{"gene":"KLHL13","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":2007,"finding":"KLHL13 forms a Cul3-based E3 ubiquitin ligase complex with KLHL9 and Cul3. Aurora B directly binds to the substrate-recognition domain of KLHL13 in vitro, coimmunoprecipitates with the Cul3 complex during mitosis, and is ubiquitylated by the reconstituted Cul3/KLHL9/KLHL13 ligase in vitro and in a Cul3-dependent manner in vivo. This complex removes Aurora B (and other chromosomal passenger complex components) from mitotic chromosomes, enabling their accumulation on the central spindle during anaphase, and is required for chromosome alignment, midzone/midbody formation, and cytokinesis completion.","method":"In vitro binding assay, co-immunoprecipitation, in vitro ubiquitylation reconstitution, in vivo ubiquitylation assay, RNAi knockdown with mitotic phenotype readout","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro ligase activity, direct binding assay, in vivo ubiquitylation, multiple orthogonal methods, replicated in subsequent publications","pmids":["17543862"],"is_preprint":false},{"year":2007,"finding":"Inactivation of the Cul3/KLHL9/KLHL13 ligase leads to premature degradation of Cyclin B and premature exit from the mitotic state in the presence of microtubule poisons, indicating this complex is required to maintain spindle assembly checkpoint (SAC) signaling.","method":"RNAi knockdown, Cyclin B degradation assay, mitotic exit assay with microtubule poisons","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean knockdown with defined cellular phenotype, single lab, two methods (Cyclin B levels + mitotic exit)","pmids":["18075312"],"is_preprint":false},{"year":2009,"finding":"KLHL9 and KLHL13 (but not KLHL21) regulate translocation of the chromosomal passenger complex (CPC) from chromosomes to the spindle midzone in anaphase; KLHL21 handles a distinct pool of Aurora B at midzone microtubules, demonstrating that different Cul3 adaptors non-redundantly regulate Aurora B at distinct subcellular localizations.","method":"RNAi knockdown, live-cell imaging, in vitro ubiquitination assay, localization studies","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal functional comparison across adaptors, in vitro ubiquitination, live imaging, replicated findings on KLHL9/KLHL13 complex from prior study","pmids":["19995937"],"is_preprint":false},{"year":2017,"finding":"The activity of the Cul3/KLHL9/KLHL13 complex was intact in SCCRO-deficient cells, demonstrating that SCCRO selectively neddylates Cul3 in a substrate-adaptor-dependent manner and does not collectively regulate all Cul3-anchored complexes; KLHL9/KLHL13-dependent activity is thus SCCRO-independent.","method":"Genetic inactivation of SCCRO, activity assay of Cul3-KLHL9/KLHL13 complex, comparison with Cul3-KLHL21 complex","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined negative result with functional readout, single lab, multiple adaptor comparisons","pmids":["28620047"],"is_preprint":false},{"year":2019,"finding":"KLHL9 and KLHL13, in complex with CUL3, act as novel E3 ubiquitin ligase adaptors for IRS1 (insulin receptor substrate-1), promoting its proteasomal degradation. Elevated KLHL9 and KLHL13 expression in ATG16L1-deficient cells drives IRS1 loss; siRNA knockdown of Klhl13 or Cul3 restores IRS1 levels and increases insulin signaling.","method":"BioID proximity labeling, co-immunoprecipitation, siRNA knockdown, proteasome inhibition rescue, insulin signaling assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — BioID plus Co-IP to identify interaction, knockdown rescue, proteasome inhibition, multiple orthogonal methods in single study","pmids":["31515271"],"is_preprint":false},{"year":2021,"finding":"In murine embryonic stem cells, Klhl13 (X-linked) promotes pluripotency factor expression and delays differentiation; together with Dusp9, it represses MAPK target gene expression, contributing to sex differences in pluripotent cells. CRISPR knockout of Klhl13 specifically impaired pluripotency factor levels distinct from Dusp9's effect on MAPK pathway intermediates.","method":"CRISPR knockout screen (primary chromosome-wide + three secondary screens), CRISPR KO validation, gene expression analysis","journal":"Genome biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic CRISPR screen with multiple secondary validation assays, single lab, functional phenotype with pathway placement","pmids":["33863351"],"is_preprint":false},{"year":2024,"finding":"The bacterial protein BipD (from B. pseudomallei) hijacks the host KLHL9-KLHL13-CUL3 E3 ubiquitin ligase complex by binding to the Back and Kelch domains of KLHL9 and KLHL13. This complex mediates K63-linked ubiquitination of the inner mitochondrial membrane protein IMMT at K211, initiating mitophagy via SQSTM1 and reducing mitochondrial ROS to promote bacterial survival.","method":"Co-immunoprecipitation, ubiquitome profiling (mass spectrometry), site-directed mutagenesis of IMMT K211, knockout/knockdown of complex