Affinage

ZNRF1

E3 ubiquitin-protein ligase ZNRF1 · UniProt Q8ND25

Length
227 aa
Mass
23.8 kDa
Annotated
2026-06-11
17 papers in source corpus 14 papers cited in narrative 14 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

ZNRF1 is an N-myristoylated RING-domain E3 ubiquitin ligase that operates on intracellular membranes to control receptor trafficking, kinase stability, and membrane-fusion events across neuronal and immune cells (PMID:22797923, PMID:22057101). Membrane targeting via N-myristoylation positions ZNRF1 to engage substrates through its UBZ domain while its RING domain recruits the E2 enzyme Ube2N (Ubc13/Uev1a) with unusually high affinity (Kd ~50 nM) to assemble Lys63-linked ubiquitin chains (PMID:22797923, PMID:29626159). A recurring activation mode involves phosphorylation at tyrosine 103 — by EGFR downstream of NADPH-oxidase-dependent oxidative stress, and by c-Src downstream of TLR3 engagement — which licenses its ligase activity (PMID:26572622, PMID:37158982). Through this activity ZNRF1 drives degradation of AKT, releasing GSK3B to phosphorylate CRMP2 and execute Wallerian axonal degeneration (PMID:22057101, PMID:26572622), a substrate axis also exploited downstream of the tumor suppressor LZTFL1 and to remodel the axon initial segment and Nav1.2 surface localization (PMID:36966254, PMID:40331626). ZNRF1 additionally ubiquitinates membrane and trafficking substrates to govern protein fate: caveolin-1 to tune TLR4 inflammatory cytokine output (PMID:28593998), EGFR at lysines distinct from those used by CBL to drive endosome-to-lysosome sorting and limit HSV-1 entry (PMID:33996800), and TLR3 at K813 to route it into multivesicular bodies and terminate interferon signaling (PMID:37158982). In macrophages its catalytic activity (requiring C184) is required for terminal trafficking and surface exposure of FasL through a Munc18-2–Syntaxin-3 docking axis (PMID:41896526), and the conserved ZNRF1/2 module regulates autophagosome–lysosome fusion via ubiquitination of HOPS complex subunits (PMID:38360932).

Mechanistic history

Synthesis pass · year-by-year structured walk · 14 steps
  1. 2009 Medium

    Establishing a physical partner and a structural requirement, this work showed ZNRF1 binds beta-tubulin and that both RING and zinc-finger domains are needed to drive neurite-like morphological change, hinting at a cytoskeletal/neuronal role.

    Evidence Yeast two-hybrid, Co-IP, colocalization, and domain-deletion analysis

    PMID:19737534

    Open questions at the time
    • No ubiquitination substrate identified
    • Tubb2 not shown to be a ubiquitination target
    • Functional consequence limited to morphology
  2. 2011 High

    This defined ZNRF1's first substrate-level mechanism: it degrades AKT via the proteasome to derepress GSK3B and drive CRMP2 phosphorylation, establishing a linear pathway for Wallerian axonal degeneration.

    Evidence Overexpression/knockdown in neurons, AKT rescue, GSK3B/CRMP2 epistasis, pharmacological GSK3B inhibition

    PMID:22057101

    Open questions at the time
    • Mechanism of substrate recognition of AKT not defined
    • Activation trigger for ZNRF1 not yet identified
    • Ubiquitin chain linkage not specified
  3. 2012 High

    Biochemical dissection established the catalytic and membrane-targeting machinery: N-myristoylation localizes ZNRF1 to membranes, the UBZ domain docks substrate (Na+/K+ATPase a1), and the RING engages Ubc13/Uev1a to build K63 chains.

    Evidence N-myristoylation assays, domain-mapping Co-IP, in vitro K63 ubiquitination, knockdown with surface biotinylation

    PMID:22797923

    Open questions at the time
    • Functional consequence of Na+/K+ATPase ubiquitination on activity not fully resolved
    • ZNRF1 vs ZNRF2 division of labor unclear
  4. 2015 High

    This identified the activating signal for ZNRF1: oxidative stress drives EGFR-dependent phosphorylation at Y103, coupling NADPH oxidase signaling to AKT degradation and neuronal death.

