{"gene":"KLHL6","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":2005,"finding":"KLHL6 is required for B-cell antigen receptor (BCR) signal transduction; KLHL6-deficient B cells show reduced proliferation, Ca2+ response, and activation of phospholipase Cγ2 upon BCR cross-linking, and display a B-cell-intrinsic defect in germinal center formation.","method":"Conditional and global knockout mouse models; BCR cross-linking assays measuring proliferation, Ca2+ flux, and PLCγ2 activation","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean B-cell-specific KO with multiple orthogonal functional readouts (proliferation, Ca2+, PLCγ2 activation), B-cell-intrinsic requirement confirmed by conditional vs global mutant comparison","pmids":["16166635"],"is_preprint":false},{"year":2017,"finding":"KLHL6 physically interacts with Cullin3 (CUL3), indicating it functions as a substrate adapter for a CUL3-RING E3 ubiquitin ligase complex. KLHL6 also binds HBXIP/Lamtor5, a protein involved in cell-cycle regulation and cytokinesis.","method":"Co-immunoprecipitation in BL2 Burkitt's lymphoma cells","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP in a cell line, single lab, corroborated by independent reports from other labs","pmids":["28807996"],"is_preprint":false},{"year":2018,"finding":"KLHL6 assembles with Cullin3 to form a functional CUL3-RING ubiquitin ligase; cancer-associated mutations in KLHL6 disrupt its interaction with Cullin3, abrogating ligase activity. The mRNA decay factor Roquin2 is a direct substrate of KLHL6: KLHL6-mediated ubiquitination and proteasomal degradation of Roquin2 is triggered by BCR activation and requires the Tyr691 residue of Roquin2 for interaction with KLHL6. Stabilized Roquin2 promotes mRNA decay of the NF-κB inhibitor TNFAIP3/A20, driving DLBCL growth and survival.","method":"Co-immunoprecipitation; ubiquitination assays; site-directed mutagenesis (Roquin2 Y691F); xenograft models; in vitro ligase activity assays","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, in vitro ubiquitination, mutagenesis, xenograft), rigorous controls, single lab with comprehensive mechanistic dissection","pmids":["29695787"],"is_preprint":false},{"year":2023,"finding":"Purified full-length KLHL6 forms a homomultimeric complex in solution, and binding to the N-terminal domain of Cullin3 (Cul3NTD) enhances KLHL6 stability and homogeneity, consistent with a stoichiometric scaffold role in the CUL3-RING ligase complex.","method":"Recombinant protein expression with SUMO-tag; gel filtration chromatography; negative-staining electron microscopy; complex reconstitution with Cul3NTD","journal":"Protein expression and purification","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — structural/biophysical characterization by EM and gel filtration, single lab, no functional mutagenesis validation","pmids":["37286065"],"is_preprint":false},{"year":2024,"finding":"KLHL6 regulates surface BCR levels by targeting its signaling subunits CD79A and CD79B for ubiquitin-mediated degradation. BTB domain mutations in KLHL6 disrupt its subcellular localization and homodimerization, increasing surface BCR levels and signaling, whereas Kelch domain mutants have the opposite effect on BCR signaling.","method":"High-throughput protein interactome screens; functional characterization including loss-of-function experiments; surface BCR level measurement; localization studies","journal":"Blood cancer discovery","confidence":"High","confidence_rationale":"Tier 2 / Strong — interactome screens combined with functional domain mutagenesis (BTB vs Kelch), loss-of-function, and surface receptor quantification across multiple DLBCL contexts","pmids":["38630892"],"is_preprint":false},{"year":2026,"finding":"In CD8+ T cells, KLHL6 promotes TOX poly-ubiquitination and proteasomal degradation, attenuating the transition from progenitor exhausted T cells to terminally exhausted T cells. Simultaneously, KLHL6 maintains mitochondrial fitness by constraining mitochondrial fission through post-translational regulation of the PGAM5–Drp1 axis. KLHL6 is naturally downregulated by TCR ligation via PI3K-AKT-mediated inhibition of FOXO1.","method":"In vivo CRISPR screens; enforced KLHL6 expression in T cells; ubiquitination assays for TOX; assessment of mitochondrial fission (PGAM5-Drp1 axis); in vivo tumor and viral infection models","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vivo CRISPR screens, mechanistic ubiquitination assays, genetic