Affinage

KLHL20

Kelch-like protein 20 · UniProt Q9Y2M5

Length
609 aa
Mass
68.0 kDa
Annotated
2026-06-10
23 papers in source corpus 15 papers cited in narrative 15 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/7 claims corpus-supported (86%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

KLHL20 is a BTB-Kelch protein that functions as the substrate-recognition subunit of a CUL3-ROC1 RING E3 ubiquitin ligase, in which its BTB domain engages Cul3 and its Kelch β-propeller domain captures substrates (PMID:20389280). A high-resolution structure of the Kelch domain bound to a DAPK1 death-domain peptide shows substrates insert an 'LPDLV' motif deep into the central pocket of the β-propeller to contact all six blades (PMID:31279627), and KLHL20 directs both degradative (K48-linked) and non-degradative (K33-linked) polyubiquitination depending on substrate and signal (PMID:24768539, PMID:35474067). Through this activity KLHL20 controls multiple cellular programs: it terminates autophagy by ubiquitinating the autophagy-initiating kinase ULK1 and governing turnover of VPS34, Beclin-1, ATG13 and ATG14 during prolonged starvation (PMID:26687681); it promotes post-Golgi vesicle trafficking from the trans-Golgi network by K33-linked ubiquitination of coronin 7, routing it via Eps15 to drive TGN F-actin assembly (PMID:24768539); and it restrains RhoA-dependent neurite outgrowth by degrading p38-phosphorylated PDZ-RhoGEF (PMID:21670212). KLHL20 activity is regulated at the level of localization and signaling: IFN-α/γ sequesters KLHL20 in PML nuclear bodies to stabilize DAPK and license IFN-induced apoptosis and autophagy (PMID:20389280), while under hypoxia KLHL20 degrades PML in a CDK1/2- and Pin1-dependent feedback loop that amplifies HIF-1α and tumor hypoxia responses (PMID:21840486). Its substrate repertoire extends to SERINC5 (controlling plasma-membrane delivery versus degradation) (PMID:35474067) and the transcriptional repressor ZBTB7A in a p53-induced axis that derepresses p21 (PMID:37011832). KLHL39, a related BTB-Kelch protein lacking Cul3-binding residues, antagonizes the ligase by occupying the Kelch domain and blocking both substrate and Cul3 engagement (PMID:25619834). De novo missense variants clustering in the substrate-binding β-propeller surface, including a recurrent Gly357Arg, cause a neurodevelopmental syndrome (PMID:36214804).

Mechanistic history

Synthesis pass · year-by-year structured walk · 11 steps
  1. 2003 Medium

    Before any enzymatic role was known, KLHL20 was placed in actin cytoskeletal dynamics, establishing an early link to Rac1-driven actin organization at nascent cell-cell contacts.

    Evidence Co-localization microscopy, E-cadherin-bead recruitment, and dominant-negative fragment expression in MDCK cells

    PMID:14668487

    Open questions at the time
    • No molecular activity or E3 ligase role identified at this stage
    • Mechanism connecting actin binding to later ubiquitin ligase function not defined
  2. 2007 Medium

    KLHL20 was tied to RhoA signaling in endothelial migration, showing it acts upstream of RhoA activation and physically associates with the GEF ECT2 during angiogenic responses.

    Evidence siRNA knockdown, sprouting angiogenesis assay, RhoA activation assays, and Co-IP of KLHL20 with ECT2 in endothelial cells

    PMID:17395875

    Open questions at the time
    • Whether KLHL20-ECT2 interaction is ubiquitination-dependent unresolved
    • Single lab, single Co-IP for the interaction
  3. 2010 High

    The defining mechanistic advance: KLHL20 was shown to be a CUL3-ROC1 substrate adaptor that degrades DAPK, and stress-induced sequestration in PML bodies emerged as a localization switch controlling substrate access.

    Evidence Reciprocal Co-IP, ubiquitination assay, BTB/Kelch domain mapping, siRNA, and IFN-induced relocalization to PML nuclear bodies

    PMID:20389280

    Open questions at the time
    • Structural basis of DAPK recognition not yet resolved
    • Generality of PML sequestration to other substrates unknown
  4. 2011 High

    KLHL20 was connected to hypoxia and neuronal morphogenesis by identifying PML and PDZ-RhoGEF as substrates, revealing kinase-gated substrate recruitment (CDK1/2-Pin1 and p38) and a HIF-1α amplification feedback loop.