components, mitophagy assays, bacterial survival assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods including interactome MS, co-IP, mutagenesis of ubiquitination site, functional rescue, replicated in companion paper","pmids":["38834545","39265641"],"is_preprint":false},{"year":2025,"finding":"KLHL13 hemizygous loss-of-function variants (frameshift and missense) in humans cause X-linked neurodevelopmental disorder with intellectual disability and macrocephaly. NDD-associated KLHL13 variants impair cell-cycle regulation during mitosis and lead to genomic instability in HEK293T cells. In zebrafish, klhl13 knockdown causes developmental deficits rescued by human KLHL13 WT mRNA but not NDD variant mRNA. Treatment with the Aurora Kinase B (AURKB) inhibitor AZD1152-HQPA rescues genomic instability in cells and neurobehavioral deficits in zebrafish, establishing KLHL13-mediated AURKB regulation as the mechanism underlying NDD.","method":"Exome sequencing, 3D protein modeling, overexpression in HEK293T cells (mitotic assay), zebrafish klhl13 morpholino knockdown, mRNA rescue, AURKB inhibitor treatment, neurobehavioral assays","journal":"Genetics in medicine : official journal of the American College of Medical Genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (cellular mitotic assay, zebrafish KD, pharmacological rescue), two independent model systems, variant-specific loss-of-function rescue experiments","pmids":["41159445"],"is_preprint":false},{"year":2025,"finding":"RHOBTB2 directly interacts with KLHL13 (validated by co-immunoprecipitation) and upregulates KLHL13 protein expression by inhibiting its proteasomal degradation (reversed by MG132 treatment), suggesting RHOBTB2 stabilizes KLHL13 protein levels.","method":"Co-immunoprecipitation, western blot, proteasome inhibitor (MG132) rescue, overexpression and knockdown experiments","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single Co-IP with proteasome inhibitor rescue, single lab, two complementary methods establishing interaction and stability regulation","pmids":["41107424"],"is_preprint":false},{"year":2025,"finding":"UBE2D3 binds KLHL13 to mediate K63-linked polyubiquitination of TAP2 at lysine 245, causing steric hindrance that blocks antigen transport and impairs MHC-I antigen presentation in pancreatic cancer cells.","method":"Co-immunoprecipitation (UBE2D3-KLHL13 interaction), ubiquitination assay (K63-linked, site-specific mutagenesis), functional antigen presentation assay, genetic/pharmacological inhibition","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — co-IP, site-specific ubiquitination mutagenesis, functional downstream assay, single lab with multiple orthogonal methods","pmids":["41315272"],"is_preprint":false},{"year":2025,"finding":"AGE treatment in colorectal cancer cells downregulates KLF5, KLHL13, and CUL3, resulting in impaired ubiquitin-mediated degradation of CEP57L1 and centrosome amplification. KLF5 was identified as a transcriptional regulator of KLHL13 and CUL3 expression, placing KLHL13 downstream of KLF5 in a pathway controlling CEP57L1 protein stability.","method":"AGE treatment of HCT116 cells, western blot, siRNA knockdown, overexpression, mouse metastasis model, patient cohort analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — knockdown and overexpression with protein stability readout, in vivo model, single lab, pathway placement via epistasis-like genetic manipulation","pmids":["41443421"],"is_preprint":false}],"current_model":"KLHL13 is a BTB-Kelch substrate adaptor protein that assembles with Cullin 3 (CUL3) and, in heterodimeric combination with KLHL9, forms an E3 ubiquitin ligase complex that directly binds and ubiquitylates Aurora B (AURKB) to remove it from mitotic chromosomes during anaphase, thereby coordinating chromosome alignment, spindle assembly checkpoint maintenance, and cytokinesis; the same KLHL9/KLHL13/CUL3 complex additionally targets IRS1 for proteasomal degradation and ubiquitinates the mitochondrial inner membrane protein IMMT (K63-linked, K211) to initiate mitophagy, while KLHL13 also partners with UBE2D3 to K63-ubiquitinate TAP2 and impair antigen presentation; loss-of-function variants in KLHL13 cause X-linked neurodevelopmental disorder through dysregulated AURKB activity, and KLHL13 protein stability is itself regulated by RHOBTB2-mediated protection from proteasomal degradation."