    Evidence Y103 mutagenesis, EGFR and NADPH oxidase inhibition, ubiquitin-proteasome assays, degeneration readouts

    PMID:26572622

    Open questions at the time
    • How Y103 phosphorylation alters ligase activity structurally not defined
    • Whether EGFR directly phosphorylates ZNRF1 not resolved
  5. 2017 High

    Extending ZNRF1 into innate immunity, this showed ZNRF1 ubiquitinates and degrades caveolin-1 downstream of TLR4 to amplify pro-inflammatory cytokine output via AKT-GSK3B.

    Evidence Reciprocal Co-IP, ubiquitination assays, hematopoietic ZNRF1 knockout mice, endotoxic shock model

    PMID:28593998

    Open questions at the time
    • Ubiquitin linkage type on CAV1 stated only as K-linked
    • Site of CAV1 ubiquitination not mapped
  6. 2018 High

    Structural work explained ZNRF1's E2 selectivity, showing an unusually tight RING–Ube2N interface (Kd ~50 nM vs ~1 uM for Ube2D2) and a concentration-dependent autoinhibition by excess ZNRF1.

    Evidence Crystal structure, ITC, site-directed mutagenesis, in vitro ubiquitination

    PMID:29626159

    Open questions at the time
    • Physiological relevance of autoinhibition not tested in cells
    • Full-length ZNRF1 structure not determined
  7. 2021 High

    This established ZNRF1 as a trafficking regulator distinct from CBL, ubiquitinating EGFR at unique lysines to drive endosome-to-lysosome sorting; its loss prolongs signaling and increases HSV-1 entry.

    Evidence ZNRF1 knockout cells, lysine-site mapping vs CBL, endosomal trafficking and degradation kinetics, HSV-1 infection assay

    PMID:33996800

    Open questions at the time
    • Specific EGFR lysines targeted not fully enumerated
    • Coordination with ESCRT machinery not defined
  8. 2023 High

    This unified the activation logic with a new substrate: TLR3 engagement activates c-Src to phosphorylate ZNRF1 at Y103, enabling K63-ubiquitination of TLR3 at K813 and its MVB/lysosomal degradation to terminate interferon signaling.

    Evidence ZNRF1 knockout mice, dual site mapping (Y103, TLR3 K813), trafficking assays, SARS-CoV-2 and EMCV infection models

    PMID:37158982

    Open questions at the time
    • Whether the same Y103-phospho mechanism applies to all substrates not tested
    • Direct c-Src–ZNRF1 contact not structurally defined
  9. 2023 Medium

    This placed ZNRF1 in a tumor-suppressive axis, showing LZTFL1 destabilizes AKT through ZNRF1 to limit kidney tumor cell proliferation.

    Evidence Gain/loss of function in kidney tumor lines, PDX model, AKT stability assays

    PMID:36966254

    Open questions at the time
    • LZTFL1-ZNRF1 physical interaction mechanism not detailed
    • Whether LZTFL1 acts as adaptor or activator unclear
  10. 2024 Medium

    Comparative genetics implicated the ZNRF1/2 module in autophagy, showing the Drosophila ortholog ubiquitinates HOPS subunits and regulates autophagosome–lysosome fusion, with mammalian knockdown altering autophagic flux.

    Evidence Drosophila detour genetics, MS interactome, HOPS subunit ubiquitination, mammalian knockdown autophagy flux

    PMID:38360932

    Open questions at the time
    • Mammalian data are knockdown only
    • Functional consequence of HOPS ubiquitination on fusion not directly shown in mammals
  11. 2024 Low

    A renal study reported ZNRF1 interacts with lipocalin-2 and preserves mitochondrial integrity and limits fibrosis, extending ZNRF1 to iron-handling and ferroptosis contexts.

    Evidence Co-IP (ZNRF1-LCN2), TFAM knockout mice, ferroptosis assays, ZNRF1 overexpression in CKD model

    PMID:39349116

    Open questions at the time
    • Single Co-IP without reciprocal validation
    • No link between ZNRF1 E3 activity and LCN2 fate established
    • Mechanism of mitochondrial protection unknown
  12. 2025 Medium

    This connected ZNRF1's AKT-degradation activity to neuronal excitability, showing it induces axon initial segment shift and Nav1.2 surface relocalization, with knockout mice showing enhanced fear memory.

