{"gene":"KLHL3","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":2012,"finding":"KLHL3 belongs to the BTB-BACK-kelch family of proteins that recruit substrates for Cullin3-based ubiquitin ligase complexes; KLHL3 is coexpressed with NCC in the distal nephron and downregulates NCC expression at the cell surface.","method":"Cell surface expression assay, co-expression analysis, linkage analysis and whole-exome sequencing identifying KLHL3 mutations in FHHt families","journal":"Nature genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional cell-surface assay plus genetic/genomic identification, single study but multiple approaches","pmids":["22406640"],"is_preprint":false},{"year":2013,"finding":"KLHL3 forms a complex with CUL3 (via its BTB-BACK domains) and directly interacts with WNK4 (and WNK1), acting as the substrate adaptor that recruits WNK kinases for CUL3-dependent ubiquitination and proteasomal degradation; disease-causing mutations in KLHL3 reduce WNK4 ubiquitination and increase WNK4 protein levels.","method":"Co-immunoprecipitation, in vitro ubiquitination assay with recombinant CUL3-KLHL3 complex, siRNA knockdown of CUL3 in HeLa cells, domain mapping","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstituted ubiquitination assay with recombinant complex plus mutagenesis; independently replicated across multiple labs (PMIDs 23387299, 23453970, 23665031)","pmids":["23387299","23453970","23665031"],"is_preprint":false},{"year":2013,"finding":"The KLHL3 BTB and BACK domains together form the CUL3 interaction surface; crystal structure of the KLHL3 BTB-BACK domain dimer in complex with the N-terminal fragment of CUL3 shows both domains contribute to binding, and disease mutations in the BTB-BACK region disrupt CUL3 association as measured by isothermal titration calorimetry.","method":"X-ray crystallography, isothermal titration calorimetry (ITC), mutagenesis","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation by ITC and disease-mutation analysis in a single rigorous study","pmids":["23573258"],"is_preprint":false},{"year":2013,"finding":"KLHL3 interacts with WNK4 through a non-catalytic region of WNK (residues 479-667 in WNK1, equivalent acidic motif in WNK4); Gordon's syndrome-causing mutations E562K and Q565E in WNK4 abolish the interaction with KLHL3.","method":"Co-immunoprecipitation, domain mapping, disease-mutant interaction studies","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — domain mapping with in vitro ubiquitination assay, interaction surface defined; replicated in subsequent structural studies","pmids":["23387299"],"is_preprint":false},{"year":2013,"finding":"Multiple disease-causing KLHL3 mutations in the BTB domain reduce CUL3 binding, mutations in the Kelch domain reduce WNK4 binding, and the S410L mutation in the Kelch domain also reduces KLHL3 intracellular stability; all lead to decreased WNK4 ubiquitination and elevated WNK4 levels.","method":"Co-immunoprecipitation, cycloheximide chase assay, in vitro and in vivo ubiquitination assays, HEK293T transient expression","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal biochemical methods in a single lab","pmids":["23962426"],"is_preprint":false},{"year":2014,"finding":"Crystal structure of the KLHL3 Kelch domain in complex with the WNK4 degron motif (acidic motif) reveals an intricate network of interactions between the Kelch β-propeller surface and WNK4; disease-causing mutations in both WNK4 and KLHL3 disrupt critical interface contacts. KLHL2 binds WNK4 similarly to KLHL3 but with distinct binding mode compared to KEAP1.","method":"X-ray crystallography of KLHL3 Kelch domain–WNK4 degron complex; also crystal structure of KLHL2–WNK4 degron complex for comparison","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation linking interface contacts to disease mutations","pmids":["24641320"],"is_preprint":false},{"year":2014,"finding":"FHHt-causing CUL3 mutant (CUL3Δ403-459) is more heavily neddylated and activated than WT CUL3; in cells it depletes KLHL3 through enhanced ubiquitylation, thereby preventing WNK degradation despite increased CUL3-mediated WNK ubiquitylation.","method":"Cell-based ubiquitylation assays, neddylation analysis, nephron-specific Cul3 knockout mouse model, Western blotting","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — mechanistic cell-based assays plus conditional knockout mouse model with defined phenotype; establishes KLHL3 as a substrate of hyperactive CUL3Δ403-459","pmids":["25250572"],"is_preprint":false},{"year":2014,"finding":"In KLHL3(R528H/+) knock-in mice, both WNK1 and WNK4 protein levels are significantly increased in the kidney; fluorescence correlation spectroscopy confirmed that the R528H mutation abolishes binding of KLHL3 to WNK1 and WNK4 peptides, establishing WNK1 and WNK4 as in vivo substrates of KLHL3-CUL3 E3 ligase.","method":"KLHL3 R528H knock-in mouse model, fluorescence correlation spectroscopy (FCS) binding assay, Western blotting, NCC phosphorylation analysis","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo knock-in model plus direct binding assay with disease mutant; multiple orthogonal methods","pmids":["24821705"],"is_preprint":false},{"year":2015,"finding":"KLHL3 directly binds claudin-8, a tight junction protein in the collecting duct, and promotes its ubiquitination and degradation, thereby regulating paracellular chloride transport; the dominant PHA-II mutation in KLHL3 impairs claudin-8 binding, ubiquitination, and degradation.","method":"Tissue-specific claudin-8 knockout mice, Co-immunoprecipitation of KLHL3 and claudin-8, KLHL3 knockdown in collecting duct cells measuring paracellular chloride permeability, ubiquitination assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, knockdown with functional permeability readout, and knockout mouse; single lab","pmids":["25831548"],"is_preprint":false},{"year":2015,"finding":"Akt (downstream of insulin signaling) and PKA (downstream of vasopressin/forskolin signaling) phosphorylate KLHL3 at serine 433 in vitro and in cells; this phosphorylation impairs the interaction between KLHL3 and WNK4, reducing WNK4 degradation and increasing WNK4 protein levels.","method":"In vitro kinase assay (Akt and PKA), mass spectrometry identification of phospho-S433, phospho-specific antibody, co-immunoprecipitation, cell-based WNK4 protein level measurement","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus mass spectrometry and cell-based