    Evidence Co-IP, ubiquitination assays, genetic epistasis, xenograft and primary neuron models with kinase inhibition

    PMID:21670212 PMID:21840486

    Open questions at the time
    • How a single adaptor coordinates context-specific substrate selection unresolved
    • Direct versus indirect degradation of some downstream factors not fully separated
  5. 2014 High

    KLHL20 was assigned a non-degradative ubiquitin function and a TGN localization, demonstrating K33-linked ubiquitination of coronin 7 to drive post-Golgi carrier biogenesis, broadening its output beyond proteolysis.

    Evidence In vitro linkage-specific ubiquitination, K33R ubiquitin mutants, Co-IP, subcellular fractionation, and enforced-targeting epistasis

    PMID:24768539

    Open questions at the time
    • How KLHL20 selects K33 versus K48 linkage at the molecular level unknown
    • Determinants of TGN recruitment of KLHL20 itself not defined
  6. 2014 Medium

    A KLHL20 knockout mouse linked it to HIF-2α stabilization and VEGF expression in lung endothelium, reinforcing a hypoxia-signaling role in vivo.

    Evidence KLEIP knockout mice, immunohistochemistry, expression analysis, and betamethasone rescue

    PMID:24785085

    Open questions at the time
    • Direct ubiquitin-mediated mechanism on HIF-2α not established
    • Relationship to the PML/HIF-1α feedback loop unclear
  7. 2015 High

    KLHL20 was established as the terminator of autophagy via ULK1 ubiquitination and turnover of multiple autophagy regulators, and a negative regulator (KLHL39) was identified that blocks the ligase by occupying its Kelch domain.

    Evidence Co-IP, ubiquitination assays, knockout mouse and diabetes models, in vitro kinase assays (ULK1); domain-mapping and rescue (KLHL39)

    PMID:25619834 PMID:26687681

    Open questions at the time
    • Which autophagy substrates are direct versus indirect not fully resolved
    • Physiological signals controlling KLHL39 expression unknown
  8. 2019 High

    A 1.1 Å crystal structure of the Kelch domain with a DAPK1 peptide defined the substrate-recognition mechanism, showing the LPDLV motif buried in the β-propeller central pocket.

    Evidence X-ray crystallography with peptide binding and mutagenesis

    PMID:31279627

    Open questions at the time
    • Whether all substrates share an LPDLV-like motif not established
    • Structure of the full CUL3-bound complex absent
  9. 2022 Medium

    Substrate scope and disease relevance expanded: KLHL20 was shown to dual-ubiquitinate SERINC5 to balance surface delivery versus degradation, and de novo Kelch-domain variants were linked to a neurodevelopmental syndrome.

    Evidence Linkage-specific ubiquitination and flow cytometry (SERINC5); patient cohort genotyping and variant mapping (neurodevelopmental syndrome)

    PMID:35474067 PMID:36214804

    Open questions at the time
    • No direct functional assay of patient variants reported
    • Which neuronal substrates underlie the disorder unidentified
  10. 2023 Medium

    KLHL20 was placed downstream of p53, mediating ZBTB7A degradation upon HSP90 inhibition and thereby derepressing p21, extending its reach into transcriptional and cell-cycle control.

    Evidence Co-IP, protein stability assays, siRNA, and HSP90 inhibitor treatment

    PMID:37011832

    Open questions at the time
    • Single lab; ubiquitination linkage on ZBTB7A not characterized
    • Direct versus indirect p21 derepression not separated
  11. 2026 Medium

    Disease-context regulation and a 'fuzzy' substrate-binding mode were probed: high-glucose suppression of KLHL20 elevates DAPK1 to drive renal tubular apoptosis, and biosensor/computational work characterized low-affinity feature-specific recognition of ZFTA.

    Evidence Co-IP, ubiquitination and half-life assays with db/db and HK-2 models (DAPK1); SPR/BLI and AlphaFold2 modeling (ZFTA)

    PMID:41637987 PMID:41762441

    Open questions at the time
    • ZFTA finding is Low confidence with no in-cell ubiquitination confirmation
    • How fuzzy binding reconciles with the defined LPDLV pocket mode unclear

Open questions

Synthesis pass · forward-looking unresolved questions
  • It remains unresolved how a single Kelch adaptor achieves context-dependent selection among its many substrates and how the choice between K33- and K48-linked chains is mechanistically encoded.
  • No unifying model for linkage-type determination
  • No structure of the assembled CUL3-KLHL20 complex with substrate
  • Patient variant consequences not functionally tested