    Evidence ZNRF1 knockout mice, AIS morphology, Nav1.2 surface localization, fear conditioning

    PMID:40331626

    Open questions at the time
    • Molecular link between AKT degradation and AIS remodeling not defined
    • Whether Nav1.2 is a direct trafficking target unclear
  13. 2025 Medium

    This showed myeloid ZNRF1 suppresses macrophage MHC class II surface expression after EAE, dampening T-cell-driven neuroinflammation.

    Evidence Myeloid-specific ZNRF1 knockout mice, EAE model, flow cytometry for MHC-II and T cell polarization

    PMID:41126242

    Open questions at the time
    • Direct ubiquitination substrate mediating MHC-II regulation not identified
    • Whether effect is trafficking- or stability-based unclear
  14. 2026 High

    This defined a catalytic-activity-dependent role in immune effector delivery, showing ZNRF1 (requiring C184) is needed for FasL surface exposure in macrophages via a Munc18-2–Syntaxin-3 docking axis controlling lysosome-related organelle fusion.

    Evidence Myeloid ZNRF1 knockout mice, C184A catalytic rescue, Stxbp2-Stx3 Co-IP, LAMP1/FasL imaging, FasL flow cytometry, T cell killing assay

    PMID:41896526

    Open questions at the time
    • Direct ubiquitination substrate within the SNARE/SM machinery not identified
    • Whether Munc18-2 or Stx3 is the ubiquitinated target unresolved

Open questions

Synthesis pass · forward-looking unresolved questions
  • How a single membrane-anchored E3 selects among its diverse substrates (AKT, CAV1, EGFR, TLR3, Na+/K+ATPase) and whether Y103 phosphorylation is the universal switch governing this selectivity remains unresolved.
  • No unified substrate-recognition model across the substrate repertoire
  • Substrate-specific adaptor proteins largely uncharacterized
  • Linkage between activation state and substrate choice not defined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140096 catalytic activity, acting on a protein 5 GO:0016874 ligase activity 4 GO:0031386 protein tag activity 3
Localization
GO:0005764 lysosome 3 GO:0005768 endosome 2 GO:0005886 plasma membrane 1
Pathway
R-HSA-392499 Metabolism of proteins 5 R-HSA-168256 Immune System 4 R-HSA-112316 Neuronal System 3 R-HSA-5653656 Vesicle-mediated transport 3 R-HSA-9612973 Autophagy 1
Partners
ATP1A1CAV1EGFRSTX3STXBP2TLR3TUBB2UBE2N

