specific ubiquitination assay plus KO functional readout, single lab","pmids":["33017719"],"is_preprint":false},{"year":2021,"finding":"Viperin binds TRAF6 and activates its ubiquitin transfer activity 2.5-fold, causing increased polyubiquitinated forms of TRAF6 important for immune signaling.","method":"Protein-protein binding assay; in vitro ubiquitin transfer assay with purified proteins","journal":"Journal of the American Chemical Society","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution with purified proteins, single lab, single study","pmids":["33779167"],"is_preprint":false},{"year":2019,"finding":"BANK1 interacts with TRAF6 via its TIR domain and via five identified TRAF6-binding motifs; the C-terminal domain of BANK1-FL and N-terminal domain of BANK1-Δ2 are required for TRAF6 binding; BANK1 Δ2 isoform lacking TIR domain shows enhanced K63-linked polyubiquitination and IL-8 production.","method":"Co-immunoprecipitation; point mutation and decoy peptide analysis; K63-ubiquitination assay; IL-8 production assay","journal":"Cellular & molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mutagenesis plus ubiquitination assay plus functional cytokine readout, single lab","pmids":["31243359"],"is_preprint":false},{"year":2019,"finding":"TRAF6 neddylation at Lys124 is essential for IL-17A-induced NF-κB activation in fibroblast-like synoviocytes; K124R mutation markedly impairs NEDD8 conjugation to TRAF6 and attenuates IL-17A-induced NF-κB activity.","method":"Site-directed mutagenesis (K124R); neddylation assay; NF-κB activation assay; collagen-induced arthritis mouse model","journal":"Laboratory investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific mutagenesis plus neddylation assay plus functional NF-κB readout, single lab","pmids":["30626891"],"is_preprint":false},{"year":2018,"finding":"YAP interacts with TRAF6 (E3 ligase), promotes TRAF6 ubiquitination and degradation, inhibiting NF-κB activation in endothelial cells; endothelial YAP KO augments inflammatory response reversed by TRAF6 depletion.","method":"Co-immunoprecipitation; ubiquitination assay; endothelial-specific YAP and TRAF6 KO mice; sepsis/LPS models","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP plus ubiquitination assay plus conditional KO mice with genetic rescue (TRAF6 depletion reverting phenotype), multiple orthogonal methods","pmids":["29794022"],"is_preprint":false},{"year":2007,"finding":"Syntenin specifically interacts with TRAF6 under physiological conditions and inhibits TRAF6-induced NF-κB and AP-1 activation; upon IL-1 stimulation, syntenin dissociates from TRAF6 as IRAK1 overexpression competes for TRAF6 binding and restores TRAF6 ubiquitination; syntenin knockdown potentiates IL-1/LPS-triggered NF-κB activation.","method":"Co-immunoprecipitation; NF-κB/AP-1 reporter assays; siRNA knockdown; competition binding assay","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus reporter assays plus siRNA functional validation, single lab","pmids":["18234474"],"is_preprint":false},{"year":2017,"finding":"SMN1 physically interacts with TRAF6 and with IKK-α, IKK-β, and IKK-γ; SMN1 inhibits TRAF6 E3 ubiquitin ligase activity and IKK kinase activity; SMN1 depletion enhances IL-1β-induced IKK activation and inflammatory mediator production; SMA patient fibroblasts show potentiated IL-1β-induced IKK activity.","method":"Co-immunoprecipitation; in vitro E3 ligase activity assay; in vitro IKK kinase assay; siRNA knockdown; patient fibroblasts","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus in vitro enzyme activity assays plus siRNA KD, single lab","pmids":["28214532"],"is_preprint":false},{"year":2008,"finding":"TRAF6 deficiency in TNF signaling leads to ROS accumulation, prolonged JNK activation, reduced NF-κB p65 phosphorylation, and accelerated cell death; TRAF6 deficiency causes constitutive phosphorylation (inactivation) of GSK3β; restoration of GSK3β activity rescues cell death in TRAF6-null cells.","method":"TRAF6-KO 3T3 cells; ROS measurement; JNK/NF-κB activation assays; GSK3β kinase assay; constitutively active GSK3β rescue","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO cells plus multiple signaling assays plus rescue experiment, single lab","pmids":["18202703"],"is_preprint":false},{"year":2022,"finding":"USP15 interacts with BECN1 (but not with TRAF6) and induces deubiquitination of BECN1, attenuating TRAF6-BECN1 axis-dependent autophagy induction and lung cancer migration/invasion.","method":"Co-immunoprecipitation; deubiquitination assay; CRISPR-KO cell lines; migration/invasion assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus deubiquitination assay plus CRISPR KO functional phenotype, single lab","pmids":["35422093"],"is_preprint":false},{"year":2024,"finding":"TRAF6 stabilizes YAP1 via K63-linked polyubiquitination, which promotes formation of YAP1/TFCP2 transcriptional complex and PD-L1 transcription in melanoma; TRAF6 inhibition with Bortezomib reduces endogenous PD-L1 and enhances CD8+ T cell cytolytic activity.","method":"CRISPR interference screening; Co-immunoprecipitation; K63-ubiquitination assay; in vitro and in vivo functional assays; flow cytometry","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus in vivo functional assays, single lab","pmids":["38583649"],"is_preprint":false},{"year":2003,"finding":"c-Src interacts with TRAF6 in response to IL-1 and this interaction is required for c-Src activity; TRAF6 and c-Src synergistically activate AP-1 through the PI3K-AKT-JNK pathway; PI3K inhibition or dominant-negative AKT downregulates TRAF6/c-Src-induced JNK activation.","method":"Co-immunoprecipitation; dominant-negative mutants; PI3K inhibition; kinase activity assays; AP-1 reporter assay","journal":"European journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus multiple kinase activity assays plus reporter, single lab","pmids":["12631284"],"is_preprint":false},{"year":2023,"finding":"Rnd3 directly interacts with the RING finger domain of TRAF6, suppressing K63-linked TRAF6 ubiquitination while promoting K48-linked ubiquitination, thereby inhibiting NF-κB activation and promoting TRAF6 degradation; TRAF6 knockdown counters Rnd3 KO-evoked EC pyroptosis in vivo.","method":"LC-MS/MS; Co-IP assay; molecular docking; endothelium-specific transgenic mice; ubiquitination assays","journal":"Clinical and translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — IP-MS plus Co-IP plus domain-specific interaction plus in vivo genetic rescue, single lab","pmids":["37743632"],"is_preprint":false}],"current_model":"TRAF6 is a RING domain E3 ubiquitin ligase and adaptor protein that functions downstream of TNF receptor superfamily members, IL-1R/TLR family receptors, TGF-β receptors, and other receptors to catalyze K63-linked polyubiquitin chain synthesis (acting as a dimer via a concerted allosteric mechanism) on itself and substrates including PINK1, HIF-1α, FOXP3, hDNA2, cGAS, ULK1, YAP1, and others, thereby activating TAK1, IKK/NF-κB, JNK/p38 MAPK, and PI3K/AKT signaling pathways; its activity is regulated by S-glutathionylation/deglutathionylation, neddylation, phosphorylation (RSK2), binding partners (PRDX1, syntenin, SMN1, NUMBL, Sufu, YAP, OTUB1), and phase separation, and is functionally redundant with Pellino E3 ligases for some K63-Ub-dependent signaling events."