{"gene":"TRAF6","run_date":"2026-04-28T21:42:59","timeline":{"discoveries":[{"year":1999,"finding":"TRAF6 is essential for osteoclast function and bone remodeling downstream of RANK/OPGL signaling, and is required for IL-1, CD40, and LPS signaling, as demonstrated by TRAF6 knockout mice showing osteopetrosis and defective signaling responses.","method":"Knockout mouse model with in vitro signaling assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — clean KO with multiple defined cellular and signaling phenotypes, highly cited foundational paper","pmids":["10215628"],"is_preprint":false},{"year":2004,"finding":"TIFA activates IKK by promoting oligomerization and Lys63-linked polyubiquitination of TRAF6; only oligomeric forms of TIFA can activate IKK, demonstrated in an in vitro reconstitution system with purified TRAF6, TAK1 complex, and Ubc13-Uev1A.","method":"In vitro reconstitution with purified proteins, cell-free extract IKK activation assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — reconstituted in vitro with purified proteins including defined E2 complex","pmids":["15492226"],"is_preprint":false},{"year":2004,"finding":"The coiled-coil domain of TRAF6 is essential for its auto-ubiquitination and NF-κB activation; it serves as a bridge to recruit Ubc13/Uev1A rather than as the target site of polyubiquitin chain attachment.","method":"Domain deletion/mutation analysis, NF-κB reporter assays, ubiquitination assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1-2 — mutagenesis with functional readout, single lab","pmids":["15465037"],"is_preprint":false},{"year":2007,"finding":"Structural and biochemical studies defined the molecular basis for TRAF6's unique binding specificity, revealing how its TRAF-C domain mediates interactions with both TNFR superfamily and IL-1R/TLR superfamily receptors.","method":"Structural and biochemical studies, receptor binding motif analysis","journal":"Advances in experimental medicine and biology","confidence":"Medium","confidence_rationale":"Tier 1-2 — structural/biochemical characterization, single lab review","pmids":["17633022"],"is_preprint":false},{"year":2008,"finding":"TRAF6 is specifically required for TGF-β-induced, Smad-independent activation of JNK and p38; the TRAF homology domain of TRAF6 physically interacts with TGF-β receptors, and TGF-β induces K63-linked ubiquitination of TRAF6 and promotes TRAF6-TAK1 association.","method":"Co-immunoprecipitation, ubiquitination assays, TRAF6-deficient MEFs with signaling readouts","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, K63-Ub assays, KO cells, multiple orthogonal methods","pmids":["18922473"],"is_preprint":false},{"year":2008,"finding":"TRAF6 is critical for IFN-α/β induction in response to viral infection and intracellular dsRNA via the RIG-I/MDA5-IPS-1 pathway, activating NF-κB, JNK, and p38 but not IRF3; MEKK1 (not TAK1) acts downstream of TRAF6 for IFN-β induction in this pathway.","method":"TRAF6-deficient and TAK1-deficient MEFs, siRNA knockdown, reporter assays, forced expression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — genetic KO epistasis combined with KD and overexpression, multiple orthogonal methods","pmids":["18984593"],"is_preprint":false},{"year":2008,"finding":"TRAF6 plays a pivotal role in IL-33/ST2L signaling; TRAF6 deficiency abolishes IRAK recruitment to ST2L and completely inhibits IL-33-induced p38, JNK, and NF-κB activation; a TRAF6 ubiquitin ligase-dead mutant (C70A) fails to restore these responses, demonstrating that E3 ligase activity is required.","method":"TRAF6-deficient MEFs, rescue with wild-type vs. C70A mutant TRAF6, signaling assays","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 1-2 — KO cells plus active-site mutagenesis, multiple pathway readouts","pmids":["18603409"],"is_preprint":false},{"year":2008,"finding":"TRAF6 deficiency leads to constitutive inactivation of GSK3β and promotes TNF-induced ROS accumulation and prolonged JNK activation leading to cell death; restoration of GSK3β activity rescues cell death in TRAF6-null cells.","method":"TRAF6-deficient 3T3 cells, reconstitution with constitutively active GSK3β, ROS and JNK assays","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 — KO cells with functional rescue, single lab","pmids":["18202703"],"is_preprint":false},{"year":2010,"finding":"TRAF6 mediates activation of JNK1/2, p38 MAPK, AMPK, and NF-κB in skeletal muscle during atrophy, and regulates expression of muscle-specific E3 ubiquitin ligases and autophagy-related molecules; muscle-restricted TRAF6 depletion rescues myofibril degradation, muscle fiber size, and prevents cancer cachexia.","method":"Muscle-specific TRAF6 knockout mice, denervation and cancer cachexia models, signaling assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — conditional KO with multiple defined phenotypic and signaling readouts","pmids":["21187332"],"is_preprint":false},{"year":2010,"finding":"Glutaredoxin-1 (GRX-1) catalyzes deglutathionylation of the RING finger motif of TRAF6; TRAF6 is S-glutathionylated under basal conditions, and deglutathionylation upon IL-1 stimulation is essential for TRAF6 auto-polyubiquitination and subsequent NF-κB activation.","method":"RNAi knockdown, S-glutathionylation assays, ubiquitination assays, NF-κB reporter","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — biochemical modification assays plus functional knockdown, single lab","pmids":["21078302"],"is_preprint":false},{"year":2010,"finding":"NUMBL directly binds TRAF6 and promotes K48-linked polyubiquitination of TRAF6, leading to proteasomal degradation of TRAF6 and inhibition of NF-κB signaling; knockdown of NUMBL increases endogenous TRAF6 protein levels in cortical neurons.","method":"Co-immunoprecipitation, in vivo ubiquitination assays (K48 vs K63 linkage), NUMBL knockdown, half-life assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus ubiquitination linkage specificity assays, single lab","pmids":["20079715"],"is_preprint":false},{"year":2011,"finding":"TRAF6 associates with TβRI and mediates Lys63-linked polyubiquitination of TβRI in a PKCζ-dependent manner, promoting cleavage of TβRI by TACE; the liberated TβRI intracellular domain then associates with p300 to activate invasion-related genes (Snail, MMP2).","method":"Co-immunoprecipitation, ubiquitination assays, TACE cleavage assays, invasion assays, tumor tissue IHC","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (Co-IP, K63-Ub, cleavage assay, gene expression), single lab but comprehensive","pmids":["21629263"],"is_preprint":false},{"year":2013,"finding":"TRAF6 binds HIF-1α and mediates its K63-linked polyubiquitination, increasing HIF-1α protein levels (independent of oxygen and mRNA levels), thereby promoting tumor angiogenesis; E3 ligase activity of TRAF6 is required for this effect.","method":"Co-immunoprecipitation, K63-linked ubiquitination assays, protein stability assays, angiogenesis assays","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus ubiquitination assays, single lab","pmids":["23722539"],"is_preprint":false},{"year":2013,"finding":"SARM1 and TRAF6 form a complex with PINK1 on depolarized mitochondria; SARM1 promotes TRAF6-mediated K63-linked ubiquitination of PINK1 at lysine 433, which is important for PINK1 stabilization, parkin recruitment, and mitophagy; some pathogenic PINK1 mutations reduce complex formation and ubiquitination.","method":"Co-immunoprecipitation, K63-linked ubiquitination assays, PINK1/parkin recruitment assays, knockdown experiments","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus site-specific ubiquitination, single lab","pmids":["23885119"],"is_preprint":false},{"year":2014,"finding":"CKIP-1 interacts with TRAF6 and terminates TRAF6-mediated K63-linked ubiquitination and plasma membrane recruitment of Akt, thereby inhibiting macrophage proliferation at late stages after M-CSF stimulation; CKIP-1 deficiency leads to macrophage-dominated splenomegaly and myeloproliferation.","method":"Co-immunoprecipitation, Akt ubiquitination assays, CKIP-1 knockout mice","journal":"Cell research","confidence":"Medium","confidence_rationale":"Tier 2 — KO mice plus biochemical Co-IP and ubiquitination assays, single lab","pmids":["24777252"],"is_preprint":false},{"year":2014,"finding":"EGFR phosphorylates DCBLD2 at Y750, which recruits TRAF6 via a TRAF6-binding motif; phospho-DCBLD2-bound TRAF6 exhibits increased E3 ubiquitin ligase activity and subsequently activates AKT to drive EGFR-dependent tumorigenesis.","method":"Co-immunoprecipitation, ubiquitination assays, mutational analysis of Y750, tumor cell lines and patient samples","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP, mutational analysis, ubiquitination assays, single lab","pmids":["25061874"],"is_preprint":false},{"year":2015,"finding":"TRAF6-mediated signaling through ERK1/2, JNK1/2, and c-JUN is critical for satellite cell homeostasis and regenerative myogenesis; TRAF6/c-JUN signaling activates Pax7 transcription and represses miR-1 and miR-206 to maintain PAX7 levels; satellite cell-specific Traf6 deletion exaggerates dystrophic phenotype in mdx mice.","method":"Satellite cell-specific TRAF6 conditional knockout mice, muscle injury models, signaling assays, ChIP, miRNA analysis","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 — conditional KO with multiple orthogonal mechanistic readouts (ChIP, miRNA, signaling), single lab but comprehensive","pmids":["26619121"],"is_preprint":false},{"year":2016,"finding":"TRAF6 ubiquitinates Basigin (BSG/CD147) with K63-linked chains, promoting BSG recruitment to the plasma membrane and BSG-dependent MMP9 induction; TRAF6 knockdown reduces plasma membrane BSG and impairs melanoma invasion and metastasis.","method":"Co-immunoprecipitation, K63-linked ubiquitination assays, shRNA knockdown, invasion assays in vitro and in vivo","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus ubiquitination and functional assays, single lab","pmids":["26769849"],"is_preprint":false},{"year":2016,"finding":"SKP2 loss upregulates TRAF6-mediated K63-linked ubiquitination of EZH2, leading to EZH2 degradation; the catalytically dead TRAF6-C70A mutant abolishes polyubiquitination of recombinant EZH2 in vitro, establishing TRAF6 as the E3 ligase for