rescue experiments, multiple orthogonal readouts across tumor and infection models","pmids":["41535474"],"is_preprint":false},{"year":2026,"finding":"KLHL6-mediated ubiquitination and degradation of CDK2 is induced by loganetin treatment in AML cells, promoting AML cell differentiation and chromosomal instability; CDK2-KLHL6 interaction was confirmed and shown to be enhanced in a dose-dependent manner.","method":"Co-immunoprecipitation; Western blot for CDK2 protein levels; in vivo AML mouse model; flow cytometry","journal":"Zhongguo shi yan xue ye xue za zhi","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP experiment, single lab, no direct ubiquitination reconstitution or mutagenesis","pmids":["42227424"],"is_preprint":false}],"current_model":"KLHL6 is a BTB-Kelch domain protein that functions as a substrate-recognition adapter for a Cullin3-RING E3 ubiquitin ligase complex, targeting diverse substrates—including Roquin2, CD79A/CD79B, TOX, CDK2, and PGAM5—for ubiquitin-mediated proteasomal degradation; in B cells it controls BCR signal transduction (PLCγ2 activation, Ca2+ flux) and surface BCR homeostasis through CD79A/B degradation, while in T cells it dually suppresses exhaustion by degrading TOX and maintains mitochondrial fitness by restraining the PGAM5–Drp1 fission axis, with its activity naturally attenuated by TCR-driven PI3K-AKT-FOXO1 signaling; cancer-associated mutations disrupt Cullin3 interaction or alter KLHL6 localization/dimerization, abrogating these tumor-suppressive and immune-regulatory functions."},"narrative":{"mechanistic_narrative":"KLHL6 is a BTB-Kelch protein that serves as the substrate-recognition adapter of a Cullin3-RING E3 ubiquitin ligase, coupling antigen-receptor signaling to targeted proteasomal degradation in lymphocytes [PMID:28807996, PMID:29695787]. It binds the N-terminal domain of Cullin3, which stabilizes KLHL6 and promotes its assembly into a homomultimeric ligase scaffold [PMID:37286065]. Through this complex KLHL6 ubiquitinates a set of substrates that tune B-cell and T-cell physiology: in B cells it is required for BCR-induced PLCγ2 activation, Ca2+ flux, proliferation, and germinal center formation [PMID:16166635], and it controls surface BCR homeostasis by degrading the signaling subunits CD79A and CD79B [PMID:38630892]; it also degrades the mRNA decay factor Roquin2 in a BCR-triggered, Roquin2-Tyr691-dependent manner, thereby relieving Roquin2-mediated decay of the NF-κB inhibitor A20/TNFAIP3 [PMID:29695787]. In CD8+ T cells KLHL6 restrains exhaustion by driving poly-ubiquitination and degradation of TOX and preserves mitochondrial fitness by limiting fission through post-translational control of the PGAM5–Drp1 axis, with KLHL6 itself downregulated by TCR-driven PI3K-AKT inhibition of FOXO1 [PMID:41535474]. Cancer-associated and domain-specific mutations alter KLHL6 function: lesions disrupting Cullin3 interaction abrogate ligase activity [PMID:29695787], while BTB-domain mutations perturb localization and homodimerization to raise surface BCR signaling and Kelch-domain mutations have the opposite effect [PMID:38630892].","teleology":[{"year":2005,"claim":"Established that KLHL6 is a B-cell-intrinsic requirement for antigen-receptor signaling, placing it upstream of the BCR signal-transduction machinery before any molecular activity was known.","evidence":"Conditional and global knockout mice with BCR cross-linking assays measuring proliferation, Ca2+ flux, and PLCγ2 activation","pmids":["16166635"],"confidence":"High","gaps":["Molecular activity of KLHL6 unknown at this stage","No substrate or biochemical mechanism linking KLHL6 to PLCγ2/Ca2+ identified","Does not explain whether the defect is signaling-proximal or receptor-level"]},{"year":2017,"claim":"Identified KLHL6 as a Cullin3 binding partner, providing the first molecular rationale that it acts as a CUL3-RING ligase substrate adapter rather than a passive signaling scaffold.","evidence":"Co-immunoprecipitation in BL2 Burkitt's lymphoma cells, with reported HBXIP/Lamtor5 binding","pmids":["28807996"],"confidence":"Medium","gaps":["Co-IP in a single cell line without reciprocal validation","No substrate identified","Functional consequence of HBXIP/Lamtor5 binding not defined"]},{"year":2018,"claim":"Demonstrated a functional CUL3-KLHL6 ligase and named its first substrate, connecting KLHL6 ubiquitination activity to NF-κB control and DLBCL biology.","evidence":"Co-IP, in vitro ubiquitination assays, Roquin2 