},"narrative":{"mechanistic_narrative":"TRAF6 is a RING-domain E3 ubiquitin ligase and signaling adaptor that operates downstream of a broad set of receptors—IL-1R/TLR, CD40, TNF receptor superfamily members, and TGF-β receptors—to translate receptor engagement into NF-κB, JNK, and p38 MAPK activation, with knockout animals exhibiting osteopetrosis, defective osteoclast function, and impaired innate and adaptive immune responses [PMID:10215628, PMID:14499111, PMID:12060722, PMID:18922473]. Its core catalytic activity is synthesis of K63-linked polyubiquitin chains via a RING dimer that uses a concerted allosteric mechanism, cooperating with the Ubc13–Uev1A E2 to build chains both on itself (auto-ubiquitination) and on substrates [PMID:15492226, PMID:29432170]. Higher-order assembly is central to TRAF6 function: oligomerization-promoting partners such as TIFA drive auto-polyubiquitination and IKK activation, and TRAF6 undergoes liquid–liquid phase separation during LPS-induced inflammation that is required for ubiquitination-driven NF-κB activation [PMID:15492226, PMID:37441604]. These K63-Ub products engage the TAK1 complex (and, in some viral pathways, MEKK1) and feed downstream kinase modules; TRAF6 also carries E3 ligase-independent scaffolding roles, since Pellino1/2 generate redundant K63-Ub chains and catalytically inactive TRAF6 partially restores IL-1 signaling [PMID:18922473, PMID:28404732, PMID:18984593, PMID:28507161]. Beyond canonical immune signaling, TRAF6 K63-ubiquitinates a diverse substrate set to control protein stability, localization, or activity—including HIF-1α stabilization for tumor angiogenesis, PINK1 for mitophagy, FOXP3 for Treg nuclear localization and suppressive function, ULK1 and BECN1 for autophagy, hDNA2 for DNA end resection and homology-directed repair, and YAP1 for PD-L1 transcription [PMID:23722539, PMID:23885119, PMID:30886050, PMID:31216032, PMID:30929559, PMID:38583649]. TRAF6 additionally drives muscle satellite cell regeneration through an ERK/JNK→c-JUN→Pax7 axis [PMID:26619121]. Its activity is tightly tuned by post-translational modification—deglutathionylation by GRX-1, neddylation at Lys124, and RSK2 phosphorylation all enhance K63 auto-ubiquitination—and by numerous binding partners that act as positive (TIFA, Cullin-5, viperin, GCA) or negative (PRDX1, OTUB1, SMN1, syntenin, Sufu, NUMBL, YAP) regulators [PMID:21078302, PMID:30626891, PMID:29563609, PMID:15492226, PMID:27233966, PMID:33779167, PMID:30929559, PMID:29929436, PMID:34591986, PMID:28214532, PMID:18234474, PMID:37441604, PMID:20079715, PMID:29794022].","teleology":[{"year":1999,"claim":"Established that TRAF6 is an essential, non-redundant in vivo signaling mediator shared across IL-1, CD40, and LPS pathways, linking it to both immunity and bone homeostasis.","evidence":"TRAF6 knockout mice with osteoclast and multi-pathway signaling assays","pmids":["10215628"],"confidence":"High","gaps":["Did not define the molecular activity of TRAF6","Mechanism connecting TRAF6 to each receptor not resolved"]},{"year":2002,"claim":"Defined receptor selectivity by showing TRAF6 associates with XEDAR and TROY/TAJ but not EDAR, connecting it to epidermal appendage development through NF-κB.","evidence":"Co-IP, TRAF6-deficient mice, NF-κB reporter assays","pmids":["12060722"],"confidence":"High","gaps":["Structural basis of receptor discrimination unresolved","Downstream substrates in this pathway not identified"]},{"year":2003,"claim":"Extended TRAF6 function to dendritic cell maturation and adaptive immune priming, showing it is required for DC antigen-presentation capacity and a specific splenic DC subset.","evidence":"TRAF6-deficient mice and bone marrow chimeras with flow cytometry/functional assays","pmids":["14499111"],"confidence":"High","gaps":["Substrates driving DC maturation not defined"]},{"year":2004,"claim":"Reconstituted the core enzymatic mechanism, showing oligomerization (driven by TIFA) triggers TRAF6 K63 auto-polyubiquitination and IKK activation using purified TRAF6, TAK1 complex, and Ubc13-Uev1A.","evidence":"In vitro reconstitution with purified proteins and cell-free IKK activation assay","pmids":["15492226"],"confidence":"High","gaps":["Did not resolve atomic RING-domain catalytic geometry","Physiological oligomerization triggers beyond TIFA not enumerated here"]},{"year":2008,"claim":"Broadened the pathway repertoire by showing TRAF6 mediates TGF-β-induced Smad-independent JNK/p38 activation and RIG-I/MDA5 antiviral signaling, the latter using MEKK1 rather than TAK1 as the MAP3K.","evidence":"Co-IP, siRNA, ubiquitination and kinase assays; TRAF6/TAK1-deficient MEFs with MEKK1 rescue","pmids":["18922473","18984593"],"confidence":"High","gaps":["How MAP3K choice (TAK1 vs MEKK1) is determined unresolved"]},{"year":2008,"claim":"Showed TRAF6 protects against cell death, with its loss causing ROS accumulation, sustained JNK, and GSK3β inactivation downstream of TNF.","evidence":"TRAF6-KO 3T3 cells with ROS/kinase assays and constitutively active GSK3β rescue","pmids":["18202703"],"confidence":"Medium","gaps":["Direct substrate linking TRAF6 to GSK3β regulation not identified"]},{"year":2013,"claim":"Demonstrated that TRAF6 K63-ubiquitination controls substrate stability and localization beyond signaling kinases, stabilizing HIF-1α (angiogenesis) and PINK1 on depolarized mitochondria (mitophagy/parkin recruitment).","evidence":"Co-IP, site-specific ubiquitination assays, E3-deficient mutant, fractionation, imaging","pmids":["23722539","23885119"],"confidence":"Medium","gaps":["Cellular signals routing TRAF6 to these non-canonical substrates not defined","PINK1 work medium-supported by single lab"]},{"year":2016,"claim":"Expanded the substrate landscape to invasion/metastasis (Basigin membrane recruitment) and to mRNA decapping/P-body formation via DCP1a, indicating roles in cancer cell behavior and RNA metabolism.","evidence":"Co-IP, ubiquitination assays, mutagenesis, invasion/metastasis and mRNA decay assays","pmids":["26769849","27315556"],"confidence":"Medium","gaps":["Direct vs indirect ubiquitination of DCP1a not fully resolved","Single-lab support for each substrate"]},{"year":2017,"claim":"Dissected catalytic versus scaffolding contributions, showing Pellino1/2 redundantly generate K63 chains and that E3-inactive TRAF6 retains partial IL-1 signaling, while TRAF6 also enables an alternative TAB1-TAK1 pathway requiring its E3 activity.","evidence":"CRISPR triple-KO cells, E3-inactive knockin mice, TAB2/3 DKO/TKO cells with rescue and TAK1 assays","pmids":["28404732","28507161"],"confidence":"High","gaps":["Which signaling outputs are E3-dependent vs scaffold-dependent not comprehensively mapped"]},{"year":2018,"claim":"Resolved the catalytic mechanism and supramolecular assembly: a concerted allosteric RING dimer mediates Ub transfer, and TRAF6 cooperatively decorates BCL10 filaments within the CBM signalosome.","evidence":"Structural modeling/mutagenesis of RING extensions; cryo-EM of BCL10 CARD filament with imaging and reconstitution","pmids":["29432170","29382759"],"confidence":"High","gaps":["Full atomic structure of an active TRAF6-E2-substrate complex not determined","How filament geometry tunes chain length unresolved"]},{"year":2019,"claim":"Established TRAF6 as a regulator of Treg function (FOXP3 nuclear localization), DNA repair (hDNA2 resection), autophagy (ULK1), and additional immune substrates, while implicating it in tumor immune evasion.","evidence":"Conditional KO mice, site-specific ubiquitination mutants, fractionation, DNA-repair, autophagy and tumor assays","pmids":["30886050","31216032","30929559"],"confidence":"High","gaps":["How substrate selection is partitioned across cellular compartments unclear","ULK1/hDNA2 findings medium-supported single-lab"]},{"year":2019,"claim":"Defined an extensive layer of post-translational and partner regulation—neddylation at Lys124, RSK2 phosphorylation, and viperin/GCA-mediated activation—that tunes TRAF6 K63 ubiquitination output.","evidence":"Site-directed mutagenesis, neddylation/kinase/ubiquitination assays, KO mice, in vitro reconstitution","pmids":["30626891","29563609","33779167"],"confidence":"Medium","gaps":["Interplay among the different PTMs on a single TRAF6 molecule not integrated","Crosstalk hierarchy among activators not established"]},{"year":2021,"claim":"Revealed bidirectional roles in T cell homeostasis—positive scaffolding for NF-κB and negative restraint of basal MALT1 protease—and identified deubiquitinase brakes (OTUB1) controlling TRAF6 in metabolic disease.","evidence":"Genetic mouse models, biochemical analyses, pharmacological MALT1 rescue; IP-MS/Co-IP, ubiquitination assays, NASH models","pmids":["34767456","34591986"],"confidence":"High","gaps":["Molecular basis of TRAF6 suppression of MALT1 protease not fully defined"]},{"year":2023,"claim":"Showed phase separation of TRAF6 is essential for ubiquitination-driven NF-κB activation, and that Sufu and Rnd3 negatively regulate oligomerization/condensate formation and K63 vs K48 chain balance.","evidence":"In vitro phase separation with purified proteins, FRAP, Native-PAGE, Co-IP, in vivo lung-injury/pyroptosis models","pmids":["37441604","37743632"],"confidence":"High","gaps":["Determinants of condensate composition and substrate enrichment unresolved","Link between condensate state and chain-linkage choice unclear"]},{"year":null,"claim":"How TRAF6 achieves substrate specificity and the appropriate K63-chain output across its very broad substrate range—and how its compartmentalization, condensate state, and competing positive/negative regulators are integrated in a single cell—remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model of substrate selection","Quantitative integration of activators and DUBs/inhibitors lacking","Tissue-specific substrate priorities not systematically mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[4,7,10,13,22]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,11,13,22,25]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[4,8,20]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[31,26,39]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4,34]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[11]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,1,2,6]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,20,45]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[11,25,9]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[22]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[13,44,32]}],"complexes":["CBM (CARMA1-BCL10-MALT1) signalosome","TAK1 (TAB1/TAB2/TAB3) complex","PINK1-SARM1-TRAF6 complex"],"partners":["TAK1","TIFA","OTUB1","YAP1","PRDX1","BCL10","RSK2","CULLIN-5"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y4K3","full_name":"TNF receptor-associated factor 6","aliases":["E3 ubiquitin-protein ligase TRAF6","Interleukin-1 signal transducer","RING finger protein 85","RING-type E3 ubiquitin transferase TRAF6"],"length_aa":522,"mass_kda":59.6,"function":"E3 ubiquitin ligase that, together with UBE2N and UBE2V1, mediates the synthesis of 'Lys-63'-linked-polyubiquitin chains conjugated to proteins, such as ECSIT, IKBKG, IRAK1, AKT1 and AKT2 (PubMed:11057907, PubMed:18347055, PubMed:19465916, PubMed:19713527, PubMed:27746020, PubMed:31620128). Also mediates ubiquitination of free/unanchored polyubiquitin chain that leads to MAP3K7 activation (PubMed:19675569). Leads to the activation of NF-kappa-B and JUN (PubMed:16378096, PubMed:17135271, PubMed:17703191). Seems to also play a role in dendritic cells (DCs) maturation and/or activation (By similarity). Represses c-Myb-mediated transactivation, in B-lymphocytes (PubMed:18093978, PubMed:18758450). Adapter protein that seems to play a role in signal transduction initiated via TNF receptor, IL-1 receptor and IL-17 receptor (PubMed:12140561, PubMed:19825828, PubMed:8837778). Regulates osteoclast differentiation by mediating the activation of adapter protein complex 1 (AP-1) and NF-kappa-B, in response to RANK-L stimulation (By similarity). Together with MAP3K8, mediates CD40 signals that activate ERK in B-cells and macrophages, and thus may play a role in the regulation of immunoglobulin production (By similarity). Acts as a regulator of the JNK and NF-kappa-B signaling pathways by initiating assembly of heterotypic 'Lys-63'-/'Lys-48'-linked branched ubiquitin chains that are then recognized by TAB2: TRAF6 catalyzes initial 'Lys-63'-linked-polyubiquitin chains that are then branched via 'Lys-48'-linked polyubiquitin by HUWE1 (PubMed:27746020). 'Lys-63'-/'Lys-48'-linked branched ubiquitin chains protect 'Lys-63'-linkages from CYLD deubiquitination (PubMed:27746020). Participates also in the TCR signaling by ubiquitinating LAT (PubMed:23514740, PubMed:25907557)","subcellular_location":"Cytoplasm; Cytoplasm, cell cortex; Nucleus; Lipid droplet","url":"https://www.uniprot.org/uniprotkb/Q9Y4K3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TRAF6","classification":"Not Classified","n_dependent_lines":46,"n_total_lines":1208,"dependency_fraction":0.0380794701986755},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TRAF6","total_profiled":1310},"omim":[{"mim_id":"620090","title":"ZINC FINGER PROTEIN 675; ZNF675","url":"https://www.omim.org/entry/620090"},{"mim_id":"619936","title":"ANKYRIN REPEAT- AND SOCS BOX-CONTAINING PROTEIN 17; ASB17","url":"https://www.omim.org/entry/619936"},{"mim_id":"619906","title":"DExD-BOX HELICASE 39A; DDX39A","url":"https://www.omim.org/entry/619906"},{"mim_id":"618491","title":"G PROTEIN-COUPLED RECEPTOR 108; GPR108","url":"https://www.omim.org/entry/618491"},{"mim_id":"617649","title":"UBIQUITIN-CONJUGATING ENZYME E2 O; UBE2O","url":"https://www.omim.org/entry/617649"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Mitochondria","reliability":"Uncertain"},{"location":"Nucleoli","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TRAF6"},"hgnc":{"alias_symbol":["RNF85"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y4K3","domains":[{"cath_id":"3.30.40.10","chopping":"67-153","consensus_level":"high","plddt":96.1998,"start":67,"end":153},{"cath_id":"2.60.210.10","chopping":"350-502","consensus_level":"high","plddt":95.0224,"start":350,"end":502}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y4K3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y4K3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y4K3-F1-predicted_aligned_error_v6.png","plddt_mean":84.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TRAF6","jax_strain_url":"https://www.jax.org/strain/search?query=TRAF6"},"sequence":{"accession":"Q9Y4K3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y4K3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y4K3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y4K3"}},"corpus_meta":[{"pmid":"10215628","id":"PMC_10215628","title":"TRAF6 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 \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with defined cellular phenotypes across multiple pathways, replicated across multiple assay readouts\",\n      \"pmids\": [\"10215628\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"TRAF6 is required for dendritic cell maturation; TRAF6-deficient DCs fail to upregulate MHCII and B7.2, produce inflammatory cytokines, or stimulate naive T cells in response to LPS or CD40L, and the CD4+CD8α− splenic DC subset is nearly absent.\",\n      \"method\": \"TRAF6-deficient mice and bone marrow chimeras; flow cytometry and functional assays\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO and chimera experiments with multiple orthogonal functional readouts\",\n      \"pmids\": [\"14499111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"TRAF6 associates with XEDAR and TROY/TAJ (EDAR-related TNFR superfamily members) and is essential for XEDAR-mediated NF-κB activation, contributing to development of epidermal appendages; TRAF6 does not associate with EDAR.\",\n      \"method\": \"Co-immunoprecipitation; TRAF6-deficient mice; NF-κB reporter assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus KO phenotype plus functional NF-κB assay, multiple orthogonal methods\",\n      \"pmids\": [\"12060722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TRAF6 is specifically required for TGF-β-induced, Smad-independent activation of JNK and p38; its C-terminal TRAF homology domain interacts with TGF-β receptors; TGF-β induces K63-linked ubiquitination of TRAF6 and promotes TRAF6–TAK1 association.