validation; single lab","pmids":["26435498"],"is_preprint":false},{"year":2015,"finding":"KLHL3 forms a complex with both WNK4 and p62/SQSTM1 via its Kelch repeat domain; under proteasome inhibition, p62-mediated selective autophagy contributes to KLHL3-dependent WNK4 degradation, with WNK4 co-localizing with KLHL3, p62, and LC3 in cytoplasmic puncta.","method":"Co-immunoprecipitation, immunofluorescence, 3-methyladenine autophagy inhibition, proteasome inhibitor (epoxomicin) treatment, p62 overexpression and knockdown in HEK293T cells","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus multiple pharmacological perturbations and imaging; single lab","pmids":["26349538"],"is_preprint":false},{"year":2016,"finding":"Phosphorylation of KLHL3 at S433 disrupts hydrogen bonds, hydrophobic, and electrostatic interactions between the Kelch domain of KLHL3 and the acidic motif of WNK4 by making the binding site more negatively charged, as demonstrated by molecular dynamics simulation corroborated by prior experimental data.","method":"Molecular dynamics simulation with energetic and structural analysis (computational study validating/explaining prior experimental findings)","journal":"Protein science","confidence":"Low","confidence_rationale":"Tier 4 / Moderate — computational/simulation study; mechanistic interpretation of prior experimental results but no new direct experimental validation","pmids":["27727489"],"is_preprint":false},{"year":2017,"finding":"KLHL3 dimerizes, and the dominant-negative effect of pathogenic KLHL3 mutations (causing autosomal dominant FHHt) is explained by dimerization of mutant KLHL3 with wild-type KLHL3, impairing the function of the wild-type subunit; heterozygous KLHL3 deletion alone is insufficient to cause PHAII, whereas homozygous deletion causes PHAII with elevated WNK1 and WNK4 only in the kidney.","method":"KLHL3 knockout mouse model (KLHL3-/- and KLHL3+/-), β-galactosidase reporter for expression, Western blot of WNK kinases in multiple organs, dimerization analysis","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo knockout mouse model with organ-specific expression analysis and mechanistic dimerization finding; single lab","pmids":["28052936"],"is_preprint":false},{"year":2017,"finding":"WNK4 is indispensable for PHAII pathogenesis caused by KLHL3 mutation; increased WNK1 protein (due to impaired KLHL3-mediated degradation) cannot compensate for WNK4 deficiency to activate SPAK/OSR1-NCC phosphorylation signaling in vivo.","method":"WNK4-/-/KLHL3R528H/+ and WNK4-/-/KLHL3R528H/R528H double-mutant mouse models, Western blot and immunofluorescence of pSPAK, pNCC in distal convoluted tubules","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via double-mutant in vivo mouse models; single lab","pmids":["28743496"],"is_preprint":false},{"year":2017,"finding":"KLHL3 ubiquitinates cardiac myosin binding protein C (cMyBP-C) via the ubiquitin-proteasome pathway; KLHL3 interacts with cMyBP-C as shown by co-immunoprecipitation and immunofluorescence, and homocysteine increases KLHL3 expression leading to decreased cMyBP-C.","method":"Co-immunoprecipitation, immunofluorescence, Western blotting, MG132 proteasome inhibitor rescue, Western blot (MRM approach)","journal":"Experimental cell research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and fluorescence study; single lab, no in vitro ubiquitination reconstitution","pmids":["28315668"],"is_preprint":false},{"year":2018,"finding":"KLHL3 BTB domain mutation M131V (M78V in human) retains intact interaction with WNK kinases but reduces binding to CUL3, leading to decreased CUL3 in distal convoluted tubule cytosol and increased WNK1/WNK4 abundance in vivo.","method":"KLHL3 M131V knock-in mouse model, in vitro co-immunoprecipitation, immunogold-labeling electron microscopy, microdissected renal tubule analysis","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knock-in mouse model combined with in vitro Co-IP and electron microscopy; single lab, multiple methods","pmids":["30148674"],"is_preprint":false},{"year":2018,"finding":"Deficiency of the COP9 signalosome (CSN) catalytic subunit Jab1 in the nephron causes increased neddylated CUL3 and near-complete loss of KLHL3, with consequent accumulation of WNK1, WNK4, and SPAK, and elevated NCC phosphorylation; this indicates that CSN-mediated deneddylation is required for normal KLHL3 stability and CUL3-KLHL3-WNK signaling.","method":"Nephron-specific Jab1 knockout mouse model (Pax8/LC1 system), Western blot, immunofluorescence","journal":"Journal of the American Society of Nephrology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional knockout mouse with multiple biochemical readouts; single lab","pmids":["30301860"],"is_preprint":false},{"year":2019,"finding":"Multiple disease-causing mutations in the Kelch domain of KLHL3 disrupt binding to the WNK4 acidic motif through two main mechanisms: altering the electrostatic potential of the binding site or disrupting Kelch-acidic motif hydrogen bonds; buried mutations can affect stability or indirect contacts. The L387P mutation causes functional impairment of WNK4 degradation by a mechanism not captured by Kelch-AM interaction simulations alone.","method":"Molecular dynamics simulations, Western blot analysis of WNK4 degradation with KLHL3 mutants","journal":"Biochemistry","confidence":"Low","confidence_rationale":"Tier 4 / Weak — primarily computational with limited Western blot corroboration; single lab","pmids":["30931564"],"is_preprint":false},{"year":2021,"finding":"The unique 30-amino acid N-terminal fragment of KS-WNK1 (kidney-specific WNK1) is required for both its activating effect on NCC and its recognition by KLHL3; specific residues in this region are critical for KLHL3 sensitivity. Full-length WNK1 is less impacted by CUL3-KLHL3-mediated degradation than KS-WNK1.","method":"KLHL3-R528H knock-in mice, mutagenesis of KS-WNK1 N-terminal residues, functional NCC activation assays, Western blotting","journal":"American journal of physiology. Renal physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knock-in mouse model plus mutagenesis and functional assays; single lab","pmids":["33682442"],"is_preprint":false},{"year":2022,"finding":"Crystal structure of the KLHL3 Kelch domain in complex with a WNK3 degron peptide reveals the complete 11-mer WNK-family degron binding mode; WNK3 Thr541 substitutes for the conserved proline, and phosphorylation of this residue abrogates KLHL3 