Y691F mutagenesis, and xenograft models","pmids":["29695787"],"confidence":"High","gaps":["Whether Roquin2 is the only relevant substrate in B cells unaddressed","Structural basis of substrate recognition not resolved","Link between this axis and the earlier PLCγ2/Ca2+ phenotype not directly tested"]},{"year":2023,"claim":"Provided biophysical evidence that KLHL6 self-assembles into a homomultimer and that Cullin3-NTD binding stabilizes it, supporting a stoichiometric scaffold role in the ligase.","evidence":"Recombinant SUMO-tagged protein, gel filtration, negative-stain EM, and reconstitution with Cul3NTD","pmids":["37286065"],"confidence":"Medium","gaps":["No high-resolution structure","No functional mutagenesis tying multimerization to ligase activity","Substrate engagement not captured in the reconstituted assembly"]},{"year":2024,"claim":"Identified CD79A/CD79B as KLHL6 degradation targets and dissected domain-specific mutation effects, explaining how KLHL6 sets surface BCR levels and how mutations rewire signaling.","evidence":"Interactome screens, loss-of-function experiments, surface BCR quantification, and BTB vs Kelch domain mutagenesis in DLBCL contexts","pmids":["38630892"],"confidence":"High","gaps":["Stoichiometry of CD79A vs CD79B targeting unresolved","How localization changes mechanistically alter substrate access not defined","Relationship between CD79A/B degradation and Roquin2 axis not integrated"]},{"year":2026,"claim":"Extended KLHL6 function to CD8+ T cells, showing dual control of exhaustion (via TOX degradation) and mitochondrial fission (via the PGAM5-Drp1 axis), and identifying TCR-PI3K-AKT-FOXO1 as the regulatory input that suppresses KLHL6.","evidence":"In vivo CRISPR screens, enforced KLHL6 expression, TOX ubiquitination assays, mitochondrial fission readouts, and tumor/viral infection models","pmids":["41535474"],"confidence":"High","gaps":["Whether PGAM5 is a direct ubiquitination substrate vs an indirect target not fully resolved","Mechanism coupling FOXO1 to KLHL6 transcription not detailed","Interplay between TOX and mitochondrial arms within the same cell unclear"]},{"year":2026,"claim":"Reported CDK2 as a drug-inducible KLHL6 degradation target in AML, suggesting a role in myeloid differentiation beyond lymphocytes.","evidence":"Co-IP, Western blot for CDK2 levels, flow cytometry, and an in vivo AML mouse model under loganetin treatment","pmids":["42227424"],"confidence":"Low","gaps":["Single Co-IP without reciprocal validation or ubiquitination reconstitution","No mutagenesis confirming direct CUL3-KLHL6-dependent CDK2 degradation","Specificity of loganetin-induced CDK2 loss to KLHL6 not established"]},{"year":null,"claim":"How a single substrate-recognition adapter selects among Roquin2, CD79A/B, TOX, PGAM5, and CDK2 in different cell types and signaling states remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of substrate degron recognition by the Kelch domain","Rules governing cell-type-specific substrate choice unknown","Whether all reported substrates are direct CUL3-KLHL6 targets not uniformly confirmed"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,4,5]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[1,2]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2,3]}],"localization":[],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,4,5]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[2,4,5]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,4]}],"complexes":["CUL3-RING E3 ubiquitin ligase (CRL3)"],"partners":["CUL3","ROQUIN2","CD79A","CD79B","TOX","PGAM5","CDK2","HBXIP/LAMTOR5"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8WZ60","full_name":"Kelch-like protein 6","aliases":[],"length_aa":621,"mass_kda":70.4,"function":"Involved in B-lymphocyte antigen receptor signaling and germinal center formation","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q8WZ60/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/KLHL6","classification":"Not Classified","n_dependent_lines":8,"n_total_lines":1208,"dependency_fraction":0.006622516556291391},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/KLHL6","total_profiled":1310},"omim":[{"mim_id":"614214","title":"KELCH-LIKE 6; KLHL6","url":"https://www.omim.org/entry/614214"},{"mim_id":"151400","title":"LEUKEMIA, CHRONIC LYMPHOCYTIC; CLL","url":"https://www.omim.org/entry/151400"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Centriolar