\",\n      \"method\": \"Co-immunoprecipitation; siRNA knockdown; ubiquitination assays; kinase activation assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus ubiquitination assay plus kinase assay, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"18922473\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TIFA (TRAF-interacting protein with a forkhead-associated domain) activates IKK by promoting oligomerization and K63-linked polyubiquitination of TRAF6; only oligomeric TIFA activates IKK; activation was reconstituted in vitro with purified TRAF6, TAK1 complex, and Ubc13-Uev1A.\",\n      \"method\": \"In vitro reconstitution with purified proteins; cell-free IKK activation assay; ubiquitination assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified proteins plus mutagenesis (TRAF6-binding-defective TIFA mutant), single lab\",\n      \"pmids\": [\"15492226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"TRAF6 associates with TβRI and mediates Lys63-linked polyubiquitination of TβRI, promoting PKCζ-dependent cleavage by TACE; the released TβRI intracellular domain translocates to the nucleus and, with p300, activates invasion-promoting genes (Snail, MMP2).\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assays; pharmacological inhibition; nuclear fractionation; cell invasion assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, ubiquitination assay, fractionation, functional assays; multiple orthogonal methods in single lab\",\n      \"pmids\": [\"21629263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRAF6 E3 ligase activity contributes to but is not solely essential for IL-1-dependent K63-Ub chain formation and TAK1 activation, because Pellino1/2 generate redundant K63-Ub chains; TRAF6/Pellino1/Pellino2 triple-KO abolishes K63-Ub formation on IRAK1, IRAK4, and MyD88. E3 ligase-inactive TRAF6 mutants partially restore IL-1 signaling, demonstrating E3 ligase-independent roles of TRAF6.\",\n      \"method\": \"CRISPR/Cas9 triple-KO cells; E3 ligase-inactive knockin mice (TRAF6[L74H]); ubiquitination assays; in vitro TAK1 activation assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple KO/knockin genetic models plus in vitro TAK1 assay plus mutagenesis, multiple orthogonal methods\",\n      \"pmids\": [\"28404732\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The TRAF6 RING dimer employs a concerted allosteric mechanism for ubiquitin transfer: both subunits cooperate, with the C-terminal extension of one TRAF6 RING subunit making trans-interactions with donor-Ub to promote Ub transfer to the E2.\",\n      \"method\": \"Structural modeling; biochemical ubiquitination assays; mutagenesis of RING domain extensions\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structural modeling combined with biochemical assay and mutagenesis, single lab\",\n      \"pmids\": [\"29432170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The CBM (CARMA1-BCL10-MALT1) signalosome recruits TRAF6, which cooperatively decorates BCL10 filaments to form higher-order assemblies; cryo-EM structure of BCL10 CARD filament at 4.0-Å resolution redefines CARD-CARD interactions; MALT1 immediately dimerizes on the BCL10 filamentous scaffold before recruiting TRAF6.\",\n      \"method\": \"Cryo-electron microscopy; time-lapse confocal imaging; biochemical reconstitution\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure plus live imaging plus reconstitution, single study with multiple orthogonal methods\",\n      \"pmids\": [\"29382759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PRDX1 interacts with the RING finger domain of TRAF6 and inhibits its E3 ubiquitin ligase activity, thereby suppressing K63-linked ubiquitination of ECSIT (required for NF-κB activation) and BECN1 (required for autophagy) in response to TLR4 stimulation.\",\n      \"method\": \"Co-immunoprecipitation; in vitro ubiquitination assay; siRNA knockdown (PRDX1KD cells); NF-κB reporter assay\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus in vitro ubiquitination assay plus multiple KD cell lines, single lab\",\n      \"pmids\": [\"29929436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TRAF6 binds HIF-1α and mediates its K63-linked polyubiquitination, increasing HIF-1α protein stability independent of oxygen levels; TRAF6 E3 ligase activity is required for this effect and for promoting tumor angiogenesis.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assay; E3 ligase-deficient mutant; cell-based HIF-1α protein stability assay; in vivo tumor angiogenesis assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus E3 ligase mutant, single lab\",\n      \"pmids\": [\"23722539\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PINK1 forms a complex with SARM1 and TRAF6; TRAF6 mediates K63-linked ubiquitination of PINK1 at Lys433, promoted by SARM1; this complex is required for PINK1 stabilization on depolarized mitochondria and subsequent parkin recruitment for mitophagy; some pathogenic PINK1 mutations reduce complex formation and ubiquitination.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assay with Lys433 mutant; siRNA knockdown; mitochondrial fractionation; fluorescence microscopy of parkin recruitment\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, site-specific ubiquitination assay with mutant, siRNA KD phenotype, localization imaging; multiple orthogonal methods, single lab\",\n      \"pmids\": [\"23885119\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TRAF6 is required for RIG-I/MDA5 pathway-dependent NF-κB, JNK, and p38 activation in response to viral infection or poly(I:C); TRAF6-deficient MEFs show impaired IRF3-independent signaling. MEKK1 (not TAK1) is the MAP3K used by TRAF6 for IFN-β induction in this pathway; forced MEKK1 + IRF3 expression induces IFN-β.\",\n      \"method\": \"TRAF6-deficient MEFs; TAK1-deficient MEFs; siRNA knockdown of MEKK1; overexpression rescue; IFN-β reporter assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO cells plus siRNA plus rescue, multiple orthogonal approaches, single lab\",\n      \"pmids\": [\"18984593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRAF6 mediates K63-linked ubiquitination of FOXP3 at Lys262, directing its proper nuclear localization; loss of TRAF6 or mutation of K262 causes perinuclear accumulation of FOXP3 and disrupted Treg suppressive function; Treg-specific TRAF6 deletion renders mice resistant to implanted tumors.\",\n      \"method\": \"Treg-specific TRAF6 conditional KO mice; site-directed mutagenesis (K262R FOXP3); ubiquitination assay; immunofluorescence localization; in vivo tumor models\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO plus site-specific ubiquitination mutagenesis plus localization imaging plus in vivo phenotype, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"30886050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"HSV-1 UL37 tegument protein activates NF-κB through direct interaction with TRAF6 via a TRAF6-binding domain in UL37; mutation of this domain reduces NF-κB activation during early infection.\",\n      \"method\": \"Co-immunoprecipitation; NF-κB reporter assay; recombinant virus with mutated TRAF6-binding site\",\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 plus reporter assay plus recombinant virus mutagenesis, single lab\",\n      \"pmids\": [\"18682563\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"T6BP specifically associates with TRAF6 via the coiled-coil region of T6BP and the N-terminal RING/zinc finger domains of TRAF6; IL-1 (not TNF) induces TRAF6–T6BP complex formation in an IRAK-dependent manner; IRAK is not present in TRAF6–T6BP complexes.