interaction (shown by fluorescence polarization), revealing isoform-specific regulation.","method":"X-ray crystallography of KLHL3 Kelch-WNK3 peptide complex, fluorescence polarization binding assay, structural modelling","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with fluorescence polarization functional validation; single lab but rigorous methods","pmids":["35179207"],"is_preprint":false},{"year":2022,"finding":"KLHL3 is recruited by KSHV vIRF1 to ubiquitinate and degrade hnRNP Q1 via the ubiquitin-proteasome pathway; vIRF1 upregulates KLHL3 expression and the KLHL3-vIRF1 complex targets hnRNP Q1, leading to destabilization of GDPD1 mRNA and induction of aerobic glycolysis.","method":"Co-immunoprecipitation, ubiquitination assay, Western blotting, metabolic assays (glucose uptake, ATP, lactate), mRNA stability assays","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with ubiquitination assay and functional metabolic readouts; single lab","pmids":["35538151"],"is_preprint":false},{"year":2023,"finding":"A novel C-terminal motif (CM, residues 1051-1075) in WNK4 rich in negatively charged residues can also mediate KLHL3-dependent WNK4 degradation; this motif responds to PHAII mutations in the KLHL3 Kelch domain similarly to the acidic motif (AM), but AM is dominant; the CM may allow WNK4 degradation when AM is mutated.","method":"Co-immunoprecipitation, deletion/mutagenesis mapping in HEK293 cell-based degradation assays","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP/cell-based assay; single lab, no in vitro reconstitution","pmids":["37285722"],"is_preprint":false},{"year":2025,"finding":"Human KLHL3 and all four WNK kinases (WNK1-4) are O-GlcNAcylated; O-GlcNAcylation affects WNK4 function in osmolarity control and ferroptosis, demonstrating functional regulation of the KLHL3/WNK axis by this post-translational modification.","method":"Glycoproteomics/mass spectrometry, biochemical assays for osmolarity control and ferroptosis, O-GlcNAc-specific detection","journal":"Glycobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — glycoproteomics plus functional assays; peer-reviewed, single lab","pmids":["40796245"],"is_preprint":false}],"current_model":"KLHL3 is a BTB-BACK-Kelch domain adaptor protein that bridges CUL3 (via its BTB-BACK domains) and substrates including WNK1, WNK4 (via the Kelch domain binding an acidic degron motif), KS-WNK1, claudin-8, cMyBP-C, and hnRNP Q1, assembling a CUL3-RING E3 ubiquitin ligase complex that ubiquitinates these substrates for proteasomal (and partially autophagic via p62) degradation; crystal structures define the KLHL3-CUL3 and KLHL3-WNK degron interfaces; KLHL3 activity is negatively regulated by phosphorylation at S433 by Akt, PKA, and PKC (which impairs WNK4 binding) and by O-GlcNAcylation, and its stability is maintained by CSN-mediated CUL3 deneddylation; loss-of-function mutations in KLHL3 cause familial hyperkalemic hypertension (PHAII/FHHt) by stabilizing WNK kinases and hyperactivating the WNK-SPAK/OSR1-NCC phosphorylation cascade in the distal nephron."},"narrative":{"mechanistic_narrative":"KLHL3 is a BTB-BACK-Kelch substrate adaptor that assembles a CUL3-RING E3 ubiquitin ligase to target the WNK kinases for ubiquitin-dependent degradation, thereby controlling the WNK-SPAK/OSR1-NCC phosphorylation cascade in the distal nephron [PMID:22406640, PMID:23387299, PMID:23453970, PMID:23665031]. Its BTB and BACK domains together form the CUL3-binding surface, while its Kelch β-propeller engages an acidic degron motif in WNK1 and WNK4; crystal structures of both the KLHL3 BTB-BACK–CUL3 and Kelch–WNK degron interfaces define these contacts at atomic resolution, and disease mutations mapped onto them abolish either CUL3 association or WNK binding [PMID:23573258, PMID:23387299, PMID:24641320]. Knock-in and knockout mouse models confirm WNK1 and WNK4 as bona fide in vivo substrates whose protein levels rise when KLHL3 binding is lost, with WNK4 being indispensable for the downstream NCC-activating phenotype [PMID:24821705, PMID:28052936, PMID:28743496]. KLHL3-directed WNK degradation proceeds primarily through the proteasome but is also routed to p62/SQSTM1-mediated selective autophagy [PMID:26349538]. The ligase is regulated at multiple levels: phosphorylation of KLHL3 at Ser433 by Akt and PKA weakens Kelch-domain binding to the WNK4 acidic motif and stabilizes WNK4 [PMID:26435498], and CSN-mediated deneddylation of CUL3 is required to maintain KLHL3 stability and proper WNK signaling [PMID:30301860]. Beyond the WNK axis, KLHL3 ubiquitinates additional substrates including the tight-junction protein claudin-8 to regulate paracellular chloride transport [PMID:25831548] and, when co-opted by KSHV vIRF1, hnRNP Q1 to reprogram glycolytic metabolism [PMID:35538151]. Loss-of-function mutations in KLHL3 cause familial hyperkalemic hypertension (PHAII/FHHt) by stabilizing WNK kinases, with dominant alleles acting through dimerization with wild-type KLHL3 [PMID:22406640, PMID:28052936].","teleology":[{"year":2012,"claim":"Linking KLHL3 to a Cullin3-based ubiquitin system and to NCC regulation established its candidacy as the distal-nephron salt-handling factor mutated in FHHt, framing the question of which substrate it controls.","evidence":"Linkage/exome sequencing in FHHt families plus cell-surface NCC expression assays","pmids":["22406640"],"confidence":"Medium","gaps":["Direct substrate not yet identified","No biochemical demonstration of ligase assembly"]},{"year":2013,"claim":"Reconstitution showed KLHL3 bridges CUL3 and WNK kinases as the substrate adaptor of a working E3 ligase, directly explaining how disease mutations elevate WNK protein.","evidence":"Co-IP, in vitro ubiquitination with recombinant CUL3-KLHL3, domain mapping, and crystallography/ITC of the BTB-BACK–CUL3 interface; replicated across labs","pmids":["23387299","23453970","23665031","23573258","23962426"],"confidence":"High","gaps":["Atomic detail of the Kelch–WNK degron contact not yet resolved","In vivo substrate identity not yet confirmed"]},{"year":2014,"claim":"Structural definition of the Kelch–WNK4 degron interface and in vivo knock-in models confirmed WNK1/WNK4 as physiological KLHL3 substrates and explained both KLHL3 and CUL3 disease alleles mechanistically.","evidence":"Crystal structure of Kelch–WNK4 degron complex, FCS binding with R528H mutant, and KLHL3 R528H knock-in plus nephron-specific Cul3 knockout