},"narrative":{"mechanistic_narrative":"KLHL13 is a BTB-Kelch substrate-recognition adaptor that, together with KLHL9, assembles a CUL3-based E3 ubiquitin ligase governing mitotic progression by directly binding and ubiquitylating Aurora B (AURKB) to extract it from chromosomes and enable chromosomal passenger complex translocation to the central spindle during anaphase, an activity required for chromosome alignment, midbody formation, cytokinesis, and spindle assembly checkpoint maintenance [PMID:17543862, PMID:18075312, PMID:19995937]. The Kelch substrate-recognition domain of KLHL13 makes the direct AURKB contact, and the reconstituted CUL3/KLHL9/KLHL13 ligase ubiquitylates AURKB both in vitro and in a CUL3-dependent manner in cells [PMID:17543862]. Beyond this mitotic role, the same KLHL9/KLHL13/CUL3 complex targets IRS1 for proteasomal degradation to dampen insulin signaling [PMID:31515271], and is hijacked by the bacterial effector BipD to drive K63-linked ubiquitination of the inner mitochondrial membrane protein IMMT at K211, initiating SQSTM1-dependent mitophagy [PMID:38834545, PMID:39265641]. KLHL13 additionally couples with the E2 enzyme UBE2D3 to mediate K63-linked ubiquitination of TAP2 at K245, impairing MHC-I antigen presentation [PMID:41315272]. Hemizygous loss-of-function variants in KLHL13 cause an X-linked neurodevelopmental disorder with intellectual disability and macrocephaly; the variants impair mitotic cell-cycle regulation and cause genomic instability that is rescued by AURKB inhibition, establishing dysregulated AURKB control as the disease mechanism [PMID:41159445]. KLHL13 protein level is itself stabilized against proteasomal degradation by RHOBTB2 [PMID:41107424] and is transcriptionally controlled downstream of KLF5 in a pathway regulating CEP57L1 stability and centrosome number [PMID:41443421].","teleology":[{"year":2007,"claim":"Established that KLHL13 is not a free-standing protein but a CUL3 ligase adaptor that directly recognizes Aurora B as substrate, defining its core molecular activity in mitosis.","evidence":"In vitro binding, co-IP, reconstituted and in vivo ubiquitylation, and RNAi mitotic phenotyping","pmids":["17543862"],"confidence":"High","gaps":["Did not resolve how KLHL9 versus KLHL13 contribute distinct substrate-recognition roles within the heterodimer","Structural basis of the Kelch domain–AURKB interaction not defined"]},{"year":2007,"claim":"Connected the ligase to checkpoint integrity, showing it is required to sustain spindle assembly checkpoint signaling rather than only to relocalize Aurora B.","evidence":"RNAi knockdown with Cyclin B degradation and mitotic-exit assays under microtubule poisons","pmids":["18075312"],"confidence":"Medium","gaps":["Whether the SAC defect is a direct consequence of AURKB mislocalization versus an independent function was not separated","Single-lab observation"]},{"year":2009,"claim":"Demonstrated spatial division of labor among CUL3 adaptors, with KLHL9/KLHL13 controlling the chromosomal pool of Aurora B distinctly from KLHL21 at the midzone.","evidence":"Reciprocal adaptor RNAi, live-cell imaging, in vitro ubiquitination, localization studies","pmids":["19995937"],"confidence":"High","gaps":["Did not define what restricts each adaptor to its subcellular pool","Relative contributions of KLHL9 and KLHL13 within the shared complex remained unresolved"]},{"year":2017,"claim":"Clarified the neddylation regulation of the complex, showing KLHL9/KLHL13-CUL3 activity is independent of the SCCRO neddylation pathway that controls other CUL3 complexes.","evidence":"SCCRO genetic inactivation with CUL3-adaptor activity assays and adaptor comparisons","pmids":["28620047"],"confidence":"Medium","gaps":["The actual neddylation route required for KLHL9/KLHL13-CUL3 activation was not identified","Negative result confined to one system"]},{"year":2019,"claim":"Extended KLHL13 substrate scope beyond mitosis by identifying IRS1 as a degradation target, linking the ligase to insulin signaling.","evidence":"BioID, co-IP, siRNA rescue, proteasome inhibition, insulin signaling assays","pmids":["31515271"],"confidence":"High","gaps":["Ubiquitination site on IRS1 and chain linkage not mapped","Whether KLHL13 directly recognizes IRS1 versus KLHL9 was not dissected"]},{"year":2021,"claim":"Revealed a developmental/pluripotency role for X-linked Klhl13 in embryonic stem cells distinct from MAPK pathway control, broadening its biology beyond E3 substrate targeting.","evidence":"Chromosome-wide CRISPR knockout screen with secondary validation and expression analysis in mouse ESCs","pmids":["33863351"],"confidence":"Medium","gaps":["The molecular substrate or mechanism underlying pluripotency-factor regulation was not identified","Relationship to its CUL3 ligase activity unclear"]},{"year":2024,"claim":"Showed the complex can be pathogen-hijacked, defining a K63-linked ubiquitination of IMMT at K211 as a trigger for mitophagy exploited for bacterial survival.","evidence":"Co-IP, ubiquitome MS, IMMT K211 mutagenesis, component knockout, mitophagy and bacterial survival assays","pmids":["38834545","39265641"],"confidence":"High","gaps":["Whether IMMT is a physiological substrate independent of bacterial BipD was not established","How K63 versus K48 linkage choice is determined for this complex unclear"]},{"year":2025,"claim":"Provided direct human genetic and cross-species causal evidence that KLHL13 loss-of-function causes an X-linked neurodevelopmental disorder via dysregulated AURKB activity.","evidence":"Exome sequencing, HEK293T mitotic/genomic-instability assays, zebrafish knockdown with WT vs