satellite","reliability":"Approved"},{"location":"Microtubules","reliability":"Additional"},{"location":"Mitotic spindle","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"lymphoid tissue","ntpm":29.1}],"url":"https://www.proteinatlas.org/search/KLHL6"},"hgnc":{"alias_symbol":["FLJ00029"],"prev_symbol":[]},"alphafold":{"accession":"Q8WZ60","domains":[{"cath_id":"3.30.710.10","chopping":"52-150","consensus_level":"medium","plddt":92.5662,"start":52,"end":150},{"cath_id":"2.120.10.80","chopping":"318-608","consensus_level":"medium","plddt":94.248,"start":318,"end":608}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WZ60","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WZ60-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WZ60-F1-predicted_aligned_error_v6.png","plddt_mean":86.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=KLHL6","jax_strain_url":"https://www.jax.org/strain/search?query=KLHL6"},"sequence":{"accession":"Q8WZ60","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8WZ60.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8WZ60/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WZ60"}},"corpus_meta":[{"pmid":"16166635","id":"PMC_16166635","title":"The BTB-kelch protein KLHL6 is involved in B-lymphocyte antigen receptor signaling and germinal center formation.","date":"2005","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/16166635","citation_count":64,"is_preprint":false},{"pmid":"29695787","id":"PMC_29695787","title":"Loss of KLHL6 promotes diffuse large B-cell lymphoma growth and survival by stabilizing the mRNA decay factor roquin2.","date":"2018","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/29695787","citation_count":46,"is_preprint":false},{"pmid":"28807996","id":"PMC_28807996","title":"Klhl6 Deficiency Impairs Transitional B Cell Survival and Differentiation.","date":"2017","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/28807996","citation_count":20,"is_preprint":false},{"pmid":"30646831","id":"PMC_30646831","title":"KLHL6 is a tumor suppressor gene in diffuse large B-cell lymphoma.","date":"2019","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/30646831","citation_count":15,"is_preprint":false},{"pmid":"29140403","id":"PMC_29140403","title":"KLHL6 Is Preferentially Expressed in Germinal Center-Derived B-Cell Lymphomas.","date":"2017","source":"American journal of clinical pathology","url":"https://pubmed.ncbi.nlm.nih.gov/29140403","citation_count":9,"is_preprint":false},{"pmid":"38630892","id":"PMC_38630892","title":"Disruption of KLHL6 Fuels Oncogenic Antigen Receptor Signaling in B-Cell Lymphoma.","date":"2024","source":"Blood cancer discovery","url":"https://pubmed.ncbi.nlm.nih.gov/38630892","citation_count":8,"is_preprint":false},{"pmid":"41535474","id":"PMC_41535474","title":"The ubiquitin ligase KLHL6 drives resistance to CD8+ T cell dysfunction.","date":"2026","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/41535474","citation_count":6,"is_preprint":false},{"pmid":"37286065","id":"PMC_37286065","title":"Expression, purification, and microscopic characterization of the tumor suppressor KLHL6.","date":"2023","source":"Protein expression and purification","url":"https://pubmed.ncbi.nlm.nih.gov/37286065","citation_count":0,"is_preprint":false},{"pmid":"41850981","id":"PMC_41850981","title":"KLHL6: a proteostatic guardian against T-cell exhaustion.","date":"2026","source":"Trends in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/41850981","citation_count":0,"is_preprint":false},{"pmid":"41797878","id":"PMC_41797878","title":"Ubiquitin E3 ligase KLHL6 brings exhausted T-cells back into action.","date":"2026","source":"Immunity & inflammation","url":"https://pubmed.ncbi.nlm.nih.gov/41797878","citation_count":0,"is_preprint":false},{"pmid":"42227424","id":"PMC_42227424","title":"[Loganetin Induces AML Cell Differentiation and Chromosomal Instability via KLHL6-Mediated CDK2 Ubiquitination Degradation].","date":"2026","source":"Zhongguo shi yan xue ye xue za zhi","url":"https://pubmed.ncbi.nlm.nih.gov/42227424","citation_count":0,"is_preprint":false},{"pmid":"41914637","id":"PMC_41914637","title":"Role of the UHRF1-KLHL6-CORO2B axis in obesity-related insulin resistance.","date":"2026","source":"Journal of diabetes investigation","url":"https://pubmed.ncbi.nlm.nih.gov/41914637","citation_count":0,"is_preprint":false},{"pmid":"41956857","id":"PMC_41956857","title":"Proteostasis regulation in T cell dysfunction: dual regulation by KLHL6.","date":"2026","source":"Trends in cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/41956857","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7687,"output_tokens":2062,"usd":0.026995,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9029,"output_tokens":3082,"usd":0.061097,"stage2_stop_reason":"end_turn"},"total_usd":0.088092,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"KLHL6 is required for B-cell antigen receptor (BCR) signal transduction; KLHL6-deficient B cells show reduced proliferation, Ca2+ response, and activation of phospholipase Cγ2 upon BCR cross-linking, and display a B-cell-intrinsic defect in germinal center formation.