mice","pmids":["24641320","24821705","25250572"],"confidence":"High","gaps":["Relative contribution of WNK1 vs WNK4 to phenotype unresolved","Regulatory inputs onto KLHL3 not yet defined"]},{"year":2015,"claim":"Discovery of phosphoregulation at Ser433 and an alternative autophagic degradation route, plus claudin-8 as a substrate, broadened KLHL3 from a single-substrate adaptor to a hormonally tuned, multi-target ligase.","evidence":"In vitro Akt/PKA kinase assays with MS and phospho-antibody, p62/autophagy perturbation with imaging, and claudin-8 Co-IP/knockout with permeability readouts","pmids":["26435498","26349538","25831548"],"confidence":"Medium","gaps":["In vivo relevance of S433 phosphorylation not established","Balance between proteasomal and autophagic routes unquantified"]},{"year":2017,"claim":"Genetic dissection in mice established the dominant-negative dimerization mechanism of pathogenic alleles and the epistatic requirement of WNK4 for the disease phenotype.","evidence":"KLHL3 knockout/heterozygote mice with dimerization analysis and WNK4-/-/KLHL3R528H double-mutant epistasis","pmids":["28052936","28743496"],"confidence":"Medium","gaps":["Stoichiometry of mutant-WT heterodimer inhibition not quantified","Why WNK1 cannot compensate for WNK4 not mechanistically resolved"]},{"year":2018,"claim":"Separation-of-function mutants and CSN manipulation showed that CUL3 binding and KLHL3 stability are distinct vulnerabilities, with deneddylation required to preserve the adaptor.","evidence":"KLHL3 M131V knock-in with Co-IP and immunogold EM, and nephron-specific Jab1 (CSN) knockout mice","pmids":["30148674","30301860"],"confidence":"Medium","gaps":["How CUL3 neddylation cycling drives KLHL3 turnover mechanistically unclear","Single-lab models"]},{"year":2021,"claim":"Mapping the KS-WNK1 N-terminal recognition element refined substrate selectivity, explaining why kidney-specific WNK1 is preferentially degraded over full-length WNK1.","evidence":"KLHL3-R528H knock-in mice with KS-WNK1 N-terminal mutagenesis and NCC functional assays","pmids":["33682442"],"confidence":"Medium","gaps":["Structural basis of KS-WNK1 N-terminal recognition not solved","Isoform competition for KLHL3 in vivo not quantified"]},{"year":2022,"claim":"A WNK3 degron co-structure defined the complete WNK-family 11-mer binding mode and phospho-degron switching, and a viral-hijack study revealed KLHL3 functions beyond ion transport.","evidence":"Crystal structure of Kelch–WNK3 peptide with fluorescence polarization, and KSHV vIRF1-directed hnRNP Q1 ubiquitination with metabolic assays","pmids":["35179207","35538151"],"confidence":"High","gaps":["Physiological signals controlling WNK degron phosphorylation unclear","Breadth of non-WNK substrate repertoire unknown"]},{"year":2025,"claim":"Identification of O-GlcNAcylation on KLHL3 and all WNK kinases added a metabolic post-translational layer regulating the axis in osmolarity and ferroptosis contexts.","evidence":"Glycoproteomics/MS with functional osmolarity and ferroptosis assays","pmids":["40796245"],"confidence":"Medium","gaps":["Specific O-GlcNAc sites and their effect on ligase activity not mapped","In vivo physiological consequence not established"]},{"year":null,"claim":"How the multiple regulatory inputs (S433 phosphorylation, O-GlcNAcylation, CUL3 neddylation, dimerization) are integrated to set KLHL3 ligase output in different tissues and physiological states remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified quantitative model of KLHL3 regulation","Full non-WNK substrate landscape uncharacterized","Tissue-specific substrate preference mechanisms unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,8,14,20]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2,3]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[1,7]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[10,15]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,7]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[0,8]}],"complexes":["CUL3-RING E3 ubiquitin ligase (CRL3)"],"partners":["CUL3","WNK4","WNK1","WNK3","CLDN8","SQSTM1","MYBPC3","SYNCRIP"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UH77","full_name":"Kelch-like protein 3","aliases":[],"length_aa":587,"mass_kda":65.0,"function":"Substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3 ubiquitin ligase complex that acts as a regulator of ion transport in the distal nephron (PubMed:14528312, PubMed:22406640, PubMed:23387299, PubMed:23453970, PubMed:23576762, PubMed:23665031, PubMed:25313067, PubMed:35093948). The BCR(KLHL3) complex acts by mediating ubiquitination and degradation of WNK1 and WNK4, two activators of Na-Cl cotransporter SLC12A3/NCC in distal convoluted tubule cells of kidney, thereby regulating NaCl reabsorption (PubMed:23387299, PubMed:23453970, PubMed:23576762, PubMed:23665031, PubMed:25313067, PubMed:35093948). The BCR(KLHL3) complex also mediates ubiquitination and degradation of WNK3 (PubMed:35179207). The BCR(KLHL3) complex also mediates ubiquitination of CLDN8, a tight-junction protein required for paracellular chloride transport in the kidney, leading to its degradation (By similarity)","subcellular_location":"Cytoplasm, cytosol; Cytoplasm, cytoskeleton","url":"https://www.uniprot.org/uniprotkb/Q9UH77/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/KLHL3","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/KLHL3","total_profiled":1310},"omim":[{"mim_id":"614496","title":"PSEUDOHYPOALDOSTERONISM, TYPE IIE; PHA2E","url":"https://www.omim.org/entry/614496"},{"mim_id":"614495","title":"PSEUDOHYPOALDOSTERONISM, TYPE IID; PHA2D","url":"https://www.omim.org/entry/614495"},{"mim_id":"614492","title":"PSEUDOHYPOALDOSTERONISM, TYPE IIC; PHA2C","url":"https://www.omim.org/entry/614492"},{"mim_id":"614491","title":"PSEUDOHYPOALDOSTERONISM, TYPE IIB; PHA2B","url":"https://www.omim.org/entry/614491"},{"mim_id":"608064","title":"KELCH-LIKE 5; KLHL5","url":"https://www.omim.org/entry/608064"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":44.9}],"url":"https://www.proteinatlas.org/search/KLHL3"},"hgnc":{"alias_symbol":["KIAA1129"],"prev_symbol":[]},"alphafold":{"accession":"Q9UH77","domains":[{"cath_id":"3.30.710.10","chopping":"32-146","consensus_level":"high","plddt":92.8791,"start":32,"end":146},{"cath_id":"1.25.40.420","chopping":"191-283","consensus_level":"high","plddt":90.439,"start":191,"end":283},{"cath_id":"2.120.10.80","chopping":"300-583","consensus_level":"medium","plddt":97.17,"start":300,"end":583}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UH77","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UH77-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UH77-F1-predicted_aligned_error_v6.png","plddt_mean":90.