variant mRNA rescue, AURKB inhibitor rescue","pmids":["41159445"],"confidence":"High","gaps":["How mitotic genomic instability translates into the macrocephaly/ID phenotype at the neuronal level not resolved","Variant-specific effects on non-AURKB substrates not examined"]},{"year":2025,"claim":"Identified an upstream stabilizer of KLHL13, showing RHOBTB2 binds KLHL13 and protects it from proteasomal degradation.","evidence":"Co-IP, western blot, MG132 rescue, overexpression and knockdown","pmids":["41107424"],"confidence":"Medium","gaps":["Single Co-IP without reciprocal structural mapping of the interaction","Whether RHOBTB2 acts as an adaptor or competitor for KLHL13 turnover unclear"]},{"year":2025,"claim":"Established an E2-partner-dependent immune function, with UBE2D3 directing KLHL13-mediated K63 ubiquitination of TAP2 to suppress antigen presentation in cancer.","evidence":"Co-IP, K63 site-specific mutagenesis at TAP2 K245, antigen presentation assays, genetic/pharmacological inhibition","pmids":["41315272"],"confidence":"High","gaps":["Whether TAP2 targeting requires the full KLHL9/CUL3 complex or KLHL13 alone not resolved","Single-lab finding in pancreatic cancer context"]},{"year":2025,"claim":"Placed KLHL13 in a transcriptional axis controlling centrosome number, downstream of KLF5 and regulating CEP57L1 stability.","evidence":"AGE treatment of HCT116, knockdown/overexpression with protein stability readout, mouse metastasis model, patient cohort","pmids":["41443421"],"confidence":"Medium","gaps":["Whether CEP57L1 is a direct KLHL13/CUL3 substrate not demonstrated","Causal contribution of KLHL13 versus CUL3 in CEP57L1 control not separated"]},{"year":null,"claim":"How KLHL13 selects among its diverse substrates (AURKB, IRS1, IMMT, TAP2, CEP57L1) and what determines K48 versus K63 chain output across these contexts remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying structural model of substrate engagement","Determinants of chain-linkage choice across substrates unknown","Relationship between mitotic, metabolic, mitophagy, and immune functions not integrated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,4,6,9]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,6,9]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,4]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[0,2]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,1,2,7]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,4,6,9]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[6]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[9]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[7]}],"complexes":["CUL3/KLHL9/KLHL13 E3 ubiquitin ligase"],"partners":["KLHL9","CUL3","AURKB","IRS1","IMMT","TAP2","UBE2D3","RHOBTB2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9P2N7","full_name":"Kelch-like protein 13","aliases":["BTB and kelch domain-containing protein 2"],"length_aa":655,"mass_kda":73.9,"function":"Substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3 ubiquitin-protein ligase complex required for mitotic progression and cytokinesis. The BCR(KLHL9-KLHL13) E3 ubiquitin ligase complex mediates the ubiquitination of AURKB and controls the dynamic behavior of AURKB on mitotic chromosomes and thereby coordinates faithful mitotic progression and completion of cytokinesis","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q9P2N7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/KLHL13","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/KLHL13","total_profiled":1310},"omim":[{"mim_id":"618020","title":"KELCH-LIKE 22; KLHL22","url":"https://www.omim.org/entry/618020"},{"mim_id":"616262","title":"KELCH-LIKE 21; KLHL21","url":"https://www.omim.org/entry/616262"},{"mim_id":"611201","title":"KELCH-LIKE 9; KLHL9","url":"https://www.omim.org/entry/611201"},{"mim_id":"603136","title":"CULLIN 3; CUL3","url":"https://www.omim.org/entry/603136"},{"mim_id":"300655","title":"KELCH-LIKE 13; KLHL13","url":"https://www.omim.org/entry/300655"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"endometrium 1","ntpm":29.2}],"url":"https://www.proteinatlas.org/search/KLHL13"},"hgnc":{"alias_symbol":["FLJ10262"],"prev_symbol":["BKLHD2","KIAA1309"]},"alphafold":{"accession":"Q9P2N7","domains":[{"cath_id":"3.30.710.10","chopping":"74-190","consensus_level":"medium","plddt":94.4252,"start":74,"end":190},{"cath_id":"2.120.10.80","chopping":"339-634","consensus_level":"high","plddt":96.329,"start":339,"end":634}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P2N7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P2N7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P2N7-F1-predicted_aligned_error_v6.png","plddt_mean":87.