\",\n      \"method\": \"Co-immunoprecipitation; yeast two-hybrid; domain-mapping pulldowns; IL-1 stimulation time course\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP with domain mapping and ligand-dependent complex formation, single lab\",\n      \"pmids\": [\"10920205\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The RING finger motif of TRAF6 is S-glutathionylated under basal conditions; upon IL-1 stimulation, GRX-1 catalyzes deglutathionylation of TRAF6, which is essential for TRAF6 K63-linked auto-polyubiquitination and subsequent NF-κB activation; GRX-1 knockdown severely reduces IL-1R/TLR4-mediated NF-κB induction.\",\n      \"method\": \"RNAi knockdown; S-glutathionylation detection assay; ubiquitination assay; NF-κB reporter assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — post-translational modification identified biochemically plus functional siRNA knockdown, single lab\",\n      \"pmids\": [\"21078302\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"p62/sequestosome-1 interacts with NEMO and is required for TRAF6-mediated K63-linked ubiquitination of NEMO; p62 depletion abolishes TRAF6's ability to ubiquitinate NEMO and severely impairs NF-κB activation following IL-1β stimulation.\",\n      \"method\": \"Co-immunoprecipitation; siRNA knockdown; ubiquitination assay; NF-κB activation assay\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus siRNA functional validation, single lab\",\n      \"pmids\": [\"24270048\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TRAF6 interacts with Basigin (BSG/CD147) and mediates its K63-linked ubiquitination, promoting BSG recruitment to the plasma membrane and BSG-dependent MMP9 induction; TRAF6 depletion reduces BSG membrane localization and impairs melanoma invasion/metastasis.\",\n      \"method\": \"Co-immunoprecipitation; shRNA knockdown; ubiquitination assay; cell invasion assays; in vivo metastasis model\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional invasion/metastasis assays, single lab\",\n      \"pmids\": [\"26769849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"NUMBL directly binds TRAF6 in vivo and in vitro, promotes K48-linked (but not K63-linked) polyubiquitination of TRAF6, reduces TRAF6 protein half-life, and thereby inhibits NF-κB signaling; NUMBL knockdown increases endogenous TRAF6 protein in neurons.\",\n      \"method\": \"Co-immunoprecipitation; in vivo ubiquitination assay with K48 and K63 mutants; siRNA knockdown; pulse-chase protein stability assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus linkage-specific ubiquitination assay plus protein stability assay, single lab\",\n      \"pmids\": [\"20079715\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"IL-1β can activate a TAB1-TAK1 heterodimer in the absence of TAB2/TAB3, but this alternative pathway requires TRAF6 expression and E3 ligase activity and depends on Ubc13-directed K63-Ub chain formation; in TAB2/3 DKO cells, IL-1β signaling is transient and JNK1/2 and p38γ activation is greatly reduced.\",\n      \"method\": \"TAB2/3 double-KO and triple-KO cell lines; TRAF6 siRNA; re-expression rescue with TAB1, TAB2, or ubiquitin-binding-defective TAB2; kinase assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with multiple KO combinations plus rescue experiments plus kinase assays, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"28507161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRAF6 K63-linked ubiquitinates cGAS, activating its enzymatic activity; TRAF6 knockdown decreases cGAS-induced IFNβ production upon HSV-1 infection.\",\n      \"method\": \"Ectopic expression and knockdown; ubiquitination assay; IFNβ reporter/expression assay; HSV-1 infection\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single method set (overexpression/KD + ubiquitination), no in vitro reconstitution or mutagenesis of ubiquitination site\",\n      \"pmids\": [\"31078259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRAF6 binds and K63-ubiquitinates hDNA2, increasing its stability and promoting its nuclear localization; TRAF6-mediated polyubiquitination of hDNA2 is required for DNA end resection and homology-directed repair; inhibiting this ubiquitination abolishes nuclear hDNA2 localization.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assay; nuclear fractionation; DNA end resection assay; HDR assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus localization and functional DNA repair assays, single lab\",\n      \"pmids\": [\"31216032\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"K63-linked ubiquitin chains and the E3 ligase TRAF6 regulate mammalian P-body formation and mRNA decapping; TRAF6 binds DCP1a and indirectly regulates its phosphorylation; mutation of six C-terminal lysines of DCP1a suppresses decapping activity and impairs interaction with DCP2, EDC4, and XRN1.\",\n      \"method\": \"Co-immunoprecipitation; DCP1a lysine-mutant analysis; P-body formation imaging; mRNA decay assays; ubiquitination assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus mutagenesis plus functional mRNA decay assays, single lab with multiple methods\",\n      \"pmids\": [\"27315556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"IL-1β stimulates a TRAF6–MLK3 interaction; TRAF6 RNAi dramatically impairs MLK3 kinase activation by IL-1β; TNF/IL-1β also stimulates K63-linked ubiquitination of MLK3 that is important for its kinase activity, while K48-linked ubiquitination directs MLK3 for proteasomal degradation.\",\n      \"method\": \"Co-immunoprecipitation; RNAi; in vitro kinase assay; linkage-specific ubiquitination analysis\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus RNAi plus kinase assay plus ubiquitination assay, single lab\",\n      \"pmids\": [\"19586614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"GCA (grancalcin) activates TRAF6 ubiquitin ligase activity to induce K63-linked ubiquitination of ULK1, resulting in ULK1 stabilization and activation and consequent autophagy induction; this GCA-TRAF6-ULK1 axis mediates imatinib resistance in CML.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assay; siRNA/overexpression; autophagy flux assays; drug resistance assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional autophagy and resistance assays, single lab\",\n      \"pmids\": [\"30929559\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRAF6 acts as both a positive regulator (scaffolding for NF-κB activation) and a negative regulator (suppressor of basal MALT1 protease activity) in T cells; loss of TRAF6-mediated homeostatic suppression of MALT1 protease leads to severe autoimmune inflammation fully reverted by genetic or pharmacological MALT1 protease inactivation.\",\n      \"method\": \"Genetically engineered mouse models; biochemical analyses; pharmacological MALT1 inhibition; epistasis experiments\",\n      \"journal\": \"Science immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic mouse models plus biochemical analysis plus pharmacological rescue, multiple orthogonal methods\",\n      \"pmids\": [\"34767456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Cullin-5 (Cul-5) directly binds TRAF6 (C-terminal domain of Cul-5 interacts with TRAF-C domain of TRAF6) and promotes TRAF6 K63-linked polyubiquitination in response to LPS; hemizygous Cul-5 KO delays NF-κB and MAPK activation in LPS-stimulated macrophages.\",\n      \"method\": \"Co-immunoprecipitation; domain-mapping pulldown; ubiquitination assay; hemizygous KO mice; macrophage signaling assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-specific Co-IP plus ubiquitination assay plus KO phenotype, single lab\",\n      \"pmids\": [\"27233966\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RSK2 directly phosphorylates TRAF6 at Ser46, Ser47, and Ser48 (in vitro kinase assay); RSK2 positively regulates TRAF6 K63-linked ubiquitination; TRAF6 is also required for RSK2 ubiquitination; RSK2 KO mice show reduced TRAF6 K63-ubiquitination and attenuated colon inflammation.