EZH2.","method":"In vitro ubiquitination assay with recombinant proteins, Co-IP, K63-Ub linkage assays, KO/KD cell lines, mouse models","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with active-site mutant control, supported by cell and in vivo data","pmids":["27869166"],"is_preprint":false},{"year":2016,"finding":"K63-linked ubiquitylation mediated by TRAF6 is required for assembly of decapping factors, P-body formation, and constitutive decay of unstable inflammatory mRNAs; TRAF6 binds DCP1a and indirectly regulates its phosphorylation and mRNA decapping activity.","method":"Co-immunoprecipitation, ubiquitination assays, DCP1a lysine mutants, P-body imaging, mRNA decay assays","journal":"Molecular cell","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus functional mutational analysis and mRNA decay readouts, single lab","pmids":["27315556"],"is_preprint":false},{"year":2017,"finding":"TRAF6 E3 ligase activity contributes to but is not essential for IL-1-dependent K63-Ub chain formation, TAK1 activation, and IL-8 production; Pellino1 and Pellino2 compensate for TRAF6 E3 activity in this pathway. TRAF6/Pellino1/2 triple-KO abolishes all K63-Ub formation and IRAK1/IRAK4/MyD88 ubiquitination. RANKL-induced osteoclastogenesis proceeds normally with E3 ligase-inactive TRAF6, identifying scaffold functions of TRAF6 independent of its E3 activity.","method":"TRAF6 KO, Pellino1/2 double-KO, triple-KO cell lines; E3 ligase-inactive TRAF6[L74H] knockin mice; in vitro TAK1 activation with purified K63-Ub chains","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro reconstitution plus multiple genetic KO models and knockin mice, multiple orthogonal methods","pmids":["28404732"],"is_preprint":false},{"year":2017,"finding":"IPMK directly binds TRAF6 (independent of IPMK catalytic activity) and stabilizes TRAF6 by blocking its K48-linked ubiquitination and proteasomal degradation, thereby sustaining TLR-induced proinflammatory signaling.","method":"Myeloid-specific IPMK KO mice, Co-immunoprecipitation, K48-Ub linkage assays, protein stability assays","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 — conditional KO plus Co-IP and ubiquitination assays, single lab","pmids":["28439546"],"is_preprint":false},{"year":2017,"finding":"RSK2 phosphorylates TRAF6 at serines 46, 47, and 48 in vitro; RSK2-mediated phosphorylation is required for TRAF6 K63-linked ubiquitination and downstream inflammatory signaling in the colon.","method":"In vitro kinase assay, phosphorylation site mutagenesis, RSK2 KO mice, colon inflammation model, Co-IP","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 1-2 — in vitro kinase assay plus KO mouse model, single lab","pmids":["29563609"],"is_preprint":false},{"year":2018,"finding":"Cryo-EM structure of BCL10 CARD filament at 4.0-Å resolution reveals that TRAF6 cooperatively decorates CBM (CARMA1-BCL10-MALT1) filaments to form higher-order assemblies, likely enabling all-or-none NF-κB activation downstream of TCR/BCR signaling.","method":"Cryo-EM structure determination, time-lapse confocal imaging, EM of MALT1-BCL10 and TRAF6-CBM assemblies","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure plus functional imaging, multiple orthogonal methods","pmids":["29382759"],"is_preprint":false},{"year":2018,"finding":"The TRAF6 RING dimer employs a concerted allosteric mechanism using both subunits to promote ubiquitin transfer; the C-terminal extension of the TRAF6 RING domain mediates trans-interactions with donor ubiquitin, and N-terminal extensions engage donor-Ub in cis.","method":"Structural modeling, biochemical analysis, comparison with E3-Ub-E2 complex structures","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 1-2 — structural modeling with biochemical validation, single lab","pmids":["29432170"],"is_preprint":false},{"year":2018,"finding":"PRDX1 interacts with the RING finger domain of TRAF6 and inhibits its ubiquitin ligase activity, suppressing ubiquitination of ECSIT (required for NF-κB activation) and BECN1 (required for autophagy); PRDX1 knockdown enhances both NF-κB activation and autophagy in response to TLR4 stimulation.","method":"Co-immunoprecipitation, RING domain interaction mapping, ubiquitination assays (ECSIT and BECN1), PRDX1 KD cell lines","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP with domain mapping plus substrate ubiquitination assays, single lab","pmids":["29929436"],"is_preprint":false},{"year":2018,"finding":"YAP interacts with TRAF6 to promote its ubiquitination and degradation, thereby inhibiting TRAF6-mediated NF-κB activation in endothelial cells; endothelial YAP deletion augments inflammatory responses, which are rescued by TRAF6 depletion.","method":"Co-immunoprecipitation, endothelial-specific YAP KO mice, TRAF6 depletion rescue experiments, NF-κB assays","journal":"Circulation research","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus conditional KO with genetic rescue, single lab","pmids":["29794022"],"is_preprint":false},{"year":2018,"finding":"TRAF6 interacts with and ubiquitinates PIK3CA (p110α) with nonproteolytic polyubiquitin chains under serum stimulation, enhancing PI3K activation and downstream AKT phosphorylation and cell growth.","method":"Co-immunoprecipitation, ubiquitination assays, overexpression and KD of TRAF6, AKT activation readouts","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus ubiquitination assays with functional signaling readout, single lab","pmids":["29729098"],"is_preprint":false},{"year":2019,"finding":"TRAF6 mediates K63-linked ubiquitination of FOXP3 at lysine 262; in the absence of TRAF6 activity or upon K262R mutation, FOXP3 shows aberrant perinuclear accumulation and disrupted regulatory T cell function; TRAF6-deficient Tregs are dysfunctional in vivo.","method":"Treg-specific TRAF6 conditional KO mice, K63-ubiquitination assays, site-directed mutagenesis of FOXP3-K262, subcellular localization imaging","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — conditional KO plus site-specific ubiquitination mutagenesis and localization experiments, multiple orthogonal methods","pmids":["30886050"],"is_preprint":false},{"year":2019,"finding":"TRAF6 binds to and mediates K63-linked polyubiquitination of hDNA2, increasing hDNA2 stability and promoting its nuclear localization; inhibiting TRAF6-mediated polyubiquitination abolishes nuclear localization of hDNA2, impairing DNA end resection and homology-directed recombination repair.","method":"Co-immunoprecipitation, K63-Ub assays, nuclear fractionation, DNA repair assays","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus ubiquitination and nuclear localization assays with functional DNA repair readout, single lab","pmids":["31216032"],"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, thereby driving autophagy and imatinib resistance in CML.","method":"Co-immunoprecipitation, K63-ubiquitination assays, ULK1 stability assays, autophagy readouts","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus ubiquitination assays, single lab","pmids":["30929559"],"is_preprint":false},{"year":2019,"finding":"TRAF6 ubiquitinates cGAS with K63-linked chains, promoting cGAS activation and downstream IFNβ production; TRAF6 knockdown reduces cGAS-induced IFNβ upon HSV-1 infection.","method":"Overexpression/knockdown of TRAF6, ubiquitination assays, IFNβ production assays, HSV-1 infection","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 — single lab, overexpression/KD approach without in vitro reconstitution","pmids":["31078259"],"is_preprint":false},{"year":2021,"finding":"OTUB1 directly binds TRAF6 and suppresses its K63-linked polyubiquitination, thereby inhibiting downstream ASK1 activation; hepatocyte-specific OTUB1 overexpression markedly alleviates NASH in mice.","method":"IP-mass spectrometry, Co-immunoprecipitation, K63-Ub assays, hepatocyte-specific overexpression mouse model","journal":"Hepatology","confidence":"Medium","confidence_rationale":"Tier 2 — IP-MS discovery plus Co-IP and ubiquitination assays with in vivo validation, single lab","pmids":["34591986"],"is_preprint":false},{"year":2022,"finding":"USP15 interacts with BECN1 (not TRAF6) and deubiquitinates BECN1, thereby attenuating TRAF6-BECN1 axis-driven autophagy induction and lung cancer migration/invasion; USP15 KO enhances autophagy in response to TLR4 stimulation.","method":"CRISPR-Cas9 USP15 KO, Co-immunoprecipitation, deubiquitination assays, autophagy and invasion assays","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — CRISPR KO plus biochemical interaction and deubiquitination assays, single lab","pmids":["35422093"],"is_preprint":false},{"year":2023,"finding":"Sufu directly interacts with TRAF6, preventing TRAF6 oligomerization and autoubiquitination; TRAF6 undergoes liquid-liquid phase separation during LPS-induced inflammation, which is essential for its ubiquitination activation and NF-κB activity; Sufu inhibits TRAF6 phase separation, reducing NF-κB activation.","method":"Co-immunoprecipitation, FRAP, Native-PAGE, in vitro phase separation assays with purified proteins, myeloid-specific Sufu KO mice, TRAF6 depletion rescue","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro phase separation with purified proteins, FRAP, Co-IP, KO mouse with genetic rescue, multiple orthogonal methods","pmids":["37441604"],"is_preprint":false},{"year":2024,"finding":"HIV-1 Tat directly interacts with TRAF6, increasing TRAF6 oligomerization and auto-ubiquitination as well as K63-linked polyubiquitin chain synthesis, thereby enhancing NF-κB pathway activation and HIV-1 transcription.","method":"Co-immunoprecipitation, ubiquitination assays, oligomerization assays, reporter assays, TRAF6 knockdown","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus ubiquitination and oligomerization assays, single lab","pmids":["38241362"],"is_preprint":false},{"year":2008,"finding":"HSV UL37 tegument protein activates NF-κB signaling through direct interaction with TRAF6 via a TRAF6-binding domain in UL37; mutation of this binding site reduces NF-κB activation during early viral infection.","method":"Transfection, Co-immunoprecipitation, NF-κB reporter assay, mutant virus with altered TRAF6-binding site","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus mutant virus