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=KLHL13","jax_strain_url":"https://www.jax.org/strain/search?query=KLHL13"},"sequence":{"accession":"Q9P2N7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9P2N7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9P2N7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P2N7"}},"corpus_meta":[{"pmid":"17543862","id":"PMC_17543862","title":"A Cul3-based E3 ligase removes Aurora B from mitotic chromosomes, regulating mitotic progression and completion of cytokinesis in human cells.","date":"2007","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/17543862","citation_count":196,"is_preprint":false},{"pmid":"19995937","id":"PMC_19995937","title":"The Cul3-KLHL21 E3 ubiquitin ligase targets aurora B to midzone microtubules in anaphase and is required for cytokinesis.","date":"2009","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/19995937","citation_count":118,"is_preprint":false},{"pmid":"24627108","id":"PMC_24627108","title":"Whole-exome sequencing in patients with inherited neuropathies: outcome and challenges.","date":"2014","source":"Journal of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/24627108","citation_count":61,"is_preprint":false},{"pmid":"31594818","id":"PMC_31594818","title":"A Genome-Wide Knockout Screen in Human Macrophages Identified Host Factors Modulating 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axis.","date":"2025","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/41443421","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11032,"output_tokens":3210,"usd":0.040623,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10696,"output_tokens":3972,"usd":0.07639,"stage2_stop_reason":"end_turn"},"total_usd":0.117013,"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\": 2007,\n      \"finding\": \"KLHL13 forms a Cul3-based E3 ubiquitin ligase complex with KLHL9 and Cul3. Aurora B directly binds to the substrate-recognition domain of KLHL13 in vitro, coimmunoprecipitates with the Cul3 complex during mitosis, and is ubiquitylated by the reconstituted Cul3/KLHL9/KLHL13 ligase in vitro and in a Cul3-dependent manner in vivo. This complex removes Aurora B (and other chromosomal passenger complex components) from mitotic chromosomes, enabling their accumulation on the central spindle during anaphase, and is required for chromosome alignment, midzone/midbody formation, and cytokinesis completion.\",\n      \"method\": \"In vitro binding assay, co-immunoprecipitation, in vitro ubiquitylation reconstitution, in vivo ubiquitylation assay, RNAi knockdown with mitotic phenotype readout\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro ligase activity, direct binding assay, in vivo ubiquitylation, multiple orthogonal methods, replicated in subsequent publications\",\n      \"pmids\": [\"17543862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Inactivation of the Cul3/KLHL9/KLHL13 ligase leads to premature degradation of Cyclin B and premature exit from the mitotic state in the presence of microtubule poisons, indicating this complex is required to maintain spindle assembly checkpoint (SAC) signaling.\",\n      \"method\": \"RNAi knockdown, Cyclin B degradation assay, mitotic exit assay with microtubule poisons\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean knockdown with defined cellular phenotype, single lab, two methods (Cyclin B levels + mitotic exit)\",\n      \"pmids\": [\"18075312\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"KLHL9 and KLHL13 (but not KLHL21) regulate translocation of the chromosomal passenger complex (CPC) from chromosomes to the spindle midzone in anaphase; KLHL21 handles a distinct pool of Aurora B at midzone microtubules, demonstrating that different Cul3 adaptors non-redundantly regulate Aurora B at distinct subcellular localizations.\",\n      \"method\": \"RNAi knockdown, live-cell imaging, in vitro ubiquitination assay, localization studies\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal functional comparison across adaptors, in vitro ubiquitination, live imaging, replicated findings on KLHL9/KLHL13 complex from prior study\",\n      \"pmids\": [\"19995937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The activity of the Cul3/KLHL9/KLHL13 complex was intact in SCCRO-deficient cells, demonstrating that SCCRO selectively neddylates Cul3 in a substrate-adaptor-dependent manner and does not collectively regulate all Cul3-anchored complexes; KLHL9/KLHL13-dependent activity is thus SCCRO-independent.\",\n      \"method\": \"Genetic inactivation of SCCRO, activity assay of Cul3-KLHL9/KLHL13 complex, comparison with Cul3-KLHL21 complex\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined negative result with functional readout, single lab, multiple adaptor comparisons\",\n      \"pmids\": [\"28620047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"KLHL9 and KLHL13, in complex with CUL3, act as novel E3 ubiquitin ligase adaptors for IRS1 (insulin receptor substrate-1), promoting its proteasomal degradation. Elevated KLHL9 and KLHL13 expression in ATG16L1-deficient cells drives IRS1 loss; siRNA knockdown of Klhl13 or Cul3 restores IRS1 levels and increases insulin signaling.