\",\n      \"method\": \"In vitro kinase assay; Co-immunoprecipitation; ubiquitination assay; RSK2 KO mice; colon inflammation model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay identifies specific phosphorylation sites, plus Co-IP, ubiquitination assay, and KO mouse phenotype, single lab\",\n      \"pmids\": [\"29563609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRAF6 directly interacts with PIK3CA (p110α catalytic subunit of PI3K) and promotes its non-proteolytic K63-linked polyubiquitination under serum stimulation; TRAF6 overexpression enhances PI3K activation and downstream AKT phosphorylation and cell growth.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assay; overexpression/knockdown; AKT phosphorylation assay; cell growth assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional signaling readout, single lab\",\n      \"pmids\": [\"29729098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HIV-1 Tat directly interacts with TRAF6, increases TRAF6 oligomerization and auto-ubiquitination, and promotes K63-linked polyubiquitin chain synthesis, thereby activating NF-κB and HIV-1 transcription; TRAF6 knockdown inhibits HIV-1 transcription.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assay; TRAF6 KD/overexpression; NF-κB reporter and HIV-1 transcription assays\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional transcription assays, single lab\",\n      \"pmids\": [\"38241362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TRAF6 binds the IL-2Rβ chain at a site overlapping the Jak1-binding site and negatively regulates IL-2-induced Jak1-Erk pathway activation; a β-chain mutation that inactivates TRAF6 binding but retains Jak1 binding abrogates TRAF6-dependent suppression of IL-2 signaling; Traf6−/− CD4+ T cells show hyperactivated IL-2 signaling.\",\n      \"method\": \"Traf6−/− mouse T cells and MEFs; β-chain binding-site mutants; IL-2 stimulation assays; Jak1 and Erk activation assays\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO plus binding-site mutagenesis plus functional signaling assays, single lab\",\n      \"pmids\": [\"21155952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Satellite cell-specific deletion of Traf6 causes profound muscle regeneration defects with reduced PAX7 and late myogenesis markers; TRAF6 activates ERK1/2 and JNK1/2 → c-JUN → Pax7 promoter binding, augmenting Pax7 expression; TRAF6/c-JUN signaling also represses miR-1 and miR-206 to maintain PAX7 levels.\",\n      \"method\": \"Conditional (satellite cell-specific) KO mice; ChIP (c-JUN at Pax7 promoter); miRNA expression analysis; kinase activation assays; mdx double-mutant model\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with specific phenotype plus ChIP plus miRNA analysis plus disease model, multiple orthogonal methods\",\n      \"pmids\": [\"26619121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTUB1 directly binds TRAF6 and suppresses its K63-linked polyubiquitination, thereby inhibiting TRAF6-mediated activation of ASK1 and downstream pathways; OTUB1 KO aggravates NASH, and hepatocyte-specific OTUB1 overexpression alleviates it.\",\n      \"method\": \"Immunoprecipitation-mass spectrometry; Co-immunoprecipitation; ubiquitination assay; OTUB1 KO and overexpression mouse models; NASH diet models\",\n      \"journal\": \"Hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — IP-MS plus Co-IP plus ubiquitination assay plus in vivo KO/OE, single lab\",\n      \"pmids\": [\"34591986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRAF6 undergoes phase separation (liquid-liquid condensate/droplet formation) during LPS-induced inflammation, which is essential for its ubiquitination-mediated NF-κB activation; Sufu directly interacts with TRAF6, prevents its oligomerization and autoubiquitination, and inhibits TRAF6 phase-separated droplet formation; Sufu deletion augments LPS/CLP-induced lung injury and is rescued by TRAF6 depletion.\",\n      \"method\": \"Co-immunoprecipitation; FRAP; Native-PAGE; in vitro phase separation assay with purified proteins; GFP-TRAF6 stable cell line; CLP sepsis mouse model\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro phase separation assay with purified proteins plus FRAP plus multiple in vivo genetic models, multiple orthogonal methods\",\n      \"pmids\": [\"37441604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TRAF6 mediates K63-linked ubiquitination of STAT6, promoting STAT6 protein stability by reducing its K48-linked ubiquitination (degradation); TRAF6's E3 ligase activity is dispensable for STAT6 stabilization despite promoting K63-ubiquitination; TRAF6 deficiency impairs IL-4-induced M2 macrophage gene expression.\",\n      \"method\": \"Co-immunoprecipitation; overexpression/knockdown/KO; ubiquitination assays (K48/K63 specific); STAT6 stability assay\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus linkage-specific ubiquitination assay plus KO functional readout, single lab\",\n      \"pmids\": [\"33017719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Viperin binds TRAF6 and activates its ubiquitin transfer activity 2.5-fold, causing increased polyubiquitinated forms of TRAF6 important for immune signaling.\",\n      \"method\": \"Protein-protein binding assay; in vitro ubiquitin transfer assay with purified proteins\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution with purified proteins, single lab, single study\",\n      \"pmids\": [\"33779167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"BANK1 interacts with TRAF6 via its TIR domain and via five identified TRAF6-binding motifs; the C-terminal domain of BANK1-FL and N-terminal domain of BANK1-Δ2 are required for TRAF6 binding; BANK1 Δ2 isoform lacking TIR domain shows enhanced K63-linked polyubiquitination and IL-8 production.\",\n      \"method\": \"Co-immunoprecipitation; point mutation and decoy peptide analysis; K63-ubiquitination assay; IL-8 production assay\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mutagenesis plus ubiquitination assay plus functional cytokine readout, single lab\",\n      \"pmids\": [\"31243359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRAF6 neddylation at Lys124 is essential for IL-17A-induced NF-κB activation in fibroblast-like synoviocytes; K124R mutation markedly impairs NEDD8 conjugation to TRAF6 and attenuates IL-17A-induced NF-κB activity.\",\n      \"method\": \"Site-directed mutagenesis (K124R); neddylation assay; NF-κB activation assay; collagen-induced arthritis mouse model\",\n      \"journal\": \"Laboratory investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific mutagenesis plus neddylation assay plus functional NF-κB readout, single lab\",\n      \"pmids\": [\"30626891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"YAP interacts with TRAF6 (E3 ligase), promotes TRAF6 ubiquitination and degradation, inhibiting NF-κB activation in endothelial cells; endothelial YAP KO augments inflammatory response reversed by TRAF6 depletion.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assay; endothelial-specific YAP and TRAF6 KO mice; sepsis/LPS models\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP plus ubiquitination assay plus conditional KO mice with genetic rescue (TRAF6 depletion reverting phenotype), multiple orthogonal methods\",\n      \"pmids\": [\"29794022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Syntenin specifically interacts with TRAF6 under physiological conditions and inhibits TRAF6-induced NF-κB and AP-1 activation; upon IL-1 stimulation, syntenin dissociates from TRAF6 as IRAK1 overexpression competes for TRAF6 binding and restores TRAF6 ubiquitination; syntenin knockdown potentiates IL-1/LPS-triggered NF-κB activation.\",\n      \"method\": \"Co-immunoprecipitation; NF-κB/AP-1 reporter assays; siRNA knockdown; competition binding assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus reporter assays plus siRNA functional validation, single lab\",\n      \"pmids\": [\"18234474\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SMN1 physically interacts with TRAF6 and with IKK-α, IKK-β, and IKK-γ; SMN1 inhibits TRAF6 E3 ubiquitin ligase activity and IKK kinase activity; SMN1 depletion enhances IL-1β-induced IKK activation and inflammatory mediator production; SMA patient fibroblasts show potentiated IL-1β-induced IKK activity.