functional assay, single lab","pmids":["18682563"],"is_preprint":false},{"year":2000,"finding":"T6BP (TRAF6-binding protein) specifically associates with TRAF6 through the coiled-coil region of T6BP and the N-terminal RING finger and zinc finger domains of TRAF6; IL-1 (but not TNF) induces TRAF6-T6BP complex formation in an IRAK-dependent manner; T6BP and IRAK exist in separate TRAF6 complexes.","method":"Co-immunoprecipitation, domain mapping, IL-1 stimulation, IKK and JNK activity assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP with domain mapping and ligand-dependent complex characterization, single lab","pmids":["10920205"],"is_preprint":false},{"year":2009,"finding":"IL-1β stimulation induces interaction between MLK3 and TRAF6; RNAi knockdown of TRAF6 dramatically impairs MLK3 activation by IL-1β; TRAF6 promotes K63-linked ubiquitination of MLK3, which is important for MLK3 kinase activity.","method":"Co-immunoprecipitation, RNAi, in vitro kinase assays, K48/K63 ubiquitination assays","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus ubiquitination and kinase assays with RNAi, single lab","pmids":["19586614"],"is_preprint":false},{"year":2017,"finding":"Syntenin interacts with TRAF6 under basal conditions and dissociates upon IL-1 stimulation; IRAK1 overexpression competes with syntenin for TRAF6 binding; syntenin inhibits TRAF6 ubiquitination and acts as a physiological suppressor of IL-1R/TLR4-mediated NF-κB activation.","method":"Co-immunoprecipitation, NF-κB reporter assays, ubiquitination assays, syntenin knockdown","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP plus functional knockdown and ubiquitination assays, single lab","pmids":["18234474"],"is_preprint":false},{"year":2019,"finding":"BANK1 interacts with TRAF6 through five identified TRAF6-binding motifs in BANK1; the C-terminal domain of BANK1-FL and N-terminal domain of BANK1-Δ2 are required for TRAF6 binding; BANK1-TIR domain is important for K63-linked polyubiquitination and IL-8 production in B cell innate immune signaling.","method":"Co-immunoprecipitation, TRAF6-binding motif point mutations, decoy peptide experiments, K63-Ub assays, TLR7/9 stimulation","journal":"Cellular & molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2-3 — Co-IP with point mutations and functional ubiquitination assays, single lab","pmids":["31243359"],"is_preprint":false},{"year":2024,"finding":"TRAF6 stabilizes YAP1 via K63-linked polyubiquitination, promoting formation of the YAP1/TFCP2 transcriptional complex and subsequent PD-L1 transcription in melanoma cells; TRAF6 inhibition reduces membrane PD-L1 expression and enhances CD8+ T cell cytolytic activity.","method":"CRISPR interference screening, Co-immunoprecipitation, K63-ubiquitination assays, transcriptional reporter assays, in vivo tumor models","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2-3 — CRISPR screen plus Co-IP, ubiquitination assays, and in vivo validation, single lab","pmids":["38583649"],"is_preprint":false}],"current_model":"TRAF6 is a RING-domain E3 ubiquitin ligase that functions as a central adaptor downstream of IL-1R/TLR and TNFR superfamily receptors; upon activation it forms homodimers/oligomers and, together with Ubc13/Uev1A, catalyzes K63-linked polyubiquitin chain synthesis on itself and diverse substrates (including IRAK1, TAK1 complex components, TβRI, HIF-1α, FOXP3, BECN1, ULK1, hDNA2, PIK3CA, EZH2, YAP1, PINK1, BSG, cGAS, and MLK3), thereby serving as a non-proteolytic scaffold that recruits and activates downstream kinase complexes (TAK1-TAB1/2, IKK, MAPKs) to drive NF-κB, JNK, and p38 activation; its activity is tightly regulated by K48-linked ubiquitination for proteasomal degradation (by NUMBL, DRAK1-autophagy), by S-glutathionylation/deglutathionylation (GRX-1), by inhibitory protein interactions (PRDX1, syntenin, IPMK-independent stabilization, Sufu), by phosphorylation (RSK2 at S46/47/48), and by liquid-liquid phase separation during inflammatory signaling."},"narrative":{"teleology":[{"year":1999,"claim":"Establishing TRAF6 as a non-redundant adaptor for multiple innate immune and TNFR superfamily pathways: TRAF6 knockout mice revealed essential roles in RANK-dependent osteoclastogenesis, IL-1, CD40, and LPS signaling, defining the breadth of TRAF6's signaling scope.","evidence":"TRAF6 knockout mice with in vitro signaling assays showing osteopetrosis and impaired cytokine responses","pmids":["10215628"],"confidence":"High","gaps":["Enzymatic mechanism of TRAF6 was unknown","Whether TRAF6 functions as scaffold versus enzyme was not resolved","Downstream kinase targets not yet mapped"]},{"year":2000,"claim":"Identifying distinct TRAF6 signaling complexes: T6BP was found to associate specifically with TRAF6 in an IL-1/IRAK-dependent manner, revealing that TRAF6 participates in stimulus-specific multiprotein complexes rather than a single uniform signaling module.","evidence":"Co-immunoprecipitation with domain mapping showing T6BP binds TRAF6 RING/zinc-finger domains, with IL-1-dependent complex formation","pmids":["10920205"],"confidence":"Medium","gaps":["T6BP function in TRAF6 signaling not defined","No structural data on the T6BP-TRAF6 interface","Stoichiometry of endogenous complexes unknown"]},{"year":2004,"claim":"Reconstituting the TRAF6 ubiquitination cascade in vitro resolved the catalytic mechanism: TIFA-induced TRAF6 oligomerization drives K63-linked polyubiquitination via the Ubc13/Uev1A E2, which activates TAK1 and IKK; the coiled-coil domain bridges TRAF6 to its E2 rather than serving as a ubiquitin acceptor.","evidence":"Cell-free reconstitution with purified TRAF6, TAK1 complex, and Ubc13-Uev1A; domain deletion/mutation analysis with NF-κB reporters","pmids":["15492226","15465037"],"confidence":"High","gaps":["Crystal structure of TRAF6 RING–E2 complex not yet available","Physiological ubiquitin acceptor sites on TRAF6 not mapped","Redundancy with other E3 ligases not tested"]},{"year":2007,"claim":"Structural characterization of the TRAF-C domain explained how TRAF6 uniquely engages both TNFR superfamily and IL-1R/TLR superfamily receptors, resolving its dual receptor specificity.","evidence":"Structural and biochemical studies of receptor-binding motifs in the TRAF-C domain","pmids":["17633022"],"confidence":"Medium","gaps":["Full-length TRAF6 structure not determined","Binding affinities for diverse receptors not systematically compared"]},{"year":2008,"claim":"TRAF6's substrate repertoire expanded beyond innate immune receptors: TRAF6 was shown to mediate TGF-β-induced Smad-independent JNK/p38 activation via K63-ubiquitination and TAK1 recruitment, to be essential for IL-33/ST2L signaling (requiring E3 ligase activity), to function in RIG-I/MDA5 antiviral IFN-α/β induction via MEKK1, and to regulate GSK3β activity controlling TNF-induced cell death.","evidence":"TRAF6-deficient MEFs with signaling readouts, C70A ligase-dead mutant rescue, epistasis with TAK1-KO, ROS and JNK assays","pmids":["18922473","18603409","18984593","18202703"],"confidence":"High","gaps":["Whether TRAF6 directly ubiquitinates TGF-β receptor components was unclear","The relative contribution of TRAF6 E3 activity versus scaffold function was unresolved across pathways"]},{"year":2009,"claim":"TRAF6-mediated K63-linked ubiquitination of MLK3 was identified as a mechanism for IL-1β-induced MLK3 kinase activation, broadening the kinase substrates of TRAF6.","evidence":"Co-immunoprecipitation, RNAi of TRAF6, in vitro kinase assays, K63-ubiquitination assays","pmids":["19586614"],"confidence":"Medium","gaps":["In vitro reconstitution of TRAF6-mediated MLK3 ubiquitination not performed","Specific ubiquitinated lysines on MLK3 not mapped"]},{"year":2010,"claim":"Multiple layers of TRAF6 regulation were uncovered: GRX-1-mediated deglutathionylation of the RING domain licenses TRAF6 autoubiquitination upon IL-1 stimulation, while NUMBL promotes K48-linked ubiquitination and degradation of TRAF6 to limit NF-κB; concurrently, muscle-specific TRAF6 deletion revealed TRAF6 as a master regulator of skeletal muscle atrophy via NF-κB, MAPK, AMPK, and autophagy.","evidence":"S-glutathionylation assays with GRX-1 RNAi; NUMBL Co-IP with K48/K63 linkage-specific Ub assays; muscle-specific TRAF6 KO mice in denervation and cancer cachexia models","pmids":["21078302","20079715","21187332"],"confidence":"High","gaps":["Glutathionylation site on TRAF6 RING domain not mapped at residue level","NUMBL-TRAF6 E3 ligase responsible for K48-Ub not identified","How TRAF6 activates AMPK in muscle was not established"]},{"year":2011,"claim":"TRAF6 was shown to directly ubiquitinate TβRI with K63-linked chains in a PKCζ-dependent manner, triggering TACE-mediated cleavage and nuclear translocation of the TβRI intracellular domain to activate invasion genes, revealing a non-canonical TGF-β signaling axis.","evidence":"Co-immunoprecipitation, K63-Ub assays, TACE cleavage assays, invasion assays, tumor tissue IHC","pmids":["21629263"],"confidence":"High","gaps":["Specific lysine residues on TβRI targeted by TRAF6 not identified","Whether this pathway operates in non-cancer contexts was unclear"]},{"year":2013,"claim":"TRAF6's K63-linked ubiquitination activity was extended to non-signaling contexts: TRAF6 ubiquitinates HIF-1α to stabilize it independently of oxygen and promotes tumor angiogenesis, and ubiquitinates PINK1 (with SARM1) on depolarized mitochondria to facilitate parkin recruitment and mitophagy.","evidence":"Co-immunoprecipitation, K63-Ub assays, protein stability assays, angiogenesis assays; SARM1-TRAF6-PINK1 complex analysis with site-specific K433 mutagenesis","pmids":["23722539","23885119"],"confidence":"Medium","gaps":["In vitro reconstitution of TRAF6-mediated HIF-1α ubiquitination not shown","Whether SARM1 is required for all TRAF6-PINK1 interactions was untested","Pathogenic PINK1 mutations reducing TRAF6 binding tested only in overexpression"]},{"year":2014,"claim":"TRAF6 emerged as a key node in growth factor signaling: CKIP-1 terminates TRAF6-dependent K63-ubiquitination and membrane recruitment of Akt to limit macrophage proliferation, while EGFR-phosphorylated DCBLD2 recruits and activates TRAF6 to drive AKT-dependent tumorigenesis.","evidence":"CKIP-1 KO mice with Akt ubiquitination