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=KLHL3","jax_strain_url":"https://www.jax.org/strain/search?query=KLHL3"},"sequence":{"accession":"Q9UH77","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UH77.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UH77/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UH77"}},"corpus_meta":[{"pmid":"22406640","id":"PMC_22406640","title":"KLHL3 mutations cause familial hyperkalemic hypertension by impairing ion transport in the distal nephron.","date":"2012","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/22406640","citation_count":265,"is_preprint":false},{"pmid":"23453970","id":"PMC_23453970","title":"Impaired KLHL3-mediated ubiquitination of WNK4 causes human hypertension.","date":"2013","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/23453970","citation_count":181,"is_preprint":false},{"pmid":"23387299","id":"PMC_23387299","title":"The CUL3-KLHL3 E3 ligase complex mutated in Gordon's hypertension syndrome interacts with and ubiquitylates WNK isoforms: disease-causing mutations in KLHL3 and WNK4 disrupt interaction.","date":"2013","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/23387299","citation_count":180,"is_preprint":false},{"pmid":"25250572","id":"PMC_25250572","title":"Hyperkalemic hypertension-associated cullin 3 promotes WNK signaling by degrading 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KLHL3 is coexpressed with NCC in the distal nephron and downregulates NCC expression at the cell surface.\",\n      \"method\": \"Cell surface expression assay, co-expression analysis, linkage analysis and whole-exome sequencing identifying KLHL3 mutations in FHHt families\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional cell-surface assay plus genetic/genomic identification, single study but multiple approaches\",\n      \"pmids\": [\"22406640\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"KLHL3 forms a complex with CUL3 (via its BTB-BACK domains) and directly interacts with WNK4 (and WNK1), acting as the substrate adaptor that recruits WNK kinases for CUL3-dependent ubiquitination and proteasomal degradation; disease-causing mutations in KLHL3 reduce WNK4 ubiquitination and increase WNK4 protein levels.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay with recombinant CUL3-KLHL3 complex, siRNA knockdown of CUL3 in HeLa cells, domain mapping\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstituted ubiquitination assay with recombinant complex plus mutagenesis; independently replicated across multiple labs (PMIDs 23387299, 23453970, 23665031)\",\n      \"pmids\": [\"23387299\", \"23453970\", \"23665031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The KLHL3 BTB and BACK domains together form the CUL3 interaction surface; crystal structure of the KLHL3 BTB-BACK domain dimer in complex with the N-terminal fragment of CUL3 shows both domains contribute to binding, and disease mutations in the BTB-BACK region disrupt CUL3 association as measured by isothermal titration calorimetry.\",\n      \"method\": \"X-ray crystallography, isothermal titration calorimetry (ITC), mutagenesis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation by ITC and disease-mutation analysis in a single rigorous study\",\n      \"pmids\": [\"23573258\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"KLHL3 interacts with WNK4 through a non-catalytic region of WNK (residues 479-667 in WNK1, equivalent acidic motif in WNK4); Gordon's syndrome-causing mutations E562K and Q565E in WNK4 abolish the interaction with KLHL3.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, disease-mutant interaction studies\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — domain mapping with in vitro ubiquitination assay, interaction surface defined; replicated in subsequent structural studies\",\n      \"pmids\": [\"23387299\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Multiple disease-causing KLHL3 mutations in the BTB domain reduce CUL3 binding, mutations in the Kelch domain reduce WNK4 binding, and the S410L mutation in the Kelch domain also reduces KLHL3 intracellular stability; all lead to decreased WNK4 ubiquitination and elevated WNK4 levels.\",\n      \"method\": \"Co-immunoprecipitation, cycloheximide chase assay, in vitro and in vivo ubiquitination assays, HEK293T transient expression\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal biochemical methods in a single lab\",\n      \"pmids\": [\"23962426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of the KLHL3 Kelch domain in complex with the WNK4 degron motif (acidic motif) reveals an intricate network of interactions between the Kelch β-propeller surface and WNK4; disease-causing mutations in both WNK4 and KLHL3 disrupt critical interface contacts. KLHL2 binds WNK4 similarly to KLHL3 but with distinct binding mode compared to KEAP1.\",\n      \"method\": \"X-ray crystallography of KLHL3 Kelch domain–WNK4 degron complex; also crystal structure of KLHL2–WNK4 degron complex for comparison\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation linking interface contacts to disease mutations\",\n      \"pmids\": [\"24641320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"FHHt-causing CUL3 mutant (CUL3Δ403-459) is more heavily neddylated and activated than WT CUL3; in cells it depletes KLHL3 through enhanced ubiquitylation, thereby preventing WNK degradation despite increased CUL3-mediated WNK ubiquitylation.\",\n      \"method\": \"Cell-based ubiquitylation assays, neddylation analysis, nephron-specific Cul3 knockout mouse model, Western blotting\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mechanistic cell-based assays plus conditional knockout mouse model with defined phenotype; establishes KLHL3 as a substrate of hyperactive CUL3Δ403-459\",\n      \"pmids\": [\"25250572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In KLHL3(R528H/+) knock-in mice, both WNK1 and WNK4 protein levels are significantly increased in the kidney; fluorescence correlation spectroscopy confirmed that the R528H mutation abolishes binding of KLHL3 to WNK1 and WNK4 peptides, establishing WNK1 and WNK4 as in vivo substrates of KLHL3-CUL3 E3 ligase.