\",\n      \"method\": \"Co-immunoprecipitation; in vitro E3 ligase activity assay; in vitro IKK kinase assay; siRNA knockdown; patient fibroblasts\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus in vitro enzyme activity assays plus siRNA KD, single lab\",\n      \"pmids\": [\"28214532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TRAF6 deficiency in TNF signaling leads to ROS accumulation, prolonged JNK activation, reduced NF-κB p65 phosphorylation, and accelerated cell death; TRAF6 deficiency causes constitutive phosphorylation (inactivation) of GSK3β; restoration of GSK3β activity rescues cell death in TRAF6-null cells.\",\n      \"method\": \"TRAF6-KO 3T3 cells; ROS measurement; JNK/NF-κB activation assays; GSK3β kinase assay; constitutively active GSK3β rescue\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO cells plus multiple signaling assays plus rescue experiment, single lab\",\n      \"pmids\": [\"18202703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP15 interacts with BECN1 (but not with TRAF6) and induces deubiquitination of BECN1, attenuating TRAF6-BECN1 axis-dependent autophagy induction and lung cancer migration/invasion.\",\n      \"method\": \"Co-immunoprecipitation; deubiquitination assay; CRISPR-KO cell lines; migration/invasion assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus deubiquitination assay plus CRISPR KO functional phenotype, single lab\",\n      \"pmids\": [\"35422093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TRAF6 stabilizes YAP1 via K63-linked polyubiquitination, which promotes formation of YAP1/TFCP2 transcriptional complex and PD-L1 transcription in melanoma; TRAF6 inhibition with Bortezomib reduces endogenous PD-L1 and enhances CD8+ T cell cytolytic activity.\",\n      \"method\": \"CRISPR interference screening; Co-immunoprecipitation; K63-ubiquitination assay; in vitro and in vivo functional assays; flow cytometry\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus in vivo functional assays, single lab\",\n      \"pmids\": [\"38583649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"c-Src interacts with TRAF6 in response to IL-1 and this interaction is required for c-Src activity; TRAF6 and c-Src synergistically activate AP-1 through the PI3K-AKT-JNK pathway; PI3K inhibition or dominant-negative AKT downregulates TRAF6/c-Src-induced JNK activation.\",\n      \"method\": \"Co-immunoprecipitation; dominant-negative mutants; PI3K inhibition; kinase activity assays; AP-1 reporter assay\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus multiple kinase activity assays plus reporter, single lab\",\n      \"pmids\": [\"12631284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Rnd3 directly interacts with the RING finger domain of TRAF6, suppressing K63-linked TRAF6 ubiquitination while promoting K48-linked ubiquitination, thereby inhibiting NF-κB activation and promoting TRAF6 degradation; TRAF6 knockdown counters Rnd3 KO-evoked EC pyroptosis in vivo.\",\n      \"method\": \"LC-MS/MS; Co-IP assay; molecular docking; endothelium-specific transgenic mice; ubiquitination assays\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — IP-MS plus Co-IP plus domain-specific interaction plus in vivo genetic rescue, single lab\",\n      \"pmids\": [\"37743632\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TRAF6 is a RING domain E3 ubiquitin ligase and adaptor protein that functions downstream of TNF receptor superfamily members, IL-1R/TLR family receptors, TGF-β receptors, and other receptors to catalyze K63-linked polyubiquitin chain synthesis (acting as a dimer via a concerted allosteric mechanism) on itself and substrates including PINK1, HIF-1α, FOXP3, hDNA2, cGAS, ULK1, YAP1, and others, thereby activating TAK1, IKK/NF-κB, JNK/p38 MAPK, and PI3K/AKT signaling pathways; its activity is regulated by S-glutathionylation/deglutathionylation, neddylation, phosphorylation (RSK2), binding partners (PRDX1, syntenin, SMN1, NUMBL, Sufu, YAP, OTUB1), and phase separation, and is functionally redundant with Pellino E3 ligases for some K63-Ub-dependent signaling events.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TRAF6 is a RING-domain E3 ubiquitin ligase and signaling adaptor that operates downstream of a broad set of receptors—IL-1R/TLR, CD40, TNF receptor superfamily members, and TGF-β receptors—to translate receptor engagement into NF-κB, JNK, and p38 MAPK activation, with knockout animals exhibiting osteopetrosis, defective osteoclast function, and impaired innate and adaptive immune responses [#0, #1, #2, #3]. Its core catalytic activity is synthesis of K63-linked polyubiquitin chains via a RING dimer that uses a concerted allosteric mechanism, cooperating with the Ubc13–Uev1A E2 to build chains both on itself (auto-ubiquitination) and on substrates [#4, #7]. Higher-order assembly is central to TRAF6 function: oligomerization-promoting partners such as TIFA drive auto-polyubiquitination and IKK activation, and TRAF6 undergoes liquid–liquid phase separation during LPS-induced inflammation that is required for ubiquitination-driven NF-κB activation [#4, #34]. These K63-Ub products engage the TAK1 complex (and, in some viral pathways, MEKK1) and feed downstream kinase modules; TRAF6 also carries E3 ligase-independent scaffolding roles, since Pellino1/2 generate redundant K63-Ub chains and catalytically inactive TRAF6 partially restores IL-1 signaling [#3, #6, #12, #20]. Beyond canonical immune signaling, TRAF6 K63-ubiquitinates a diverse substrate set to control protein stability, localization, or activity—including HIF-1α stabilization for tumor angiogenesis, PINK1 for mitophagy, FOXP3 for Treg nuclear localization and suppressive function, ULK1 and BECN1 for autophagy, hDNA2 for DNA end resection and homology-directed repair, and YAP1 for PD-L1 transcription [#10, #11, #13, #22, #25, #44]. TRAF6 additionally drives muscle satellite cell regeneration through an ERK/JNK→c-JUN→Pax7 axis [#32]. Its activity is tightly tuned by post-translational modification—deglutathionylation by GRX-1, neddylation at Lys124, and RSK2 phosphorylation all enhance K63 auto-ubiquitination—and by numerous binding partners that act as positive (TIFA, Cullin-5, viperin, GCA) or negative (PRDX1, OTUB1, SMN1, syntenin, Sufu, NUMBL, YAP) regulators [#16, #38, #28, #4, #27, #36, #25, #9, #33, #41, #40, #34, #19, #39].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established that TRAF6 is an essential, non-redundant in vivo signaling mediator shared across IL-1, CD40, and LPS pathways, linking it to both immunity and bone homeostasis.\",\n      \"evidence\": \"TRAF6 knockout mice with osteoclast and multi-pathway signaling assays\",\n      \"pmids\": [\"10215628\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the molecular activity of TRAF6\", \"Mechanism connecting TRAF6 to each receptor not resolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined receptor selectivity by showing TRAF6 associates with XEDAR and TROY/TAJ but not EDAR, connecting it to epidermal appendage development through NF-κB.\",\n      \"evidence\": \"Co-IP, TRAF6-deficient mice, NF-κB reporter assays\",\n      \"pmids\": [\"12060722\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of receptor discrimination unresolved\", \"Downstream substrates in this pathway not identified\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Extended TRAF6 function to dendritic cell maturation and adaptive immune priming, showing it is required for DC antigen-presentation capacity and a specific splenic DC subset.