assays; DCBLD2 Y750 mutagenesis with Co-IP and tumor studies","pmids":["24777252","25061874"],"confidence":"Medium","gaps":["Direct substrate relationship between TRAF6 and Akt ubiquitination not reconstituted in vitro","DCBLD2-TRAF6 structural basis not determined"]},{"year":2016,"claim":"The substrate landscape of TRAF6 broadened to include chromatin regulators, membrane trafficking targets, and mRNA decay machinery: TRAF6 ubiquitinates EZH2 with K63 chains leading to its degradation, ubiquitinates Basigin/CD147 promoting its plasma membrane localization and MMP9-driven invasion, and ubiquitinates DCP1a-associated factors to drive P-body assembly and constitutive inflammatory mRNA decay.","evidence":"In vitro ubiquitination with recombinant EZH2 and TRAF6-C70A control; BSG K63-Ub assays with invasion readouts; DCP1a Co-IP and mRNA decay assays","pmids":["27869166","26769849","27315556"],"confidence":"High","gaps":["Whether TRAF6 directly ubiquitinates DCP1a or acts indirectly was ambiguous","Mechanism connecting K63-Ub of EZH2 to its degradation (normally K48-mediated) unclear"]},{"year":2017,"claim":"A critical revision of the TRAF6 model showed that Pellino E3 ligases compensate for TRAF6 E3 activity in IL-1 signaling (triple-KO required to abolish K63-Ub), while RANKL-induced osteoclastogenesis proceeds with E3 ligase-inactive TRAF6, formally separating TRAF6's scaffold and catalytic functions; additionally, IPMK and syntenin were identified as regulators that stabilize and sequester TRAF6, respectively, and RSK2 phosphorylation at S46/47/48 licenses TRAF6 activation.","evidence":"Pellino1/2 double-KO plus TRAF6-KO triple cell lines; TRAF6[L74H] knockin mice; IPMK myeloid-KO with K48-Ub assays; syntenin Co-IP/competition; in vitro RSK2 kinase assay","pmids":["28404732","28439546","18234474","29563609"],"confidence":"High","gaps":["Structural basis for TRAF6 scaffold function independent of E3 activity not resolved","Whether Pellino compensation occurs in all TLR pathways untested","RSK2 phosphorylation sites validated in vitro but not confirmed with phospho-specific antibodies in vivo"]},{"year":2018,"claim":"Structural and biophysical studies revealed how TRAF6 functions in higher-order signaling assemblies: cryo-EM showed TRAF6 cooperatively decorates BCL10-MALT1 filaments enabling threshold NF-κB activation downstream of antigen receptors; structural modeling revealed a concerted allosteric mechanism in the TRAF6 RING dimer for ubiquitin transfer; and multiple negative regulators (PRDX1, YAP) were shown to restrain TRAF6 activity.","evidence":"Cryo-EM of BCL10 CARD filament–TRAF6 assemblies; structural modeling of RING dimer–Ub–E2 complex; PRDX1 Co-IP with ECSIT/BECN1 ubiquitination assays; endothelial YAP KO mice with TRAF6 rescue","pmids":["29382759","29432170","29929436","29794022"],"confidence":"High","gaps":["Atomic-resolution structure of full-length TRAF6 in a signalosome not available","How TRAF6 transitions from resting to active state on filaments not resolved"]},{"year":2019,"claim":"TRAF6-mediated K63 ubiquitination was shown to regulate nuclear trafficking and function of key transcription factors and DNA repair enzymes: TRAF6 ubiquitinates FOXP3 at K262 to maintain its nuclear localization and Treg function, ubiquitinates hDNA2 to promote its nuclear import and homology-directed repair, and ubiquitinates ULK1 to drive autophagy; BANK1 was identified as a TRAF6 adapter in B cell TLR signaling.","evidence":"Treg-specific TRAF6 conditional KO with FOXP3-K262R mutagenesis; hDNA2 nuclear fractionation and DNA repair assays; ULK1 stability and autophagy readouts; BANK1 binding motif mutations and K63-Ub assays","pmids":["30886050","31216032","30929559","31243359"],"confidence":"High","gaps":["Whether TRAF6-mediated FOXP3 ubiquitination is direct (in vitro reconstitution) was not shown","Physiological signals activating GCA-TRAF6-ULK1 axis beyond CML not characterized"]},{"year":2021,"claim":"OTUB1 was identified as a deubiquitinase that directly binds TRAF6 and suppresses its K63-linked polyubiquitination, attenuating downstream ASK1 signaling in hepatocytes and protecting against NASH.","evidence":"IP-mass spectrometry, Co-immunoprecipitation, K63-Ub assays, hepatocyte-specific OTUB1 overexpression mouse model","pmids":["34591986"],"confidence":"Medium","gaps":["Whether OTUB1 acts on TRAF6 directly or via K63-chain editing not distinguished","Catalytic versus non-catalytic mechanism of OTUB1 on TRAF6 not resolved"]},{"year":2022,"claim":"USP15 was shown to oppose the TRAF6-BECN1 autophagy axis by deubiquitinating BECN1, revealing how TRAF6-dependent K63 ubiquitination of BECN1 is reversibly regulated to tune autophagy and cell invasion.","evidence":"CRISPR KO of USP15, Co-IP, deubiquitination assays, autophagy and invasion readouts","pmids":["35422093"],"confidence":"Medium","gaps":["Direct in vitro reconstitution of USP15-mediated BECN1 deubiquitination not performed","Whether USP15 competes with TRAF6 for BECN1 binding not tested"]},{"year":2023,"claim":"A biophysical mechanism for TRAF6 activation was established: TRAF6 undergoes liquid–liquid phase separation during LPS-induced inflammation, which is essential for its oligomerization, autoubiquitination, and NF-κB activation; the inhibitor Sufu blocks TRAF6 phase separation and oligomerization.","evidence":"In vitro phase separation with purified TRAF6, FRAP, Native-PAGE, myeloid-specific Sufu KO mice with TRAF6 depletion rescue","pmids":["37441604"],"confidence":"High","gaps":["Molecular determinants (IDRs, post-translational modifications) driving TRAF6 phase separation not mapped","Whether phase separation occurs for all TRAF6 signaling contexts or only TLR4 not tested"]},{"year":2024,"claim":"TRAF6 was linked to immune evasion via K63-linked ubiquitination of YAP1 promoting PD-L1 transcription in melanoma, and to viral exploitation by HIV-1 Tat which directly enhances TRAF6 oligomerization and ubiquitination to activate NF-κB-driven viral transcription.","evidence":"CRISPR interference screen with Co-IP and K63-Ub assays for YAP1-PD-L1 axis; Tat-TRAF6 Co-IP with oligomerization and ubiquitination assays","pmids":["38583649","38241362"],"confidence":"Medium","gaps":["Whether Tat-TRAF6 interaction is direct or bridged not confirmed with purified proteins","TRAF6-YAP1 ubiquitination sites not mapped","Therapeutic potential of TRAF6 inhibition for PD-L1 regulation not validated in clinical models"]},{"year":null,"claim":"Key unresolved questions include: the full-length atomic structure of TRAF6 in active signalosome complexes, the molecular determinants governing TRAF6 liquid–liquid phase separation across diverse signaling contexts, systematic identification of all direct TRAF6 substrates versus indirect targets, and the in vivo significance of separable scaffold versus E3 ligase functions across the full range of TRAF6-dependent pathways.","evidence":"","pmids":[],"confidence":"High","gaps":["No full-length TRAF6 structure in a signalosome complex","Phase separation determinants unmapped","Systematic substrate identification lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2,6,11,18,20,24,28,29,30,34]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[1,6,18,20,28]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,4,20,23]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,34]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[14,17]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[13]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,5,6,23,28,40]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,4,8,11,15,27]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[25,30,33]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[7]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[29]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,18,20,28]}],"complexes":["CBM (CARMA1-BCL10-MALT1) signalosomes","TRAF6-Ubc13/Uev1A E2-E3 complex","IRAK1-MyD88-TRAF6 myddosome"],"partners":["UBE2N","UBE2V1","TAK1","IRAK1","BECN1","FOXP3","EZH2","TGFBR1"],"other_free_text":[]},"mechanistic_narrative":"TRAF6 is a RING-domain E3 ubiquitin ligase that functions as a central signaling hub downstream of IL-1R/TLR, TNFR superfamily, TGF-β, and antigen receptors, coupling receptor activation to NF-κB, JNK, and p38 MAPK pathways. Upon stimulation, TRAF6 oligomerizes and, together with the Ubc13/Uev1A E2 complex, catalyzes K63-linked polyubiquitin chain synthesis on itself and a broad array of substrates—including TβRI, FOXP3, EZH2, BECN1, ULK1, hDNA2, cGAS, PINK1, and PIK3CA—thereby serving as a non-proteolytic scaffold that recruits and activates TAK1-TAB and IKK kinase complexes [PMID:15492226, PMID:18922473, PMID:30886050, PMID:27869166]. TRAF6 E3 ligase activity is essential for some pathways (IL-33/ST2L signaling, FOXP3 ubiquitination in Tregs) but is partially redundant with Pellino E3 ligases in IL-1-induced NF-κB activation, revealing separable scaffold and catalytic functions [PMID:18603409, PMID:28404732]. TRAF6 activity is regulated by S-glutathionylation of its RING domain, RSK2-mediated phosphorylation at S46/47/48, inhibitory interactions (PRDX1, syntenin, Sufu, OTUB1), K48-linked ubiquitination-dependent degradation (counteracted by IPMK), and liquid–liquid phase separation that promotes its oligomerization and autoubiquitination during inflammatory signaling [PMID:21078302, PMID:29563609, PMID:37441604, PMID:28439546]."},"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_rationale\": \"Tier 2 — clean KO with multiple defined cellular and signaling phenotypes, highly cited foundational paper\",\n      \"pmids\": [\"10215628\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TIFA activates IKK by promoting oligomerization and Lys63-linked polyubiquitination of TRAF6; only oligomeric forms of TIFA can activate IKK, demonstrated in an in vitro reconstitution system with purified TRAF6, TAK1 complex, and Ubc13-Uev1A.\",\n      \"method\": \"In vitro reconstitution with purified proteins, cell-free extract IKK 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 — reconstituted in vitro with purified proteins including defined E2 complex\",\n      \"pmids\": [\"15492226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The coiled-coil domain of TRAF6 is essential for its auto-ubiquitination and NF-κB activation; it serves as a bridge to recruit Ubc13/Uev1A rather than as the target site of polyubiquitin chain attachment.