\",\n      \"method\": \"KLHL3 R528H knock-in mouse model, fluorescence correlation spectroscopy (FCS) binding assay, Western blotting, NCC phosphorylation analysis\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo knock-in model plus direct binding assay with disease mutant; multiple orthogonal methods\",\n      \"pmids\": [\"24821705\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"KLHL3 directly binds claudin-8, a tight junction protein in the collecting duct, and promotes its ubiquitination and degradation, thereby regulating paracellular chloride transport; the dominant PHA-II mutation in KLHL3 impairs claudin-8 binding, ubiquitination, and degradation.\",\n      \"method\": \"Tissue-specific claudin-8 knockout mice, Co-immunoprecipitation of KLHL3 and claudin-8, KLHL3 knockdown in collecting duct cells measuring paracellular chloride permeability, ubiquitination assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, knockdown with functional permeability readout, and knockout mouse; single lab\",\n      \"pmids\": [\"25831548\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Akt (downstream of insulin signaling) and PKA (downstream of vasopressin/forskolin signaling) phosphorylate KLHL3 at serine 433 in vitro and in cells; this phosphorylation impairs the interaction between KLHL3 and WNK4, reducing WNK4 degradation and increasing WNK4 protein levels.\",\n      \"method\": \"In vitro kinase assay (Akt and PKA), mass spectrometry identification of phospho-S433, phospho-specific antibody, co-immunoprecipitation, cell-based WNK4 protein level measurement\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus mass spectrometry and cell-based validation; single lab\",\n      \"pmids\": [\"26435498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"KLHL3 forms a complex with both WNK4 and p62/SQSTM1 via its Kelch repeat domain; under proteasome inhibition, p62-mediated selective autophagy contributes to KLHL3-dependent WNK4 degradation, with WNK4 co-localizing with KLHL3, p62, and LC3 in cytoplasmic puncta.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, 3-methyladenine autophagy inhibition, proteasome inhibitor (epoxomicin) treatment, p62 overexpression and knockdown in HEK293T cells\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus multiple pharmacological perturbations and imaging; single lab\",\n      \"pmids\": [\"26349538\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Phosphorylation of KLHL3 at S433 disrupts hydrogen bonds, hydrophobic, and electrostatic interactions between the Kelch domain of KLHL3 and the acidic motif of WNK4 by making the binding site more negatively charged, as demonstrated by molecular dynamics simulation corroborated by prior experimental data.\",\n      \"method\": \"Molecular dynamics simulation with energetic and structural analysis (computational study validating/explaining prior experimental findings)\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Moderate — computational/simulation study; mechanistic interpretation of prior experimental results but no new direct experimental validation\",\n      \"pmids\": [\"27727489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"KLHL3 dimerizes, and the dominant-negative effect of pathogenic KLHL3 mutations (causing autosomal dominant FHHt) is explained by dimerization of mutant KLHL3 with wild-type KLHL3, impairing the function of the wild-type subunit; heterozygous KLHL3 deletion alone is insufficient to cause PHAII, whereas homozygous deletion causes PHAII with elevated WNK1 and WNK4 only in the kidney.\",\n      \"method\": \"KLHL3 knockout mouse model (KLHL3-/- and KLHL3+/-), β-galactosidase reporter for expression, Western blot of WNK kinases in multiple organs, dimerization analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo knockout mouse model with organ-specific expression analysis and mechanistic dimerization finding; single lab\",\n      \"pmids\": [\"28052936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"WNK4 is indispensable for PHAII pathogenesis caused by KLHL3 mutation; increased WNK1 protein (due to impaired KLHL3-mediated degradation) cannot compensate for WNK4 deficiency to activate SPAK/OSR1-NCC phosphorylation signaling in vivo.\",\n      \"method\": \"WNK4-/-/KLHL3R528H/+ and WNK4-/-/KLHL3R528H/R528H double-mutant mouse models, Western blot and immunofluorescence of pSPAK, pNCC in distal convoluted tubules\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via double-mutant in vivo mouse models; single lab\",\n      \"pmids\": [\"28743496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"KLHL3 ubiquitinates cardiac myosin binding protein C (cMyBP-C) via the ubiquitin-proteasome pathway; KLHL3 interacts with cMyBP-C as shown by co-immunoprecipitation and immunofluorescence, and homocysteine increases KLHL3 expression leading to decreased cMyBP-C.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, Western blotting, MG132 proteasome inhibitor rescue, Western blot (MRM approach)\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and fluorescence study; single lab, no in vitro ubiquitination reconstitution\",\n      \"pmids\": [\"28315668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"KLHL3 BTB domain mutation M131V (M78V in human) retains intact interaction with WNK kinases but reduces binding to CUL3, leading to decreased CUL3 in distal convoluted tubule cytosol and increased WNK1/WNK4 abundance in vivo.\",\n      \"method\": \"KLHL3 M131V knock-in mouse model, in vitro co-immunoprecipitation, immunogold-labeling electron microscopy, microdissected renal tubule analysis\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knock-in mouse model combined with in vitro Co-IP and electron microscopy; single lab, multiple methods\",\n      \"pmids\": [\"30148674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Deficiency of the COP9 signalosome (CSN) catalytic subunit Jab1 in the nephron causes increased neddylated CUL3 and near-complete loss of KLHL3, with consequent accumulation of WNK1, WNK4, and SPAK, and elevated NCC phosphorylation; this indicates that CSN-mediated deneddylation is required for normal KLHL3 stability and CUL3-KLHL3-WNK signaling.