\",\n      \"method\": \"Conditional and global knockout mouse models; BCR cross-linking assays measuring proliferation, Ca2+ flux, and PLCγ2 activation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean B-cell-specific KO with multiple orthogonal functional readouts (proliferation, Ca2+, PLCγ2 activation), B-cell-intrinsic requirement confirmed by conditional vs global mutant comparison\",\n      \"pmids\": [\"16166635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"KLHL6 physically interacts with Cullin3 (CUL3), indicating it functions as a substrate adapter for a CUL3-RING E3 ubiquitin ligase complex. KLHL6 also binds HBXIP/Lamtor5, a protein involved in cell-cycle regulation and cytokinesis.\",\n      \"method\": \"Co-immunoprecipitation in BL2 Burkitt's lymphoma cells\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP in a cell line, single lab, corroborated by independent reports from other labs\",\n      \"pmids\": [\"28807996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"KLHL6 assembles with Cullin3 to form a functional CUL3-RING ubiquitin ligase; cancer-associated mutations in KLHL6 disrupt its interaction with Cullin3, abrogating ligase activity. The mRNA decay factor Roquin2 is a direct substrate of KLHL6: KLHL6-mediated ubiquitination and proteasomal degradation of Roquin2 is triggered by BCR activation and requires the Tyr691 residue of Roquin2 for interaction with KLHL6. Stabilized Roquin2 promotes mRNA decay of the NF-κB inhibitor TNFAIP3/A20, driving DLBCL growth and survival.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assays; site-directed mutagenesis (Roquin2 Y691F); xenograft models; in vitro ligase activity assays\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, in vitro ubiquitination, mutagenesis, xenograft), rigorous controls, single lab with comprehensive mechanistic dissection\",\n      \"pmids\": [\"29695787\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Purified full-length KLHL6 forms a homomultimeric complex in solution, and binding to the N-terminal domain of Cullin3 (Cul3NTD) enhances KLHL6 stability and homogeneity, consistent with a stoichiometric scaffold role in the CUL3-RING ligase complex.\",\n      \"method\": \"Recombinant protein expression with SUMO-tag; gel filtration chromatography; negative-staining electron microscopy; complex reconstitution with Cul3NTD\",\n      \"journal\": \"Protein expression and purification\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — structural/biophysical characterization by EM and gel filtration, single lab, no functional mutagenesis validation\",\n      \"pmids\": [\"37286065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"KLHL6 regulates surface BCR levels by targeting its signaling subunits CD79A and CD79B for ubiquitin-mediated degradation. BTB domain mutations in KLHL6 disrupt its subcellular localization and homodimerization, increasing surface BCR levels and signaling, whereas Kelch domain mutants have the opposite effect on BCR signaling.\",\n      \"method\": \"High-throughput protein interactome screens; functional characterization including loss-of-function experiments; surface BCR level measurement; localization studies\",\n      \"journal\": \"Blood cancer discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — interactome screens combined with functional domain mutagenesis (BTB vs Kelch), loss-of-function, and surface receptor quantification across multiple DLBCL contexts\",\n      \"pmids\": [\"38630892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"In CD8+ T cells, KLHL6 promotes TOX poly-ubiquitination and proteasomal degradation, attenuating the transition from progenitor exhausted T cells to terminally exhausted T cells. Simultaneously, KLHL6 maintains mitochondrial fitness by constraining mitochondrial fission through post-translational regulation of the PGAM5–Drp1 axis. KLHL6 is naturally downregulated by TCR ligation via PI3K-AKT-mediated inhibition of FOXO1.