\",\n      \"method\": \"Domain deletion/mutation analysis, NF-κB reporter assays, ubiquitination assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis with functional readout, single lab\",\n      \"pmids\": [\"15465037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Structural and biochemical studies defined the molecular basis for TRAF6's unique binding specificity, revealing how its TRAF-C domain mediates interactions with both TNFR superfamily and IL-1R/TLR superfamily receptors.\",\n      \"method\": \"Structural and biochemical studies, receptor binding motif analysis\",\n      \"journal\": \"Advances in experimental medicine and biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — structural/biochemical characterization, single lab review\",\n      \"pmids\": [\"17633022\"],\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; the TRAF homology domain of TRAF6 physically interacts with TGF-β receptors, and TGF-β induces K63-linked ubiquitination of TRAF6 and promotes TRAF6-TAK1 association.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, TRAF6-deficient MEFs with signaling readouts\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, K63-Ub assays, KO cells, multiple orthogonal methods\",\n      \"pmids\": [\"18922473\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TRAF6 is critical for IFN-α/β induction in response to viral infection and intracellular dsRNA via the RIG-I/MDA5-IPS-1 pathway, activating NF-κB, JNK, and p38 but not IRF3; MEKK1 (not TAK1) acts downstream of TRAF6 for IFN-β induction in this pathway.\",\n      \"method\": \"TRAF6-deficient and TAK1-deficient MEFs, siRNA knockdown, reporter assays, forced expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO epistasis combined with KD and overexpression, multiple orthogonal methods\",\n      \"pmids\": [\"18984593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TRAF6 plays a pivotal role in IL-33/ST2L signaling; TRAF6 deficiency abolishes IRAK recruitment to ST2L and completely inhibits IL-33-induced p38, JNK, and NF-κB activation; a TRAF6 ubiquitin ligase-dead mutant (C70A) fails to restore these responses, demonstrating that E3 ligase activity is required.\",\n      \"method\": \"TRAF6-deficient MEFs, rescue with wild-type vs. C70A mutant TRAF6, signaling assays\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — KO cells plus active-site mutagenesis, multiple pathway readouts\",\n      \"pmids\": [\"18603409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TRAF6 deficiency leads to constitutive inactivation of GSK3β and promotes TNF-induced ROS accumulation and prolonged JNK activation leading to cell death; restoration of GSK3β activity rescues cell death in TRAF6-null cells.\",\n      \"method\": \"TRAF6-deficient 3T3 cells, reconstitution with constitutively active GSK3β, ROS and JNK assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO cells with functional rescue, single lab\",\n      \"pmids\": [\"18202703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TRAF6 mediates activation of JNK1/2, p38 MAPK, AMPK, and NF-κB in skeletal muscle during atrophy, and regulates expression of muscle-specific E3 ubiquitin ligases and autophagy-related molecules; muscle-restricted TRAF6 depletion rescues myofibril degradation, muscle fiber size, and prevents cancer cachexia.\",\n      \"method\": \"Muscle-specific TRAF6 knockout mice, denervation and cancer cachexia models, signaling assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with multiple defined phenotypic and signaling readouts\",\n      \"pmids\": [\"21187332\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Glutaredoxin-1 (GRX-1) catalyzes deglutathionylation of the RING finger motif of TRAF6; TRAF6 is S-glutathionylated under basal conditions, and deglutathionylation upon IL-1 stimulation is essential for TRAF6 auto-polyubiquitination and subsequent NF-κB activation.\",\n      \"method\": \"RNAi knockdown, S-glutathionylation assays, ubiquitination assays, NF-κB reporter\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — biochemical modification assays plus functional knockdown, single lab\",\n      \"pmids\": [\"21078302\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"NUMBL directly binds TRAF6 and promotes K48-linked polyubiquitination of TRAF6, leading to proteasomal degradation of TRAF6 and inhibition of NF-κB signaling; knockdown of NUMBL increases endogenous TRAF6 protein levels in cortical neurons.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assays (K48 vs K63 linkage), NUMBL knockdown, half-life assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus ubiquitination linkage specificity assays, single lab\",\n      \"pmids\": [\"20079715\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"TRAF6 associates with TβRI and mediates Lys63-linked polyubiquitination of TβRI in a PKCζ-dependent manner, promoting cleavage of TβRI by TACE; the liberated TβRI intracellular domain then associates with p300 to activate invasion-related genes (Snail, MMP2).\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, TACE cleavage assays, invasion assays, tumor tissue IHC\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Co-IP, K63-Ub, cleavage assay, gene expression), single lab but comprehensive\",\n      \"pmids\": [\"21629263\"],\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 levels (independent of oxygen and mRNA levels), thereby promoting tumor angiogenesis; E3 ligase activity of TRAF6 is required for this effect.\",\n      \"method\": \"Co-immunoprecipitation, K63-linked ubiquitination assays, protein stability assays, angiogenesis assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus ubiquitination assays, single lab\",\n      \"pmids\": [\"23722539\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"SARM1 and TRAF6 form a complex with PINK1 on depolarized mitochondria; SARM1 promotes TRAF6-mediated K63-linked ubiquitination of PINK1 at lysine 433, which is important for PINK1 stabilization, parkin recruitment, and mitophagy; some pathogenic PINK1 mutations reduce complex formation and ubiquitination.\",\n      \"method\": \"Co-immunoprecipitation, K63-linked ubiquitination assays, PINK1/parkin recruitment assays, knockdown experiments\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus site-specific ubiquitination, single lab\",\n      \"pmids\": [\"23885119\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CKIP-1 interacts with TRAF6 and terminates TRAF6-mediated K63-linked ubiquitination and plasma membrane recruitment of Akt, thereby inhibiting macrophage proliferation at late stages after M-CSF stimulation; CKIP-1 deficiency leads to macrophage-dominated splenomegaly and myeloproliferation.\",\n      \"method\": \"Co-immunoprecipitation, Akt ubiquitination assays, CKIP-1 knockout mice\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mice plus biochemical Co-IP and ubiquitination assays, single lab\",\n      \"pmids\": [\"24777252\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"EGFR phosphorylates DCBLD2 at Y750, which recruits TRAF6 via a TRAF6-binding motif; phospho-DCBLD2-bound TRAF6 exhibits increased E3 ubiquitin ligase activity and subsequently activates AKT to drive EGFR-dependent tumorigenesis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, mutational analysis of Y750, tumor cell lines and patient samples\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP, mutational analysis, ubiquitination assays, single lab\",\n      \"pmids\": [\"25061874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TRAF6-mediated signaling through ERK1/2, JNK1/2, and c-JUN is critical for satellite cell homeostasis and regenerative myogenesis; TRAF6/c-JUN signaling activates Pax7 transcription and represses miR-1 and miR-206 to maintain PAX7 levels; satellite cell-specific Traf6 deletion exaggerates dystrophic phenotype in mdx mice.\",\n      \"method\": \"Satellite cell-specific TRAF6 conditional knockout mice, muscle injury models, signaling assays, ChIP, miRNA analysis\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with multiple orthogonal mechanistic readouts (ChIP, miRNA, signaling), single lab but comprehensive\",\n      \"pmids\": [\"26619121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TRAF6 ubiquitinates Basigin (BSG/CD147) with K63-linked chains, promoting BSG recruitment to the plasma membrane and BSG-dependent MMP9 induction; TRAF6 knockdown reduces plasma membrane BSG and impairs melanoma invasion and metastasis.\",\n      \"method\": \"Co-immunoprecipitation, K63-linked ubiquitination assays, shRNA knockdown, invasion assays in vitro and in vivo\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus ubiquitination and functional assays, single lab\",\n      \"pmids\": [\"26769849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SKP2 loss upregulates TRAF6-mediated K63-linked ubiquitination of EZH2, leading to EZH2 degradation; the catalytically dead TRAF6-C70A mutant abolishes polyubiquitination of recombinant EZH2 in vitro, establishing TRAF6 as the E3 ligase for EZH2.\",\n      \"method\": \"In vitro ubiquitination assay with recombinant proteins, Co-IP, K63-Ub linkage assays, KO/KD cell lines, mouse models\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with active-site mutant control, supported by cell and in vivo data\",\n      \"pmids\": [\"27869166\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"K63-linked ubiquitylation mediated by TRAF6 is required for assembly of decapping factors, P-body formation, and constitutive decay of unstable inflammatory mRNAs; TRAF6 binds DCP1a and indirectly regulates its phosphorylation and mRNA decapping activity.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, DCP1a lysine mutants, P-body imaging, mRNA decay assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus functional mutational analysis and mRNA decay readouts, single lab\",\n      \"pmids\": [\"27315556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRAF6 E3 ligase activity contributes to but is not essential for IL-1-dependent K63-Ub chain formation, TAK1 activation, and IL-8 production; Pellino1 and Pellino2 compensate for TRAF6 E3 activity in this pathway. TRAF6/Pellino1/2 triple-KO abolishes all K63-Ub formation and IRAK1/IRAK4/MyD88 ubiquitination. RANKL-induced osteoclastogenesis proceeds normally with E3 ligase-inactive TRAF6, identifying scaffold functions of TRAF6 independent of its E3 activity.