\",\n      \"evidence\": \"TRAF6-deficient mice and bone marrow chimeras with flow cytometry/functional assays\",\n      \"pmids\": [\"14499111\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrates driving DC maturation not defined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Reconstituted the core enzymatic mechanism, showing oligomerization (driven by TIFA) triggers TRAF6 K63 auto-polyubiquitination and IKK activation using purified TRAF6, TAK1 complex, and Ubc13-Uev1A.\",\n      \"evidence\": \"In vitro reconstitution with purified proteins and cell-free IKK activation assay\",\n      \"pmids\": [\"15492226\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve atomic RING-domain catalytic geometry\", \"Physiological oligomerization triggers beyond TIFA not enumerated here\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Broadened the pathway repertoire by showing TRAF6 mediates TGF-β-induced Smad-independent JNK/p38 activation and RIG-I/MDA5 antiviral signaling, the latter using MEKK1 rather than TAK1 as the MAP3K.\",\n      \"evidence\": \"Co-IP, siRNA, ubiquitination and kinase assays; TRAF6/TAK1-deficient MEFs with MEKK1 rescue\",\n      \"pmids\": [\"18922473\", \"18984593\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How MAP3K choice (TAK1 vs MEKK1) is determined unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showed TRAF6 protects against cell death, with its loss causing ROS accumulation, sustained JNK, and GSK3β inactivation downstream of TNF.\",\n      \"evidence\": \"TRAF6-KO 3T3 cells with ROS/kinase assays and constitutively active GSK3β rescue\",\n      \"pmids\": [\"18202703\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct substrate linking TRAF6 to GSK3β regulation not identified\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated that TRAF6 K63-ubiquitination controls substrate stability and localization beyond signaling kinases, stabilizing HIF-1α (angiogenesis) and PINK1 on depolarized mitochondria (mitophagy/parkin recruitment).\",\n      \"evidence\": \"Co-IP, site-specific ubiquitination assays, E3-deficient mutant, fractionation, imaging\",\n      \"pmids\": [\"23722539\", \"23885119\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cellular signals routing TRAF6 to these non-canonical substrates not defined\", \"PINK1 work medium-supported by single lab\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Expanded the substrate landscape to invasion/metastasis (Basigin membrane recruitment) and to mRNA decapping/P-body formation via DCP1a, indicating roles in cancer cell behavior and RNA metabolism.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, mutagenesis, invasion/metastasis and mRNA decay assays\",\n      \"pmids\": [\"26769849\", \"27315556\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect ubiquitination of DCP1a not fully resolved\", \"Single-lab support for each substrate\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Dissected catalytic versus scaffolding contributions, showing Pellino1/2 redundantly generate K63 chains and that E3-inactive TRAF6 retains partial IL-1 signaling, while TRAF6 also enables an alternative TAB1-TAK1 pathway requiring its E3 activity.\",\n      \"evidence\": \"CRISPR triple-KO cells, E3-inactive knockin mice, TAB2/3 DKO/TKO cells with rescue and TAK1 assays\",\n      \"pmids\": [\"28404732\", \"28507161\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which signaling outputs are E3-dependent vs scaffold-dependent not comprehensively mapped\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Resolved the catalytic mechanism and supramolecular assembly: a concerted allosteric RING dimer mediates Ub transfer, and TRAF6 cooperatively decorates BCL10 filaments within the CBM signalosome.\",\n      \"evidence\": \"Structural modeling/mutagenesis of RING extensions; cryo-EM of BCL10 CARD filament with imaging and reconstitution\",\n      \"pmids\": [\"29432170\", \"29382759\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full atomic structure of an active TRAF6-E2-substrate complex not determined\", \"How filament geometry tunes chain length unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established TRAF6 as a regulator of Treg function (FOXP3 nuclear localization), DNA repair (hDNA2 resection), autophagy (ULK1), and additional immune substrates, while implicating it in tumor immune evasion.\",\n      \"evidence\": \"Conditional KO mice, site-specific ubiquitination mutants, fractionation, DNA-repair, autophagy and tumor assays\",\n      \"pmids\": [\"30886050\", \"31216032\", \"30929559\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How substrate selection is partitioned across cellular compartments unclear\", \"ULK1/hDNA2 findings medium-supported single-lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined an extensive layer of post-translational and partner regulation—neddylation at Lys124, RSK2 phosphorylation, and viperin/GCA-mediated activation—that tunes TRAF6 K63 ubiquitination output.\",\n      \"evidence\": \"Site-directed mutagenesis, neddylation/kinase/ubiquitination assays, KO mice, in vitro reconstitution\",\n      \"pmids\": [\"30626891\", \"29563609\", \"33779167\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interplay among the different PTMs on a single TRAF6 molecule not integrated\", \"Crosstalk hierarchy among activators not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Revealed bidirectional roles in T cell homeostasis—positive scaffolding for NF-κB and negative restraint of basal MALT1 protease—and identified deubiquitinase brakes (OTUB1) controlling TRAF6 in metabolic disease.\",\n      \"evidence\": \"Genetic mouse models, biochemical analyses, pharmacological MALT1 rescue; IP-MS/Co-IP, ubiquitination assays, NASH models\",\n      \"pmids\": [\"34767456\", \"34591986\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of TRAF6 suppression of MALT1 protease not fully defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed phase separation of TRAF6 is essential for ubiquitination-driven NF-κB activation, and that Sufu and Rnd3 negatively regulate oligomerization/condensate formation and K63 vs K48 chain balance.\",\n      \"evidence\": \"In vitro phase separation with purified proteins, FRAP, Native-PAGE, Co-IP, in vivo lung-injury/pyroptosis models\",\n      \"pmids\": [\"37441604\", \"37743632\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants of condensate composition and substrate enrichment unresolved\", \"Link between condensate state and chain-linkage choice unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TRAF6 achieves substrate specificity and the appropriate K63-chain output across its very broad substrate range—and how its compartmentalization, condensate state, and competing positive/negative regulators are integrated in a single cell—remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model of substrate selection\", \"Quantitative integration of activators and DUBs/inhibitors lacking\", \"Tissue-specific substrate priorities not systematically mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [4, 7, 10, 13, 22]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 11, 13, 22, 25]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [4, 8, 20]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [31, 26, 39]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4, 34]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 1, 2, 6]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 20, 45]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [11, 25, 9]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [22]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [13, 44, 32]}\n    ],\n    \"complexes\": [\n      \"CBM (CARMA1-BCL10-MALT1) signalosome\",\n      \"TAK1 (TAB1/TAB2/TAB3) complex\",\n      \"PINK1-SARM1-TRAF6 complex\"\n    ],\n    \"partners\": [\n      \"TAK1\",\n      \"TIFA\",\n      \"OTUB1\",\n      \"YAP1\",\n      \"PRDX1\",\n      \"BCL10\",\n      \"RSK2\",\n      \"Cullin-5\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}