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140096 catalytic activity, acting on a protein 6 GO:0016874 ligase activity 4 GO:0060090 molecular adaptor activity 3 GO:0008092 cytoskeletal protein binding 1
Localization
GO:0005794 Golgi apparatus 2 GO:0005829 cytosol 1
Pathway
R-HSA-392499 Metabolism of proteins 6 R-HSA-8953897 Cellular responses to stimuli 3 R-HSA-5357801 Programmed Cell Death 2 R-HSA-5653656 Vesicle-mediated transport 2 R-HSA-9612973 Autophagy 1
Partners
CUL3DAPK1ECT2KLHL39PDZ-RHOGEF (ARHGEF11)PMLSERINC5ULK1
Complex memberships
CUL3-ROC1 (Cullin-RING) E3 ubiquitin ligase

Evidence

Reading pass · 15 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2003 KLEIP (KLHL20) is an actin-binding protein that localizes transiently to cell-cell contact sites during induction of cell-cell contact (not at mature junctions), colocalizing with F-actin. Constitutively active Rac1 enhanced KLEIP and F-actin recruitment to adhesion sites, while the N-terminal half of KLEIP (lacking the actin-binding site) inhibited Rac1-induced actin assembly at contact sites, indicating KLEIP participates in Rac1-induced actin organization. Co-localization microscopy, E-cadherin-bead recruitment assay, cytochalasin B treatment, constitutively active Rac1 overexpression, dominant-negative fragment expression in MDCK cells Molecular biology of the cell Medium 14668487
2007 KLEIP (KLHL20) acts as an essential downstream regulator of VEGF- and bFGF-induced endothelial cell migration and sprouting angiogenesis. KLEIP depletion blunted VEGF-induced RhoA activation (but not ERK1/2 activation), and VEGF induced physical association of KLEIP with the guanine nucleotide exchange factor ECT2. siRNA knockdown in endothelial cells, in-gel sprouting angiogenesis assay, RhoA and ERK1/2 activation assays, co-immunoprecipitation of KLEIP with ECT2 Circulation research Medium 17395875
2010 KLHL20 forms a Cul3-ROC1 E3 ligase complex (via its BTB domain binding Cul3 and Kelch-repeat domain binding DAPK) that promotes DAPK polyubiquitination and proteasomal degradation. IFN-alpha/gamma induces sequestration of KLHL20 in PML nuclear bodies, separating it from DAPK and thereby stabilizing DAPK to enable IFN-induced apoptosis and autophagy. Co-immunoprecipitation, ubiquitination assay, siRNA depletion of KLHL20, domain-mapping experiments (Kelch vs. BTB), immunofluorescence showing KLHL20 redistribution to PML nuclear bodies upon IFN treatment The EMBO journal High 20389280
2011 KLHL20 functions as a Cul3 substrate adaptor that targets PML for proteasomal degradation through a pathway requiring CDK1/2-mediated and Pin1-mediated modifications of PML. This KLHL20-mediated PML destruction acts in a positive feedback loop to maximize HIF-1α induction under hypoxia, potentiating tumor hypoxia responses including metabolic reprogramming, EMT, migration, angiogenesis, and chemoresistance. KLHL20 expression is itself induced by HIF-1. Co-immunoprecipitation, ubiquitination assays, siRNA knockdown, dominant-negative constructs, in vivo tumor xenograft models, epistasis experiments placing CDK1/2 and Pin1 upstream of KLHL20-mediated PML degradation Cancer cell High 21840486
2011 The Cul3-KLHL20 ubiquitin ligase complex targets PDZ-RhoGEF for ubiquitin-dependent proteolysis, restricting RhoA activity and facilitating growth cone spreading and neurite outgrowth. PDZ-RhoGEF phosphorylation by p38 MAPK is required for its recruitment to KLHL20, and neurotrophins (BDNF, NT-3) activate p38 to potentiate this degradation pathway. Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, primary hippocampal/cortical neuron culture, neurite outgrowth/arborization measurements, p38 inhibition and phosphorylation analysis The Journal of cell biology High 21670212
2014 KLHL20 localizes to the trans-Golgi network (TGN) and promotes post-Golgi trafficking by catalyzing K33-linked (non-degradative) polyubiquitination of coronin 7 (Crn7) via the Cul3-KLHL20 E3 ligase. K33-ubiquitinated Crn7 is targeted to the TGN through a ubiquitin-dependent interaction with Eps15, where it promotes TGN-pool F-actin assembly required for transport carrier biogenesis. Co-immunoprecipitation, in vitro ubiquitination assay with linkage-specific analysis, siRNA knockdown, subcellular fractionation/immunofluorescence, dominant-negative ubiquitin mutants (K33R), enforced Crn7 TGN targeting as epistasis Molecular cell High 24768539