\",\n      \"method\": \"TRAF6 KO, Pellino1/2 double-KO, triple-KO cell lines; E3 ligase-inactive TRAF6[L74H] knockin mice; in vitro TAK1 activation with purified K63-Ub chains\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro reconstitution plus multiple genetic KO models and knockin mice, multiple orthogonal methods\",\n      \"pmids\": [\"28404732\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"IPMK directly binds TRAF6 (independent of IPMK catalytic activity) and stabilizes TRAF6 by blocking its K48-linked ubiquitination and proteasomal degradation, thereby sustaining TLR-induced proinflammatory signaling.\",\n      \"method\": \"Myeloid-specific IPMK KO mice, Co-immunoprecipitation, K48-Ub linkage assays, protein stability assays\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO plus Co-IP and ubiquitination assays, single lab\",\n      \"pmids\": [\"28439546\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RSK2 phosphorylates TRAF6 at serines 46, 47, and 48 in vitro; RSK2-mediated phosphorylation is required for TRAF6 K63-linked ubiquitination and downstream inflammatory signaling in the colon.\",\n      \"method\": \"In vitro kinase assay, phosphorylation site mutagenesis, RSK2 KO mice, colon inflammation model, Co-IP\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay plus KO mouse model, single lab\",\n      \"pmids\": [\"29563609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cryo-EM structure of BCL10 CARD filament at 4.0-Å resolution reveals that TRAF6 cooperatively decorates CBM (CARMA1-BCL10-MALT1) filaments to form higher-order assemblies, likely enabling all-or-none NF-κB activation downstream of TCR/BCR signaling.\",\n      \"method\": \"Cryo-EM structure determination, time-lapse confocal imaging, EM of MALT1-BCL10 and TRAF6-CBM assemblies\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure plus functional imaging, multiple orthogonal methods\",\n      \"pmids\": [\"29382759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The TRAF6 RING dimer employs a concerted allosteric mechanism using both subunits to promote ubiquitin transfer; the C-terminal extension of the TRAF6 RING domain mediates trans-interactions with donor ubiquitin, and N-terminal extensions engage donor-Ub in cis.\",\n      \"method\": \"Structural modeling, biochemical analysis, comparison with E3-Ub-E2 complex structures\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — structural modeling with biochemical validation, single lab\",\n      \"pmids\": [\"29432170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PRDX1 interacts with the RING finger domain of TRAF6 and inhibits its ubiquitin ligase activity, suppressing ubiquitination of ECSIT (required for NF-κB activation) and BECN1 (required for autophagy); PRDX1 knockdown enhances both NF-κB activation and autophagy in response to TLR4 stimulation.\",\n      \"method\": \"Co-immunoprecipitation, RING domain interaction mapping, ubiquitination assays (ECSIT and BECN1), PRDX1 KD cell lines\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP with domain mapping plus substrate ubiquitination assays, single lab\",\n      \"pmids\": [\"29929436\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"YAP interacts with TRAF6 to promote its ubiquitination and degradation, thereby inhibiting TRAF6-mediated NF-κB activation in endothelial cells; endothelial YAP deletion augments inflammatory responses, which are rescued by TRAF6 depletion.\",\n      \"method\": \"Co-immunoprecipitation, endothelial-specific YAP KO mice, TRAF6 depletion rescue experiments, NF-κB assays\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus conditional KO with genetic rescue, single lab\",\n      \"pmids\": [\"29794022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TRAF6 interacts with and ubiquitinates PIK3CA (p110α) with nonproteolytic polyubiquitin chains under serum stimulation, enhancing PI3K activation and downstream AKT phosphorylation and cell growth.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, overexpression and KD of TRAF6, AKT activation readouts\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus ubiquitination assays with functional signaling readout, single lab\",\n      \"pmids\": [\"29729098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRAF6 mediates K63-linked ubiquitination of FOXP3 at lysine 262; in the absence of TRAF6 activity or upon K262R mutation, FOXP3 shows aberrant perinuclear accumulation and disrupted regulatory T cell function; TRAF6-deficient Tregs are dysfunctional in vivo.\",\n      \"method\": \"Treg-specific TRAF6 conditional KO mice, K63-ubiquitination assays, site-directed mutagenesis of FOXP3-K262, subcellular localization imaging\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO plus site-specific ubiquitination mutagenesis and localization experiments, multiple orthogonal methods\",\n      \"pmids\": [\"30886050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRAF6 binds to and mediates K63-linked polyubiquitination of hDNA2, increasing hDNA2 stability and promoting its nuclear localization; inhibiting TRAF6-mediated polyubiquitination abolishes nuclear localization of hDNA2, impairing DNA end resection and homology-directed recombination repair.\",\n      \"method\": \"Co-immunoprecipitation, K63-Ub assays, nuclear fractionation, DNA repair assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus ubiquitination and nuclear localization assays with functional DNA repair readout, single lab\",\n      \"pmids\": [\"31216032\"],\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, thereby driving autophagy and imatinib resistance in CML.\",\n      \"method\": \"Co-immunoprecipitation, K63-ubiquitination assays, ULK1 stability assays, autophagy readouts\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus ubiquitination assays, single lab\",\n      \"pmids\": [\"30929559\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRAF6 ubiquitinates cGAS with K63-linked chains, promoting cGAS activation and downstream IFNβ production; TRAF6 knockdown reduces cGAS-induced IFNβ upon HSV-1 infection.\",\n      \"method\": \"Overexpression/knockdown of TRAF6, ubiquitination assays, IFNβ production assays, HSV-1 infection\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, overexpression/KD approach without in vitro reconstitution\",\n      \"pmids\": [\"31078259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTUB1 directly binds TRAF6 and suppresses its K63-linked polyubiquitination, thereby inhibiting downstream ASK1 activation; hepatocyte-specific OTUB1 overexpression markedly alleviates NASH in mice.\",\n      \"method\": \"IP-mass spectrometry, Co-immunoprecipitation, K63-Ub assays, hepatocyte-specific overexpression mouse model\",\n      \"journal\": \"Hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — IP-MS discovery plus Co-IP and ubiquitination assays with in vivo validation, single lab\",\n      \"pmids\": [\"34591986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"USP15 interacts with BECN1 (not TRAF6) and deubiquitinates BECN1, thereby attenuating TRAF6-BECN1 axis-driven autophagy induction and lung cancer migration/invasion; USP15 KO enhances autophagy in response to TLR4 stimulation.\",\n      \"method\": \"CRISPR-Cas9 USP15 KO, Co-immunoprecipitation, deubiquitination assays, autophagy and invasion assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO plus biochemical interaction and deubiquitination assays, single lab\",\n      \"pmids\": [\"35422093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Sufu directly interacts with TRAF6, preventing TRAF6 oligomerization and autoubiquitination; TRAF6 undergoes liquid-liquid phase separation during LPS-induced inflammation, which is essential for its ubiquitination activation and NF-κB activity; Sufu inhibits TRAF6 phase separation, reducing NF-κB activation.\",\n      \"method\": \"Co-immunoprecipitation, FRAP, Native-PAGE, in vitro phase separation assays with purified proteins, myeloid-specific Sufu KO mice, TRAF6 depletion rescue\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro phase separation with purified proteins, FRAP, Co-IP, KO mouse with genetic rescue, multiple orthogonal methods\",\n      \"pmids\": [\"37441604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HIV-1 Tat directly interacts with TRAF6, increasing TRAF6 oligomerization and auto-ubiquitination as well as K63-linked polyubiquitin chain synthesis, thereby enhancing NF-κB pathway activation and HIV-1 transcription.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, oligomerization assays, reporter assays, TRAF6 knockdown\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus ubiquitination and oligomerization assays, single lab\",\n      \"pmids\": [\"38241362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"HSV UL37 tegument protein activates NF-κB signaling through direct interaction with TRAF6 via a TRAF6-binding domain in UL37; mutation of this binding site reduces NF-κB activation during early viral infection.\",\n      \"method\": \"Transfection, Co-immunoprecipitation, NF-κB reporter assay, mutant virus with altered 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 2-3 — Co-IP plus mutant virus functional assay, single lab\",\n      \"pmids\": [\"18682563\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"T6BP (TRAF6-binding protein) specifically associates with TRAF6 through the coiled-coil region of T6BP and the N-terminal RING finger and zinc finger domains of TRAF6; IL-1 (but not TNF) induces TRAF6-T6BP complex formation in an IRAK-dependent manner; T6BP and IRAK exist in separate TRAF6 complexes.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, IL-1 stimulation, IKK and JNK activity assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP with domain mapping and ligand-dependent complex characterization, single lab\",\n      \"pmids\": [\"10920205\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"IL-1β stimulation induces interaction between MLK3 and TRAF6; RNAi knockdown of TRAF6 dramatically impairs MLK3 activation by IL-1β; TRAF6 promotes K63-linked ubiquitination of MLK3, which is important for MLK3 kinase activity.