\",\n      \"method\": \"BioID proximity labeling, co-immunoprecipitation, siRNA knockdown, proteasome inhibition rescue, insulin signaling assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — BioID plus Co-IP to identify interaction, knockdown rescue, proteasome inhibition, multiple orthogonal methods in single study\",\n      \"pmids\": [\"31515271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In murine embryonic stem cells, Klhl13 (X-linked) promotes pluripotency factor expression and delays differentiation; together with Dusp9, it represses MAPK target gene expression, contributing to sex differences in pluripotent cells. CRISPR knockout of Klhl13 specifically impaired pluripotency factor levels distinct from Dusp9's effect on MAPK pathway intermediates.\",\n      \"method\": \"CRISPR knockout screen (primary chromosome-wide + three secondary screens), CRISPR KO validation, gene expression analysis\",\n      \"journal\": \"Genome biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic CRISPR screen with multiple secondary validation assays, single lab, functional phenotype with pathway placement\",\n      \"pmids\": [\"33863351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The bacterial protein BipD (from B. pseudomallei) hijacks the host KLHL9-KLHL13-CUL3 E3 ubiquitin ligase complex by binding to the Back and Kelch domains of KLHL9 and KLHL13. This complex mediates K63-linked ubiquitination of the inner mitochondrial membrane protein IMMT at K211, initiating mitophagy via SQSTM1 and reducing mitochondrial ROS to promote bacterial survival.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitome profiling (mass spectrometry), site-directed mutagenesis of IMMT K211, knockout/knockdown of complex components, mitophagy assays, bacterial survival assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods including interactome MS, co-IP, mutagenesis of ubiquitination site, functional rescue, replicated in companion paper\",\n      \"pmids\": [\"38834545\", \"39265641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"KLHL13 hemizygous loss-of-function variants (frameshift and missense) in humans cause X-linked neurodevelopmental disorder with intellectual disability and macrocephaly. NDD-associated KLHL13 variants impair cell-cycle regulation during mitosis and lead to genomic instability in HEK293T cells. In zebrafish, klhl13 knockdown causes developmental deficits rescued by human KLHL13 WT mRNA but not NDD variant mRNA. Treatment with the Aurora Kinase B (AURKB) inhibitor AZD1152-HQPA rescues genomic instability in cells and neurobehavioral deficits in zebrafish, establishing KLHL13-mediated AURKB regulation as the mechanism underlying NDD.\",\n      \"method\": \"Exome sequencing, 3D protein modeling, overexpression in HEK293T cells (mitotic assay), zebrafish klhl13 morpholino knockdown, mRNA rescue, AURKB inhibitor treatment, neurobehavioral assays\",\n      \"journal\": \"Genetics in medicine : official journal of the American College of Medical Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (cellular mitotic assay, zebrafish KD, pharmacological rescue), two independent model systems, variant-specific loss-of-function rescue experiments\",\n      \"pmids\": [\"41159445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RHOBTB2 directly interacts with KLHL13 (validated by co-immunoprecipitation) and upregulates KLHL13 protein expression by inhibiting its proteasomal degradation (reversed by MG132 treatment), suggesting RHOBTB2 stabilizes KLHL13 protein levels.\",\n      \"method\": \"Co-immunoprecipitation, western blot, proteasome inhibitor (MG132) rescue, overexpression and knockdown experiments\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single Co-IP with proteasome inhibitor rescue, single lab, two complementary methods establishing interaction and stability regulation\",\n      \"pmids\": [\"41107424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"UBE2D3 binds KLHL13 to mediate K63-linked polyubiquitination of TAP2 at lysine 245, causing steric hindrance that blocks antigen transport and impairs MHC-I antigen presentation in pancreatic cancer cells.\",\n      \"method\": \"Co-immunoprecipitation (UBE2D3-KLHL13 interaction), ubiquitination assay (K63-linked, site-specific mutagenesis), functional antigen presentation assay, genetic/pharmacological inhibition\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — co-IP, site-specific ubiquitination mutagenesis, functional downstream assay, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"41315272\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"AGE treatment in colorectal cancer cells downregulates KLF5, KLHL13, and CUL3, resulting in impaired ubiquitin-mediated degradation of CEP57L1 and centrosome amplification. KLF5 was identified as a transcriptional regulator of KLHL13 and CUL3 expression, placing KLHL13 downstream of KLF5 in a pathway controlling CEP57L1 protein stability.