2015 KLHL39, another BTB-Kelch protein, acts as a negative regulator of Cul3-KLHL20 E3 ligase by binding to the substrate-binding (Kelch) domain of KLHL20, thereby disrupting both substrate (PML, DAPK) binding to KLHL20 and KLHL20 binding to Cul3. This dual blockade increases PML and DAPK stability. KLHL39 itself lacks Cul3-binding ability due to absent conserved BTB-domain residues and is not a KLHL20 substrate. Co-immunoprecipitation, domain-mapping, ubiquitination assays, siRNA knockdown, in vitro migration/invasion assays, in vivo metastasis model Oncogene High 25619834
2015 Cul3-KLHL20 E3 ligase ubiquitinates ULK1 (an autophagy-initiating kinase) to promote its proteasomal degradation. ULK1 autophosphorylation upon autophagy induction facilitates its recruitment to KLHL20. KLHL20 also governs degradation of ATG13, VPS34, Beclin-1, and ATG14 during prolonged starvation (directly or indirectly), thereby terminating autophagy. Impairment of this pathway potentiates starvation-induced cell death and aggravates diabetes-associated muscle atrophy. Co-immunoprecipitation, ubiquitination assays, siRNA/shRNA knockdown and knockout mouse models, in vitro kinase assays, autophagic flux assays, diabetes mouse model Molecular cell High 26687681
2014 KLEIP (KLHL20) regulates HIF-2α stabilization and transcriptional activation in lung endothelial cells. KLEIP knockout mice show strongly reduced Hif-2α and VEGF mRNA/protein levels in embryonic lungs, associated with endothelial apoptosis; betamethasone treatment rescues this by increasing Hif-2α expression. KLEIP expression is controlled by two hypoxia response elements. KLEIP knockout mouse generation, immunohistochemistry, Western blotting, mRNA expression analysis, betamethasone rescue experiment Disease models & mechanisms Medium 24785085
2019 Crystal structure (1.1 Å) of the KLHL20 Kelch domain bound to a DAPK1 death-domain peptide reveals that DAPK1 binds via an 'LPDLV' motif as a loose helical turn inserting deeply into the central pocket of the β-propeller to contact all six blades. KLHL20 engages DAPK1 through salt-bridge and hydrophobic interactions involving tryptophan and cysteine residues. X-ray crystallography at 1.1 Å resolution, peptide binding assays, mutagenesis to identify critical residues Structure High 31279627
2022 Cul3-KLHL20 (localized at the TGN) polyubiquitinates SERINC5 at lysine 130 via K33/K48-linked chains. K33-linked polyubiquitination determines SERINC5 expression on the plasma membrane (post-Golgi trafficking), while K48-linked polyubiquitination contributes to SERINC5 downregulation/degradation from the cell surface. HIV-1 Nef counteracts SERINC5 restriction independently of this pathway. Co-immunoprecipitation, ubiquitination assay with linkage-specific analysis, site-directed mutagenesis (K130R SERINC5), flow cytometry, siRNA knockdown Nature communications High 35474067
2022 De novo missense variants in KLHL20 cause a neurodevelopmental syndrome. All 14 patient variants clustered in the Kelch-type β-propeller domain (substrate-binding surface), with a recurrent variant Gly357Arg found in 11 patients, suggesting that disruption of substrate recognition by KLHL20 underlies this disorder. Patient cohort genotyping via Matchmaker Exchange, variant mapping onto known KLHL20 domain structure Genetics in medicine Medium 36214804
2023 p53 upregulates KLHL20 expression, and the resulting CUL3-KLHL20 E3 ligase complex mediates proteasomal degradation of ZBTB7A. HSP90 inhibition (by 17-AAG) triggers p53-dependent upregulation of KLHL20, leading to ZBTB7A proteolysis and derepression of p21/CDKN1A. Co-immunoprecipitation, protein stability assays, siRNA knockdown, HSP90 inhibitor treatment, Western blotting Biochimica et biophysica acta. Gene regulatory mechanisms Medium 37011832
2026 KLHL20 interacts with ZFTA (zinc finger translocation associated protein) via its Kelch domain with an estimated KD ~35 µM, exhibiting 'fuzzy binding' through feature-specific rather than sequence-specific recognition. The interaction specificity for KLHL20 vs. KLHL12 was confirmed. This mechanism reflects KLHL20's role as an adaptor for disordered substrate recognition. Biosensor (SPR/BLI) analysis, AlphaFold2-based structural modeling, truncation peptide binding experiments Chembiochem Low 41762441
2026 Under high glucose conditions, KLHL20 expression is transcriptionally suppressed, leading to reduced DAPK1 ubiquitination and elevated DAPK1 protein levels that promote mitochondrial apoptosis in renal tubular cells. KLHL20 overexpression restores DAPK1 ubiquitination and reduces protein levels (without affecting mRNA), confirming KLHL20 as an E3 adaptor for DAPK1 post-translational regulation in this context. Co-immunoprecipitation, ubiquitination assay, KLHL20 overexpression, DAPK1 knockdown, protein stability/half-life assay, db/db mouse model, HK-2 cell model Biochemical and biophysical research communications Medium 41637987