\",\n      \"method\": \"In vivo CRISPR screens; enforced KLHL6 expression in T cells; ubiquitination assays for TOX; assessment of mitochondrial fission (PGAM5-Drp1 axis); in vivo tumor and viral infection models\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vivo CRISPR screens, mechanistic ubiquitination assays, genetic rescue experiments, multiple orthogonal readouts across tumor and infection models\",\n      \"pmids\": [\"41535474\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"KLHL6-mediated ubiquitination and degradation of CDK2 is induced by loganetin treatment in AML cells, promoting AML cell differentiation and chromosomal instability; CDK2-KLHL6 interaction was confirmed and shown to be enhanced in a dose-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation; Western blot for CDK2 protein levels; in vivo AML mouse model; flow cytometry\",\n      \"journal\": \"Zhongguo shi yan xue ye xue za zhi\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP experiment, single lab, no direct ubiquitination reconstitution or mutagenesis\",\n      \"pmids\": [\"42227424\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"KLHL6 is a BTB-Kelch domain protein that functions as a substrate-recognition adapter for a Cullin3-RING E3 ubiquitin ligase complex, targeting diverse substrates—including Roquin2, CD79A/CD79B, TOX, CDK2, and PGAM5—for ubiquitin-mediated proteasomal degradation; in B cells it controls BCR signal transduction (PLCγ2 activation, Ca2+ flux) and surface BCR homeostasis through CD79A/B degradation, while in T cells it dually suppresses exhaustion by degrading TOX and maintains mitochondrial fitness by restraining the PGAM5–Drp1 fission axis, with its activity naturally attenuated by TCR-driven PI3K-AKT-FOXO1 signaling; cancer-associated mutations disrupt Cullin3 interaction or alter KLHL6 localization/dimerization, abrogating these tumor-suppressive and immune-regulatory functions.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"KLHL6 is a BTB-Kelch protein that serves as the substrate-recognition adapter of a Cullin3-RING E3 ubiquitin ligase, coupling antigen-receptor signaling to targeted proteasomal degradation in lymphocytes [#1, #2]. It binds the N-terminal domain of Cullin3, which stabilizes KLHL6 and promotes its assembly into a homomultimeric ligase scaffold [#3]. Through this complex KLHL6 ubiquitinates a set of substrates that tune B-cell and T-cell physiology: in B cells it is required for BCR-induced PLCγ2 activation, Ca2+ flux, proliferation, and germinal center formation [#0], and it controls surface BCR homeostasis by degrading the signaling subunits CD79A and CD79B [#4]; it also degrades the mRNA decay factor Roquin2 in a BCR-triggered, Roquin2-Tyr691-dependent manner, thereby relieving Roquin2-mediated decay of the NF-κB inhibitor A20/TNFAIP3 [#2]. In CD8+ T cells KLHL6 restrains exhaustion by driving poly-ubiquitination and degradation of TOX and preserves mitochondrial fitness by limiting fission through post-translational control of the PGAM5–Drp1 axis, with KLHL6 itself downregulated by TCR-driven PI3K-AKT inhibition of FOXO1 [#5]. Cancer-associated and domain-specific mutations alter KLHL6 function: lesions disrupting Cullin3 interaction abrogate ligase activity [#2], while BTB-domain mutations perturb localization and homodimerization to raise surface BCR signaling and Kelch-domain mutations have the opposite effect [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established that KLHL6 is a B-cell-intrinsic requirement for antigen-receptor signaling, placing it upstream of the BCR signal-transduction machinery before any molecular activity was known.\",\n      \"evidence\": \"Conditional and global knockout mice with BCR cross-linking assays measuring proliferation, Ca2+ flux, and PLCγ2 activation\",\n      \"pmids\": [\"16166635\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular activity of KLHL6 unknown at this stage\", \"No substrate or biochemical mechanism linking KLHL6 to PLCγ2/Ca2+ identified\", \"Does not explain whether the defect is signaling-proximal or receptor-level\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified KLHL6 as a Cullin3 binding partner, providing the first molecular rationale that it acts as a CUL3-RING ligase substrate adapter rather than a passive signaling scaffold.