\",\n      \"method\": \"Co-immunoprecipitation, RNAi, in vitro kinase assays, K48/K63 ubiquitination assays\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus ubiquitination and kinase assays with RNAi, single lab\",\n      \"pmids\": [\"19586614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Syntenin interacts with TRAF6 under basal conditions and dissociates upon IL-1 stimulation; IRAK1 overexpression competes with syntenin for TRAF6 binding; syntenin inhibits TRAF6 ubiquitination and acts as a physiological suppressor of IL-1R/TLR4-mediated NF-κB activation.\",\n      \"method\": \"Co-immunoprecipitation, NF-κB reporter assays, ubiquitination assays, syntenin knockdown\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP plus functional knockdown and ubiquitination assays, single lab\",\n      \"pmids\": [\"18234474\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"BANK1 interacts with TRAF6 through five identified TRAF6-binding motifs in BANK1; the C-terminal domain of BANK1-FL and N-terminal domain of BANK1-Δ2 are required for TRAF6 binding; BANK1-TIR domain is important for K63-linked polyubiquitination and IL-8 production in B cell innate immune signaling.\",\n      \"method\": \"Co-immunoprecipitation, TRAF6-binding motif point mutations, decoy peptide experiments, K63-Ub assays, TLR7/9 stimulation\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — Co-IP with point mutations and functional ubiquitination assays, single lab\",\n      \"pmids\": [\"31243359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TRAF6 stabilizes YAP1 via K63-linked polyubiquitination, promoting formation of the YAP1/TFCP2 transcriptional complex and subsequent PD-L1 transcription in melanoma cells; TRAF6 inhibition reduces membrane PD-L1 expression and enhances CD8+ T cell cytolytic activity.\",\n      \"method\": \"CRISPR interference screening, Co-immunoprecipitation, K63-ubiquitination assays, transcriptional reporter assays, in vivo tumor models\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — CRISPR screen plus Co-IP, ubiquitination assays, and in vivo validation, single lab\",\n      \"pmids\": [\"38583649\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TRAF6 is a RING-domain E3 ubiquitin ligase that functions as a central adaptor downstream of IL-1R/TLR and TNFR superfamily receptors; upon activation it forms homodimers/oligomers and, together with Ubc13/Uev1A, catalyzes K63-linked polyubiquitin chain synthesis on itself and diverse substrates (including IRAK1, TAK1 complex components, TβRI, HIF-1α, FOXP3, BECN1, ULK1, hDNA2, PIK3CA, EZH2, YAP1, PINK1, BSG, cGAS, and MLK3), thereby serving as a non-proteolytic scaffold that recruits and activates downstream kinase complexes (TAK1-TAB1/2, IKK, MAPKs) to drive NF-κB, JNK, and p38 activation; its activity is tightly regulated by K48-linked ubiquitination for proteasomal degradation (by NUMBL, DRAK1-autophagy), by S-glutathionylation/deglutathionylation (GRX-1), by inhibitory protein interactions (PRDX1, syntenin, IPMK-independent stabilization, Sufu), by phosphorylation (RSK2 at S46/47/48), and by liquid-liquid phase separation during inflammatory signaling.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TRAF6 is a RING-domain E3 ubiquitin ligase that functions as a central signaling hub downstream of IL-1R/TLR, TNFR superfamily, TGF-β, and antigen receptors, coupling receptor activation to NF-κB, JNK, and p38 MAPK pathways. Upon stimulation, TRAF6 oligomerizes and, together with the Ubc13/Uev1A E2 complex, catalyzes K63-linked polyubiquitin chain synthesis on itself and a broad array of substrates—including TβRI, FOXP3, EZH2, BECN1, ULK1, hDNA2, cGAS, PINK1, and PIK3CA—thereby serving as a non-proteolytic scaffold that recruits and activates TAK1-TAB and IKK kinase complexes [PMID:15492226, PMID:18922473, PMID:30886050, PMID:27869166]. TRAF6 E3 ligase activity is essential for some pathways (IL-33/ST2L signaling, FOXP3 ubiquitination in Tregs) but is partially redundant with Pellino E3 ligases in IL-1-induced NF-κB activation, revealing separable scaffold and catalytic functions [PMID:18603409, PMID:28404732]. TRAF6 activity is regulated by S-glutathionylation of its RING domain, RSK2-mediated phosphorylation at S46/47/48, inhibitory interactions (PRDX1, syntenin, Sufu, OTUB1), K48-linked ubiquitination-dependent degradation (counteracted by IPMK), and liquid–liquid phase separation that promotes its oligomerization and autoubiquitination during inflammatory signaling [PMID:21078302, PMID:29563609, PMID:37441604, PMID:28439546].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Establishing TRAF6 as a non-redundant adaptor for multiple innate immune and TNFR superfamily pathways: TRAF6 knockout mice revealed essential roles in RANK-dependent osteoclastogenesis, IL-1, CD40, and LPS signaling, defining the breadth of TRAF6's signaling scope.\",\n      \"evidence\": \"TRAF6 knockout mice with in vitro signaling assays showing osteopetrosis and impaired cytokine responses\",\n      \"pmids\": [\"10215628\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Enzymatic mechanism of TRAF6 was unknown\", \"Whether TRAF6 functions as scaffold versus enzyme was not resolved\", \"Downstream kinase targets not yet mapped\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Identifying distinct TRAF6 signaling complexes: T6BP was found to associate specifically with TRAF6 in an IL-1/IRAK-dependent manner, revealing that TRAF6 participates in stimulus-specific multiprotein complexes rather than a single uniform signaling module.\",\n      \"evidence\": \"Co-immunoprecipitation with domain mapping showing T6BP binds TRAF6 RING/zinc-finger domains, with IL-1-dependent complex formation\",\n      \"pmids\": [\"10920205\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"T6BP function in TRAF6 signaling not defined\", \"No structural data on the T6BP-TRAF6 interface\", \"Stoichiometry of endogenous complexes unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Reconstituting the TRAF6 ubiquitination cascade in vitro resolved the catalytic mechanism: TIFA-induced TRAF6 oligomerization drives K63-linked polyubiquitination via the Ubc13/Uev1A E2, which activates TAK1 and IKK; the coiled-coil domain bridges TRAF6 to its E2 rather than serving as a ubiquitin acceptor.\",\n      \"evidence\": \"Cell-free reconstitution with purified TRAF6, TAK1 complex, and Ubc13-Uev1A; domain deletion/mutation analysis with NF-κB reporters\",\n      \"pmids\": [\"15492226\", \"15465037\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crystal structure of TRAF6 RING–E2 complex not yet available\", \"Physiological ubiquitin acceptor sites on TRAF6 not mapped\", \"Redundancy with other E3 ligases not tested\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Structural characterization of the TRAF-C domain explained how TRAF6 uniquely engages both TNFR superfamily and IL-1R/TLR superfamily receptors, resolving its dual receptor specificity.\",\n      \"evidence\": \"Structural and biochemical studies of receptor-binding motifs in the TRAF-C domain\",\n      \"pmids\": [\"17633022\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Full-length TRAF6 structure not determined\", \"Binding affinities for diverse receptors not systematically compared\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"TRAF6's substrate repertoire expanded beyond innate immune receptors: TRAF6 was shown to mediate TGF-β-induced Smad-independent JNK/p38 activation via K63-ubiquitination and TAK1 recruitment, to be essential for IL-33/ST2L signaling (requiring E3 ligase activity), to function in RIG-I/MDA5 antiviral IFN-α/β induction via MEKK1, and to regulate GSK3β activity controlling TNF-induced cell death.\",\n      \"evidence\": \"TRAF6-deficient MEFs with signaling readouts, C70A ligase-dead mutant rescue, epistasis with TAK1-KO, ROS and JNK assays\",\n      \"pmids\": [\"18922473\", \"18603409\", \"18984593\", \"18202703\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TRAF6 directly ubiquitinates TGF-β receptor components was unclear\", \"The relative contribution of TRAF6 E3 activity versus scaffold function was unresolved across pathways\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"TRAF6-mediated K63-linked ubiquitination of MLK3 was identified as a mechanism for IL-1β-induced MLK3 kinase activation, broadening the kinase substrates of TRAF6.\",\n      \"evidence\": \"Co-immunoprecipitation, RNAi of TRAF6, in vitro kinase assays, K63-ubiquitination assays\",\n      \"pmids\": [\"19586614\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro reconstitution of TRAF6-mediated MLK3 ubiquitination not performed\", \"Specific ubiquitinated lysines on MLK3 not mapped\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Multiple layers of TRAF6 regulation were uncovered: GRX-1-mediated deglutathionylation of the RING domain licenses TRAF6 autoubiquitination upon IL-1 stimulation, while NUMBL promotes K48-linked ubiquitination and degradation of TRAF6 to limit NF-κB; concurrently, muscle-specific TRAF6 deletion revealed TRAF6 as a master regulator of skeletal muscle atrophy via NF-κB, MAPK, AMPK, and autophagy.\",\n      \"evidence\": \"S-glutathionylation assays with GRX-1 RNAi; NUMBL Co-IP with K48/K63 linkage-specific Ub assays; muscle-specific TRAF6 KO mice in denervation and cancer cachexia models\",\n      \"pmids\": [\"21078302\", \"20079715\", \"21187332\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Glutathionylation site on TRAF6 RING domain not mapped at residue level\", \"NUMBL-TRAF6 E3 ligase responsible for K48-Ub not identified\", \"How TRAF6 activates AMPK in muscle was not established\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"TRAF6 was shown to directly ubiquitinate TβRI with K63-linked chains in a PKCζ-dependent manner, triggering TACE-mediated cleavage and nuclear translocation of the TβRI intracellular domain to activate invasion genes, revealing a non-canonical TGF-β signaling axis.