\",\n      \"method\": \"AGE treatment of HCT116 cells, western blot, siRNA knockdown, overexpression, mouse metastasis model, patient cohort analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — knockdown and overexpression with protein stability readout, in vivo model, single lab, pathway placement via epistasis-like genetic manipulation\",\n      \"pmids\": [\"41443421\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"KLHL13 is a BTB-Kelch substrate adaptor protein that assembles with Cullin 3 (CUL3) and, in heterodimeric combination with KLHL9, forms an E3 ubiquitin ligase complex that directly binds and ubiquitylates Aurora B (AURKB) to remove it from mitotic chromosomes during anaphase, thereby coordinating chromosome alignment, spindle assembly checkpoint maintenance, and cytokinesis; the same KLHL9/KLHL13/CUL3 complex additionally targets IRS1 for proteasomal degradation and ubiquitinates the mitochondrial inner membrane protein IMMT (K63-linked, K211) to initiate mitophagy, while KLHL13 also partners with UBE2D3 to K63-ubiquitinate TAP2 and impair antigen presentation; loss-of-function variants in KLHL13 cause X-linked neurodevelopmental disorder through dysregulated AURKB activity, and KLHL13 protein stability is itself regulated by RHOBTB2-mediated protection from proteasomal degradation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"KLHL13 is a BTB-Kelch substrate-recognition adaptor that, together with KLHL9, assembles a CUL3-based E3 ubiquitin ligase governing mitotic progression by directly binding and ubiquitylating Aurora B (AURKB) to extract it from chromosomes and enable chromosomal passenger complex translocation to the central spindle during anaphase, an activity required for chromosome alignment, midbody formation, cytokinesis, and spindle assembly checkpoint maintenance [#0, #1, #2]. The Kelch substrate-recognition domain of KLHL13 makes the direct AURKB contact, and the reconstituted CUL3/KLHL9/KLHL13 ligase ubiquitylates AURKB both in vitro and in a CUL3-dependent manner in cells [#0]. Beyond this mitotic role, the same KLHL9/KLHL13/CUL3 complex targets IRS1 for proteasomal degradation to dampen insulin signaling [#4], and is hijacked by the bacterial effector BipD to drive K63-linked ubiquitination of the inner mitochondrial membrane protein IMMT at K211, initiating SQSTM1-dependent mitophagy [#6]. KLHL13 additionally couples with the E2 enzyme UBE2D3 to mediate K63-linked ubiquitination of TAP2 at K245, impairing MHC-I antigen presentation [#9]. Hemizygous loss-of-function variants in KLHL13 cause an X-linked neurodevelopmental disorder with intellectual disability and macrocephaly; the variants impair mitotic cell-cycle regulation and cause genomic instability that is rescued by AURKB inhibition, establishing dysregulated AURKB control as the disease mechanism [#7]. KLHL13 protein level is itself stabilized against proteasomal degradation by RHOBTB2 [#8] and is transcriptionally controlled downstream of KLF5 in a pathway regulating CEP57L1 stability and centrosome number [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established that KLHL13 is not a free-standing protein but a CUL3 ligase adaptor that directly recognizes Aurora B as substrate, defining its core molecular activity in mitosis.\",\n      \"evidence\": \"In vitro binding, co-IP, reconstituted and in vivo ubiquitylation, and RNAi mitotic phenotyping\",\n      \"pmids\": [\"17543862\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how KLHL9 versus KLHL13 contribute distinct substrate-recognition roles within the heterodimer\", \"Structural basis of the Kelch domain–AURKB interaction not defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Connected the ligase to checkpoint integrity, showing it is required to sustain spindle assembly checkpoint signaling rather than only to relocalize Aurora B.\",\n      \"evidence\": \"RNAi knockdown with Cyclin B degradation and mitotic-exit assays under microtubule poisons\",\n      \"pmids\": [\"18075312\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the SAC defect is a direct consequence of AURKB mislocalization versus an independent function was not separated\", \"Single-lab observation\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstrated spatial division of labor among CUL3 adaptors, with KLHL9/KLHL13 controlling the chromosomal pool of Aurora B distinctly from KLHL21 at the midzone.\",\n      \"evidence\": \"Reciprocal adaptor RNAi, live-cell imaging, in vitro ubiquitination, localization studies\",\n      \"pmids\": [\"19995937\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define what restricts each adaptor to its subcellular pool\", \"Relative contributions of KLHL9 and KLHL13 within the shared complex remained unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Clarified the neddylation regulation of the complex, showing KLHL9/KLHL13-CUL3 activity is independent of the SCCRO neddylation pathway that controls other CUL3 complexes.