\",\n      \"evidence\": \"Co-immunoprecipitation in BL2 Burkitt's lymphoma cells, with reported HBXIP/Lamtor5 binding\",\n      \"pmids\": [\"28807996\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Co-IP in a single cell line without reciprocal validation\", \"No substrate identified\", \"Functional consequence of HBXIP/Lamtor5 binding not defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrated a functional CUL3-KLHL6 ligase and named its first substrate, connecting KLHL6 ubiquitination activity to NF-κB control and DLBCL biology.\",\n      \"evidence\": \"Co-IP, in vitro ubiquitination assays, Roquin2 Y691F mutagenesis, and xenograft models\",\n      \"pmids\": [\"29695787\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Roquin2 is the only relevant substrate in B cells unaddressed\", \"Structural basis of substrate recognition not resolved\", \"Link between this axis and the earlier PLCγ2/Ca2+ phenotype not directly tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Provided biophysical evidence that KLHL6 self-assembles into a homomultimer and that Cullin3-NTD binding stabilizes it, supporting a stoichiometric scaffold role in the ligase.\",\n      \"evidence\": \"Recombinant SUMO-tagged protein, gel filtration, negative-stain EM, and reconstitution with Cul3NTD\",\n      \"pmids\": [\"37286065\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution structure\", \"No functional mutagenesis tying multimerization to ligase activity\", \"Substrate engagement not captured in the reconstituted assembly\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified CD79A/CD79B as KLHL6 degradation targets and dissected domain-specific mutation effects, explaining how KLHL6 sets surface BCR levels and how mutations rewire signaling.\",\n      \"evidence\": \"Interactome screens, loss-of-function experiments, surface BCR quantification, and BTB vs Kelch domain mutagenesis in DLBCL contexts\",\n      \"pmids\": [\"38630892\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of CD79A vs CD79B targeting unresolved\", \"How localization changes mechanistically alter substrate access not defined\", \"Relationship between CD79A/B degradation and Roquin2 axis not integrated\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Extended KLHL6 function to CD8+ T cells, showing dual control of exhaustion (via TOX degradation) and mitochondrial fission (via the PGAM5-Drp1 axis), and identifying TCR-PI3K-AKT-FOXO1 as the regulatory input that suppresses KLHL6.\",\n      \"evidence\": \"In vivo CRISPR screens, enforced KLHL6 expression, TOX ubiquitination assays, mitochondrial fission readouts, and tumor/viral infection models\",\n      \"pmids\": [\"41535474\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PGAM5 is a direct ubiquitination substrate vs an indirect target not fully resolved\", \"Mechanism coupling FOXO1 to KLHL6 transcription not detailed\", \"Interplay between TOX and mitochondrial arms within the same cell unclear\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Reported CDK2 as a drug-inducible KLHL6 degradation target in AML, suggesting a role in myeloid differentiation beyond lymphocytes.\",\n      \"evidence\": \"Co-IP, Western blot for CDK2 levels, flow cytometry, and an in vivo AML mouse model under loganetin treatment\",\n      \"pmids\": [\"42227424\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP without reciprocal validation or ubiquitination reconstitution\", \"No mutagenesis confirming direct CUL3-KLHL6-dependent CDK2 degradation\", \"Specificity of loganetin-induced CDK2 loss to KLHL6 not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single substrate-recognition adapter selects among Roquin2, CD79A/B, TOX, PGAM5, and CDK2 in different cell types and signaling states remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of substrate degron recognition by the Kelch domain\", \"Rules governing cell-type-specific substrate choice unknown\", \"Whether all reported substrates are direct CUL3-KLHL6 targets not uniformly confirmed\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 4, 5]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2, 3]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 4, 5]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [2, 4, 5]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"complexes\": [\"CUL3-RING E3 ubiquitin ligase (CRL3)\"],\n    \"partners\": [\"CUL3\", \"Roquin2\", \"CD79A\", \"CD79B\", \"TOX\", \"PGAM5\", \"CDK2\", \"HBXIP/LAMTOR5\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}