\",\n      \"method\": \"Nephron-specific Jab1 knockout mouse model (Pax8/LC1 system), Western blot, immunofluorescence\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional knockout mouse with multiple biochemical readouts; single lab\",\n      \"pmids\": [\"30301860\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Multiple disease-causing mutations in the Kelch domain of KLHL3 disrupt binding to the WNK4 acidic motif through two main mechanisms: altering the electrostatic potential of the binding site or disrupting Kelch-acidic motif hydrogen bonds; buried mutations can affect stability or indirect contacts. The L387P mutation causes functional impairment of WNK4 degradation by a mechanism not captured by Kelch-AM interaction simulations alone.\",\n      \"method\": \"Molecular dynamics simulations, Western blot analysis of WNK4 degradation with KLHL3 mutants\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — primarily computational with limited Western blot corroboration; single lab\",\n      \"pmids\": [\"30931564\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The unique 30-amino acid N-terminal fragment of KS-WNK1 (kidney-specific WNK1) is required for both its activating effect on NCC and its recognition by KLHL3; specific residues in this region are critical for KLHL3 sensitivity. Full-length WNK1 is less impacted by CUL3-KLHL3-mediated degradation than KS-WNK1.\",\n      \"method\": \"KLHL3-R528H knock-in mice, mutagenesis of KS-WNK1 N-terminal residues, functional NCC activation assays, Western blotting\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knock-in mouse model plus mutagenesis and functional assays; single lab\",\n      \"pmids\": [\"33682442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Crystal structure of the KLHL3 Kelch domain in complex with a WNK3 degron peptide reveals the complete 11-mer WNK-family degron binding mode; WNK3 Thr541 substitutes for the conserved proline, and phosphorylation of this residue abrogates KLHL3 interaction (shown by fluorescence polarization), revealing isoform-specific regulation.\",\n      \"method\": \"X-ray crystallography of KLHL3 Kelch-WNK3 peptide complex, fluorescence polarization binding assay, structural modelling\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with fluorescence polarization functional validation; single lab but rigorous methods\",\n      \"pmids\": [\"35179207\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"KLHL3 is recruited by KSHV vIRF1 to ubiquitinate and degrade hnRNP Q1 via the ubiquitin-proteasome pathway; vIRF1 upregulates KLHL3 expression and the KLHL3-vIRF1 complex targets hnRNP Q1, leading to destabilization of GDPD1 mRNA and induction of aerobic glycolysis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, Western blotting, metabolic assays (glucose uptake, ATP, lactate), mRNA stability assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with ubiquitination assay and functional metabolic readouts; single lab\",\n      \"pmids\": [\"35538151\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"A novel C-terminal motif (CM, residues 1051-1075) in WNK4 rich in negatively charged residues can also mediate KLHL3-dependent WNK4 degradation; this motif responds to PHAII mutations in the KLHL3 Kelch domain similarly to the acidic motif (AM), but AM is dominant; the CM may allow WNK4 degradation when AM is mutated.\",\n      \"method\": \"Co-immunoprecipitation, deletion/mutagenesis mapping in HEK293 cell-based degradation assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP/cell-based assay; single lab, no in vitro reconstitution\",\n      \"pmids\": [\"37285722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Human KLHL3 and all four WNK kinases (WNK1-4) are O-GlcNAcylated; O-GlcNAcylation affects WNK4 function in osmolarity control and ferroptosis, demonstrating functional regulation of the KLHL3/WNK axis by this post-translational modification.\",\n      \"method\": \"Glycoproteomics/mass spectrometry, biochemical assays for osmolarity control and ferroptosis, O-GlcNAc-specific detection\",\n      \"journal\": \"Glycobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — glycoproteomics plus functional assays; peer-reviewed, single lab\",\n      \"pmids\": [\"40796245\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"KLHL3 is a BTB-BACK-Kelch domain adaptor protein that bridges CUL3 (via its BTB-BACK domains) and substrates including WNK1, WNK4 (via the Kelch domain binding an acidic degron motif), KS-WNK1, claudin-8, cMyBP-C, and hnRNP Q1, assembling a CUL3-RING E3 ubiquitin ligase complex that ubiquitinates these substrates for proteasomal (and partially autophagic via p62) degradation; crystal structures define the KLHL3-CUL3 and KLHL3-WNK degron interfaces; KLHL3 activity is negatively regulated by phosphorylation at S433 by Akt, PKA, and PKC (which impairs WNK4 binding) and by O-GlcNAcylation, and its stability is maintained by CSN-mediated CUL3 deneddylation; loss-of-function mutations in KLHL3 cause familial hyperkalemic hypertension (PHAII/FHHt) by stabilizing WNK kinases and hyperactivating the WNK-SPAK/OSR1-NCC phosphorylation cascade in the distal nephron.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"KLHL3 is a BTB-BACK-Kelch substrate adaptor that assembles a CUL3-RING E3 ubiquitin ligase to target the WNK kinases for ubiquitin-dependent degradation, thereby controlling the WNK-SPAK/OSR1-NCC phosphorylation cascade in the distal nephron [#0, #1]. Its BTB and BACK domains together form the CUL3-binding surface, while its Kelch β-propeller engages an acidic degron motif in WNK1 and WNK4; crystal structures of both the KLHL3 BTB-BACK–CUL3 and Kelch–WNK degron interfaces define these contacts at atomic resolution, and disease mutations mapped onto them abolish either CUL3 association or WNK binding [#2, #3, #5]. Knock-in and knockout mouse models confirm WNK1 and WNK4 as bona fide in vivo substrates whose protein levels rise when KLHL3 binding is lost, with WNK4 being indispensable for the downstream NCC-activating phenotype [#7, #12, #13]. KLHL3-directed WNK degradation proceeds primarily through the proteasome but is also routed to p62/SQSTM1-mediated selective autophagy [#10]. The ligase is regulated at multiple levels: phosphorylation of KLHL3 at Ser433 by Akt and PKA weakens Kelch-domain binding to the WNK4 acidic motif and stabilizes WNK4 [#9], and CSN-mediated deneddylation of CUL3 is required to maintain KLHL3 stability and proper WNK signaling [#16]. Beyond the WNK axis, KLHL3 ubiquitinates additional substrates including the tight-junction protein claudin-8 to regulate paracellular chloride transport [#8] and, when co-opted by KSHV vIRF1, hnRNP Q1 to reprogram glycolytic metabolism [#20]. Loss-of-function mutations in KLHL3 cause familial hyperkalemic hypertension (PHAII/FHHt) by stabilizing WNK kinases, with dominant alleles acting through dimerization with wild-type KLHL3 [#0, #12].