\",\n      \"evidence\": \"Co-immunoprecipitation, K63-Ub assays, TACE cleavage assays, invasion assays, tumor tissue IHC\",\n      \"pmids\": [\"21629263\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific lysine residues on TβRI targeted by TRAF6 not identified\", \"Whether this pathway operates in non-cancer contexts was unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"TRAF6's K63-linked ubiquitination activity was extended to non-signaling contexts: TRAF6 ubiquitinates HIF-1α to stabilize it independently of oxygen and promotes tumor angiogenesis, and ubiquitinates PINK1 (with SARM1) on depolarized mitochondria to facilitate parkin recruitment and mitophagy.\",\n      \"evidence\": \"Co-immunoprecipitation, K63-Ub assays, protein stability assays, angiogenesis assays; SARM1-TRAF6-PINK1 complex analysis with site-specific K433 mutagenesis\",\n      \"pmids\": [\"23722539\", \"23885119\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro reconstitution of TRAF6-mediated HIF-1α ubiquitination not shown\", \"Whether SARM1 is required for all TRAF6-PINK1 interactions was untested\", \"Pathogenic PINK1 mutations reducing TRAF6 binding tested only in overexpression\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"TRAF6 emerged as a key node in growth factor signaling: CKIP-1 terminates TRAF6-dependent K63-ubiquitination and membrane recruitment of Akt to limit macrophage proliferation, while EGFR-phosphorylated DCBLD2 recruits and activates TRAF6 to drive AKT-dependent tumorigenesis.\",\n      \"evidence\": \"CKIP-1 KO mice with Akt ubiquitination assays; DCBLD2 Y750 mutagenesis with Co-IP and tumor studies\",\n      \"pmids\": [\"24777252\", \"25061874\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct substrate relationship between TRAF6 and Akt ubiquitination not reconstituted in vitro\", \"DCBLD2-TRAF6 structural basis not determined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"The substrate landscape of TRAF6 broadened to include chromatin regulators, membrane trafficking targets, and mRNA decay machinery: TRAF6 ubiquitinates EZH2 with K63 chains leading to its degradation, ubiquitinates Basigin/CD147 promoting its plasma membrane localization and MMP9-driven invasion, and ubiquitinates DCP1a-associated factors to drive P-body assembly and constitutive inflammatory mRNA decay.\",\n      \"evidence\": \"In vitro ubiquitination with recombinant EZH2 and TRAF6-C70A control; BSG K63-Ub assays with invasion readouts; DCP1a Co-IP and mRNA decay assays\",\n      \"pmids\": [\"27869166\", \"26769849\", \"27315556\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TRAF6 directly ubiquitinates DCP1a or acts indirectly was ambiguous\", \"Mechanism connecting K63-Ub of EZH2 to its degradation (normally K48-mediated) unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"A critical revision of the TRAF6 model showed that Pellino E3 ligases compensate for TRAF6 E3 activity in IL-1 signaling (triple-KO required to abolish K63-Ub), while RANKL-induced osteoclastogenesis proceeds with E3 ligase-inactive TRAF6, formally separating TRAF6's scaffold and catalytic functions; additionally, IPMK and syntenin were identified as regulators that stabilize and sequester TRAF6, respectively, and RSK2 phosphorylation at S46/47/48 licenses TRAF6 activation.\",\n      \"evidence\": \"Pellino1/2 double-KO plus TRAF6-KO triple cell lines; TRAF6[L74H] knockin mice; IPMK myeloid-KO with K48-Ub assays; syntenin Co-IP/competition; in vitro RSK2 kinase assay\",\n      \"pmids\": [\"28404732\", \"28439546\", \"18234474\", \"29563609\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for TRAF6 scaffold function independent of E3 activity not resolved\", \"Whether Pellino compensation occurs in all TLR pathways untested\", \"RSK2 phosphorylation sites validated in vitro but not confirmed with phospho-specific antibodies in vivo\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Structural and biophysical studies revealed how TRAF6 functions in higher-order signaling assemblies: cryo-EM showed TRAF6 cooperatively decorates BCL10-MALT1 filaments enabling threshold NF-κB activation downstream of antigen receptors; structural modeling revealed a concerted allosteric mechanism in the TRAF6 RING dimer for ubiquitin transfer; and multiple negative regulators (PRDX1, YAP) were shown to restrain TRAF6 activity.\",\n      \"evidence\": \"Cryo-EM of BCL10 CARD filament–TRAF6 assemblies; structural modeling of RING dimer–Ub–E2 complex; PRDX1 Co-IP with ECSIT/BECN1 ubiquitination assays; endothelial YAP KO mice with TRAF6 rescue\",\n      \"pmids\": [\"29382759\", \"29432170\", \"29929436\", \"29794022\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution structure of full-length TRAF6 in a signalosome not available\", \"How TRAF6 transitions from resting to active state on filaments not resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"TRAF6-mediated K63 ubiquitination was shown to regulate nuclear trafficking and function of key transcription factors and DNA repair enzymes: TRAF6 ubiquitinates FOXP3 at K262 to maintain its nuclear localization and Treg function, ubiquitinates hDNA2 to promote its nuclear import and homology-directed repair, and ubiquitinates ULK1 to drive autophagy; BANK1 was identified as a TRAF6 adapter in B cell TLR signaling.\",\n      \"evidence\": \"Treg-specific TRAF6 conditional KO with FOXP3-K262R mutagenesis; hDNA2 nuclear fractionation and DNA repair assays; ULK1 stability and autophagy readouts; BANK1 binding motif mutations and K63-Ub assays\",\n      \"pmids\": [\"30886050\", \"31216032\", \"30929559\", \"31243359\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TRAF6-mediated FOXP3 ubiquitination is direct (in vitro reconstitution) was not shown\", \"Physiological signals activating GCA-TRAF6-ULK1 axis beyond CML not characterized\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"OTUB1 was identified as a deubiquitinase that directly binds TRAF6 and suppresses its K63-linked polyubiquitination, attenuating downstream ASK1 signaling in hepatocytes and protecting against NASH.\",\n      \"evidence\": \"IP-mass spectrometry, Co-immunoprecipitation, K63-Ub assays, hepatocyte-specific OTUB1 overexpression mouse model\",\n      \"pmids\": [\"34591986\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether OTUB1 acts on TRAF6 directly or via K63-chain editing not distinguished\", \"Catalytic versus non-catalytic mechanism of OTUB1 on TRAF6 not resolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"USP15 was shown to oppose the TRAF6-BECN1 autophagy axis by deubiquitinating BECN1, revealing how TRAF6-dependent K63 ubiquitination of BECN1 is reversibly regulated to tune autophagy and cell invasion.\",\n      \"evidence\": \"CRISPR KO of USP15, Co-IP, deubiquitination assays, autophagy and invasion readouts\",\n      \"pmids\": [\"35422093\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct in vitro reconstitution of USP15-mediated BECN1 deubiquitination not performed\", \"Whether USP15 competes with TRAF6 for BECN1 binding not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A biophysical mechanism for TRAF6 activation was established: TRAF6 undergoes liquid–liquid phase separation during LPS-induced inflammation, which is essential for its oligomerization, autoubiquitination, and NF-κB activation; the inhibitor Sufu blocks TRAF6 phase separation and oligomerization.\",\n      \"evidence\": \"In vitro phase separation with purified TRAF6, FRAP, Native-PAGE, myeloid-specific Sufu KO mice with TRAF6 depletion rescue\",\n      \"pmids\": [\"37441604\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular determinants (IDRs, post-translational modifications) driving TRAF6 phase separation not mapped\", \"Whether phase separation occurs for all TRAF6 signaling contexts or only TLR4 not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"TRAF6 was linked to immune evasion via K63-linked ubiquitination of YAP1 promoting PD-L1 transcription in melanoma, and to viral exploitation by HIV-1 Tat which directly enhances TRAF6 oligomerization and ubiquitination to activate NF-κB-driven viral transcription.\",\n      \"evidence\": \"CRISPR interference screen with Co-IP and K63-Ub assays for YAP1-PD-L1 axis; Tat-TRAF6 Co-IP with oligomerization and ubiquitination assays\",\n      \"pmids\": [\"38583649\", \"38241362\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Tat-TRAF6 interaction is direct or bridged not confirmed with purified proteins\", \"TRAF6-YAP1 ubiquitination sites not mapped\", \"Therapeutic potential of TRAF6 inhibition for PD-L1 regulation not validated in clinical models\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: the full-length atomic structure of TRAF6 in active signalosome complexes, the molecular determinants governing TRAF6 liquid–liquid phase separation across diverse signaling contexts, systematic identification of all direct TRAF6 substrates versus indirect targets, and the in vivo significance of separable scaffold versus E3 ligase functions across the full range of TRAF6-dependent pathways.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No full-length TRAF6 structure in a signalosome complex\", \"Phase separation determinants unmapped\", \"Systematic substrate identification lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 6, 11, 18, 20, 24, 28, 29, 30, 34]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [1, 6, 18, 20, 28]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 4, 20, 23]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 34]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [14, 17]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 5, 6, 23, 28, 40]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 4, 8, 11, 15, 27]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [25, 30, 33]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [29]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 18, 20, 28]}\n    ],\n    \"complexes\": [\n      \"CBM (CARMA1-BCL10-MALT1) signalosomes\",\n      \"TRAF6-Ubc13/Uev1A E2-E3 complex\",\n      \"IRAK1-MyD88-TRAF6 myddosome\"\n    ],\n    \"partners\": [\n      \"UBE2N\",\n      \"UBE2V1\",\n      \"TAK1\",\n      \"IRAK1\",\n      \"BECN1\",\n      \"FOXP3\",\n      \"EZH2\",\n      \"TGFBR1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}