Source papers

Stage 0 corpus · 23 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2015 Cul3-KLHL20 Ubiquitin Ligase Governs the Turnover of ULK1 and VPS34 Complexes to Control Autophagy Termination. Molecular cell 188 26687681
2011 A Cullin3-KLHL20 Ubiquitin ligase-dependent pathway targets PML to potentiate HIF-1 signaling and prostate cancer progression. Cancer cell 151 21840486
2014 K33-Linked Polyubiquitination of Coronin 7 by Cul3-KLHL20 Ubiquitin E3 Ligase Regulates Protein Trafficking. Molecular cell 139 24768539
2010 The Cullin 3 substrate adaptor KLHL20 mediates DAPK ubiquitination to control interferon responses. The EMBO journal 104 20389280
2011 PDZ-RhoGEF ubiquitination by Cullin3-KLHL20 controls neurotrophin-induced neurite outgrowth. The Journal of cell biology 44 21670212
2019 Structural Basis for Recruitment of DAPK1 to the KLHL20 E3 Ligase. Structure (London, England : 1993) 33 31279627
2015 KLHL39 suppresses colon cancer metastasis by blocking KLHL20-mediated PML and DAPK ubiquitination. Oncogene 33 25619834
2003 Novel kelch-like protein, KLEIP, is involved in actin assembly at cell-cell contact sites of Madin-Darby canine kidney cells. Molecular biology of the cell 32 14668487
2007 The BTB-Kelch protein KLEIP controls endothelial migration and sprouting angiogenesis. Circulation research 29 17395875
2014 Angiopoietin-1 is regulated by miR-204 and contributes to corneal neovascularization in KLEIP-deficient mice. Investigative ophthalmology & visual science 27 24917145
2022 A synthetic KLHL20 ligand to validate CUL3KLHL20 as a potent E3 ligase for targeted protein degradation. Genes & development 19 36328355
2016 Cul3-KLHL20 ubiquitin ligase: physiological functions, stress responses, and disease implications. Cell division 19 27042198
2022 Cul3-KLHL20 E3 ubiquitin ligase plays a key role in the arms race between HIV-1 Nef and host SERINC5 restriction. Nature communications 17 35474067
2012 KLEIP deficiency in mice causes progressive corneal neovascular dystrophy. Investigative ophthalmology & visual science 15 22511632
2016 KLHL20 links the ubiquitin-proteasome system to autophagy termination. Autophagy 13 26985984
2014 Kelch-like ECT2-interacting protein KLEIP regulates late-stage pulmonary maturation via Hif-2α in mice. Disease models & mechanisms 11 24785085
2022 De novo missense variants in the E3 ubiquitin ligase adaptor KLHL20 cause a developmental disorder with intellectual disability, epilepsy, and autism spectrum disorder. Genetics in medicine : official journal of the American College of Medical Genetics 7 36214804
2024 KLHL20 and its role in cell homeostasis: A new perspective and therapeutic potential. Life sciences 3 39233199
2023 Post-translational regulation of proto-oncogene ZBTB7A expression by p53 status in cancer cells: HSP90-dependent stabilization vs. p53-KLHL20-ubiquitin proteasomal degradation. Biochimica et biophysica acta. Gene regulatory mechanisms 3 37011832
2025 Temporal and Spatial Characterization of CUL3KLHL20-Driven Targeted Degradation of BET Family BRD Proteins by the Macrocycle-Based Degrader BTR2004. ACS chemical biology 1 40891966
2024 Temporal and Spatial Characterization of CUL3KLHL20-driven Targeted Degradation of BET family, BRD Proteins by the Macrocycle-based Degrader BTR2004. bioRxiv : the preprint server for biology 1 39677683
2026 KLHL20 alleviates high glucose-induced mitochondrial apoptosis in renal tubular cells by targeting DAPK1 for ubiquitination and degradation. Biochemical and biophysical research communications 0 41637987
2026 Identification of ZFTA as a Novel KLHL20 Substrate and Mechanistic Insights Into Fuzzy Binding of Disordered Peptides via Biosensor Analysis and Computational Modelling. Chembiochem : a European journal of chemical biology 0 41762441

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