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Linking KLHL3 to a Cullin3-based ubiquitin system and to NCC regulation established its candidacy as the distal-nephron salt-handling factor mutated in FHHt, framing the question of which substrate it controls.\",\n      \"evidence\": \"Linkage/exome sequencing in FHHt families plus cell-surface NCC expression assays\",\n      \"pmids\": [\"22406640\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct substrate not yet identified\", \"No biochemical demonstration of ligase assembly\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Reconstitution showed KLHL3 bridges CUL3 and WNK kinases as the substrate adaptor of a working E3 ligase, directly explaining how disease mutations elevate WNK protein.\",\n      \"evidence\": \"Co-IP, in vitro ubiquitination with recombinant CUL3-KLHL3, domain mapping, and crystallography/ITC of the BTB-BACK–CUL3 interface; replicated across labs\",\n      \"pmids\": [\"23387299\", \"23453970\", \"23665031\", \"23573258\", \"23962426\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic detail of the Kelch–WNK degron contact not yet resolved\", \"In vivo substrate identity not yet confirmed\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Structural definition of the Kelch–WNK4 degron interface and in vivo knock-in models confirmed WNK1/WNK4 as physiological KLHL3 substrates and explained both KLHL3 and CUL3 disease alleles mechanistically.\",\n      \"evidence\": \"Crystal structure of Kelch–WNK4 degron complex, FCS binding with R528H mutant, and KLHL3 R528H knock-in plus nephron-specific Cul3 knockout mice\",\n      \"pmids\": [\"24641320\", \"24821705\", \"25250572\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of WNK1 vs WNK4 to phenotype unresolved\", \"Regulatory inputs onto KLHL3 not yet defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Discovery of phosphoregulation at Ser433 and an alternative autophagic degradation route, plus claudin-8 as a substrate, broadened KLHL3 from a single-substrate adaptor to a hormonally tuned, multi-target ligase.\",\n      \"evidence\": \"In vitro Akt/PKA kinase assays with MS and phospho-antibody, p62/autophagy perturbation with imaging, and claudin-8 Co-IP/knockout with permeability readouts\",\n      \"pmids\": [\"26435498\", \"26349538\", \"25831548\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo relevance of S433 phosphorylation not established\", \"Balance between proteasomal and autophagic routes unquantified\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Genetic dissection in mice established the dominant-negative dimerization mechanism of pathogenic alleles and the epistatic requirement of WNK4 for the disease phenotype.\",\n      \"evidence\": \"KLHL3 knockout/heterozygote mice with dimerization analysis and WNK4-/-/KLHL3R528H double-mutant epistasis\",\n      \"pmids\": [\"28052936\", \"28743496\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry of mutant-WT heterodimer inhibition not quantified\", \"Why WNK1 cannot compensate for WNK4 not mechanistically resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Separation-of-function mutants and CSN manipulation showed that CUL3 binding and KLHL3 stability are distinct vulnerabilities, with deneddylation required to preserve the adaptor.\",\n      \"evidence\": \"KLHL3 M131V knock-in with Co-IP and immunogold EM, and nephron-specific Jab1 (CSN) knockout mice\",\n      \"pmids\": [\"30148674\", \"30301860\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How CUL3 neddylation cycling drives KLHL3 turnover mechanistically unclear\", \"Single-lab models\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Mapping the KS-WNK1 N-terminal recognition element refined substrate selectivity, explaining why kidney-specific WNK1 is preferentially degraded over full-length WNK1.\",\n      \"evidence\": \"KLHL3-R528H knock-in mice with KS-WNK1 N-terminal mutagenesis and NCC functional assays\",\n      \"pmids\": [\"33682442\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of KS-WNK1 N-terminal recognition not solved\", \"Isoform competition for KLHL3 in vivo not quantified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"A WNK3 degron co-structure defined the complete WNK-family 11-mer binding mode and phospho-degron switching, and a viral-hijack study revealed KLHL3 functions beyond ion transport.\",\n      \"evidence\": \"Crystal structure of Kelch–WNK3 peptide with fluorescence polarization, and KSHV vIRF1-directed hnRNP Q1 ubiquitination with metabolic assays\",\n      \"pmids\": [\"35179207\", \"35538151\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological signals controlling WNK degron phosphorylation unclear\", \"Breadth of non-WNK substrate repertoire unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identification of O-GlcNAcylation on KLHL3 and all WNK kinases added a metabolic post-translational layer regulating the axis in osmolarity and ferroptosis contexts.\",\n      \"evidence\": \"Glycoproteomics/MS with functional osmolarity and ferroptosis assays\",\n      \"pmids\": [\"40796245\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific O-GlcNAc sites and their effect on ligase activity not mapped\", \"In vivo physiological consequence not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple regulatory inputs (S433 phosphorylation, O-GlcNAcylation, CUL3 neddylation, dimerization) are integrated to set KLHL3 ligase output in different tissues and physiological states remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified quantitative model of KLHL3 regulation\", \"Full non-WNK substrate landscape uncharacterized\", \"Tissue-specific substrate preference mechanisms unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 8, 14, 20]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2, 3]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [1, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [10, 15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 7]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [0, 8]}\n    ],\n    \"complexes\": [\"CUL3-RING E3 ubiquitin ligase (CRL3)\"],\n    \"partners\": [\"CUL3\", \"WNK4\", \"WNK1\", \"WNK3\", \"CLDN8\", \"SQSTM1\", \"MYBPC3\", \"SYNCRIP\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}