\",\n      \"evidence\": \"SCCRO genetic inactivation with CUL3-adaptor activity assays and adaptor comparisons\",\n      \"pmids\": [\"28620047\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The actual neddylation route required for KLHL9/KLHL13-CUL3 activation was not identified\", \"Negative result confined to one system\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended KLHL13 substrate scope beyond mitosis by identifying IRS1 as a degradation target, linking the ligase to insulin signaling.\",\n      \"evidence\": \"BioID, co-IP, siRNA rescue, proteasome inhibition, insulin signaling assays\",\n      \"pmids\": [\"31515271\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitination site on IRS1 and chain linkage not mapped\", \"Whether KLHL13 directly recognizes IRS1 versus KLHL9 was not dissected\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Revealed a developmental/pluripotency role for X-linked Klhl13 in embryonic stem cells distinct from MAPK pathway control, broadening its biology beyond E3 substrate targeting.\",\n      \"evidence\": \"Chromosome-wide CRISPR knockout screen with secondary validation and expression analysis in mouse ESCs\",\n      \"pmids\": [\"33863351\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The molecular substrate or mechanism underlying pluripotency-factor regulation was not identified\", \"Relationship to its CUL3 ligase activity unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed the complex can be pathogen-hijacked, defining a K63-linked ubiquitination of IMMT at K211 as a trigger for mitophagy exploited for bacterial survival.\",\n      \"evidence\": \"Co-IP, ubiquitome MS, IMMT K211 mutagenesis, component knockout, mitophagy and bacterial survival assays\",\n      \"pmids\": [\"38834545\", \"39265641\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether IMMT is a physiological substrate independent of bacterial BipD was not established\", \"How K63 versus K48 linkage choice is determined for this complex unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided direct human genetic and cross-species causal evidence that KLHL13 loss-of-function causes an X-linked neurodevelopmental disorder via dysregulated AURKB activity.\",\n      \"evidence\": \"Exome sequencing, HEK293T mitotic/genomic-instability assays, zebrafish knockdown with WT vs variant mRNA rescue, AURKB inhibitor rescue\",\n      \"pmids\": [\"41159445\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How mitotic genomic instability translates into the macrocephaly/ID phenotype at the neuronal level not resolved\", \"Variant-specific effects on non-AURKB substrates not examined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified an upstream stabilizer of KLHL13, showing RHOBTB2 binds KLHL13 and protects it from proteasomal degradation.\",\n      \"evidence\": \"Co-IP, western blot, MG132 rescue, overexpression and knockdown\",\n      \"pmids\": [\"41107424\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP without reciprocal structural mapping of the interaction\", \"Whether RHOBTB2 acts as an adaptor or competitor for KLHL13 turnover unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established an E2-partner-dependent immune function, with UBE2D3 directing KLHL13-mediated K63 ubiquitination of TAP2 to suppress antigen presentation in cancer.\",\n      \"evidence\": \"Co-IP, K63 site-specific mutagenesis at TAP2 K245, antigen presentation assays, genetic/pharmacological inhibition\",\n      \"pmids\": [\"41315272\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TAP2 targeting requires the full KLHL9/CUL3 complex or KLHL13 alone not resolved\", \"Single-lab finding in pancreatic cancer context\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Placed KLHL13 in a transcriptional axis controlling centrosome number, downstream of KLF5 and regulating CEP57L1 stability.\",\n      \"evidence\": \"AGE treatment of HCT116, knockdown/overexpression with protein stability readout, mouse metastasis model, patient cohort\",\n      \"pmids\": [\"41443421\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CEP57L1 is a direct KLHL13/CUL3 substrate not demonstrated\", \"Causal contribution of KLHL13 versus CUL3 in CEP57L1 control not separated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How KLHL13 selects among its diverse substrates (AURKB, IRS1, IMMT, TAP2, CEP57L1) and what determines K48 versus K63 chain output across these contexts remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying structural model of substrate engagement\", \"Determinants of chain-linkage choice across substrates unknown\", \"Relationship between mitotic, metabolic, mitophagy, and immune functions not integrated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 4, 6, 9]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 6, 9]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 1, 2, 7]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 4, 6, 9]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [\"CUL3/KLHL9/KLHL13 E3 ubiquitin ligase\"],\n    \"partners\": [\"KLHL9\", \"CUL3\", \"AURKB\", \"IRS1\", \"IMMT\", \"TAP2\", \"UBE2D3\", \"RHOBTB2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}