Evidence

Reading pass · 14 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2011 ZNRF1, an E3 ubiquitin ligase, promotes Wallerian degeneration by targeting AKT for degradation via the ubiquitin-proteasome system. AKT degradation releases its inhibitory phosphorylation of GSK3B, allowing active GSK3B to phosphorylate CRMP2, which is required for microtubule reorganization in degenerating axons. Overexpression and knockdown experiments in neurons, AKT overexpression rescue assay, active GSK3B overexpression, CRMP2 phosphorylation assays, pharmacological GSK3B inhibition Nature cell biology High 22057101
2015 Oxidative stress activates ZNRF1 E3 ligase activity through EGFR-mediated phosphorylation at tyrosine 103. NADPH oxidase activity is required for EGFR-dependent phosphorylation of ZNRF1, which then drives AKT degradation via the ubiquitin-proteasome system, leading to neuronal apoptosis and Wallerian degeneration. Phosphorylation site mutagenesis (Y103), EGFR inhibition, NADPH oxidase inhibition, ubiquitin-proteasome assays, neuronal degeneration readouts The Journal of cell biology High 26572622
2017 ZNRF1 physically interacts with caveolin-1 (CAV1) in response to LPS and mediates K-linked ubiquitination and proteasomal degradation of CAV1. This ZNRF1-CAV1 axis regulates AKT-GSK3β activity downstream of TLR4 activation to enhance pro-inflammatory cytokine production and inhibit IL-10. Co-immunoprecipitation, ubiquitination assays, ZNRF1 knockout mice (hematopoietic deletion), cytokine measurements, endotoxic shock model Nature communications High 28593998
2012 ZNRF1 and ZNRF2 are N-myristoylated, localizing them to intracellular membranes where they interact with the Na+/K+ATPase α1 subunit via their UBZ domains. Their RING domains interact with E2 Ubc13/Uev1a to mediate Lys63-linked ubiquitination of the cytoplasmic loop of Na+/K+ATPase α1. Ouabain decreases ZNRF1 protein levels; ZNRF2 knockdown inhibits ouabain-induced decrease of Na+/K+ATPase surface levels. N-myristoylation assays, Co-immunoprecipitation (UBZ-Na+/K+ATPase interaction), in vitro ubiquitination assays with Lys63-linkage analysis, ZNRF2 knockdown, cell surface biotinylation Journal of cell science High 22797923
2018 The crystal structure of ZNRF1 C-terminal domain in complex with Ube2N reveals an unusually high-affinity interaction (Kd ~50 nM) between the ZNRF1 RING domain and Ube2N, compared to ~1 µM for Ube2D2. Mutational analyses confirmed the molecular basis of this affinity. Excess ZNRF1 (≥500 nM) inhibits Ube2N-mediated ubiquitination, suggesting a concentration-dependent autoregulatory mechanism. Crystal structure determination, isothermal titration calorimetry (Kd measurement), site-directed mutagenesis, in vitro ubiquitination assays The Biochemical journal High 29626159
2021 ZNRF1 mediates ligand-induced EGFR ubiquitination at distinct lysine residues from those targeted by CBL. ZNRF1 deletion inhibits endosome-to-lysosome sorting of EGFR, resulting in delayed receptor degradation and prolonged downstream signaling. Loss of ZNRF1 increases susceptibility to HSV-1 infection due to enhanced EGFR-dependent viral entry. ZNRF1 knockout cells, EGFR ubiquitination assays, endosomal trafficking assays, receptor degradation kinetics, lysine-site mapping comparing ZNRF1 vs. CBL, HSV-1 infection assay Frontiers in cell and developmental biology High 33996800
2009 ZNRF1 interacts with beta-tubulin type 2 (Tubb2) identified by yeast two-hybrid screening and confirmed by in vivo co-immunoprecipitation. ZNRF1 colocalizes with Tubb2, and both the RING finger domain and zinc finger domain are required for ZNRF1-induced morphological changes (neurite-like elongation). Yeast two-hybrid screening, in vivo co-immunoprecipitation, immunofluorescence colocalization, domain deletion analysis Biochemical and biophysical research communications Medium 19737534
2023 c-Src kinase, activated by TLR3 engagement, phosphorylates ZNRF1 at tyrosine 103, enabling ZNRF1 to mediate K63-linked ubiquitination of TLR3 at lysine 813, which promotes TLR3 sorting into multivesicular bodies/lysosomes and its degradation, terminating type I interferon signaling. ZNRF1 knockout mice and cells, phosphorylation site mapping (Y103), ubiquitination site mapping (K813 of TLR3), TLR3 lysosomal trafficking assays, SARS-CoV-2 and EMCV infection models The Journal of experimental medicine High 37158982
2024 In Drosophila, the ZNRF1/2 homologue detour interacts with HOPS complex subunits (VPS18/dor, VPS16A, VPS41/lt) and promotes their ubiquitination, regulating autophagosome-lysosome fusion. In mammalian cells, ablation of ZNRF1 or ZNRF2 increased basal autophagy; overexpression increased autophagic vesicle size. Drosophila genetic model (detour depletion/overexpression), mass spectrometry interactome, ubiquitination assays of HOPS subunits, mammalian ZNRF1/2 knockdown with autophagy flux assays Communications biology Medium 38360932
2023 LZTFL1 tumor suppressor inhibits kidney tumor cell proliferation by destabilizing AKT through a ZNRF1-mediated ubiquitin-proteasome pathway, placing LZTFL1 upstream of ZNRF1 in AKT regulation. Gain- and loss-of-function studies in kidney tumor cell lines, patient-derived xenograft model, AKT protein stability assays with ZNRF1 Oncogene Medium 36966254
2024 ZNRF1 interacts with lipocalin-2 (LCN2), an iron transport-related protein, and this interaction is disrupted by TFAM deficiency or ferroptosis. Overexpression of ZNRF1 maintains mitochondrial integrity and inhibits renal fibrosis. Co-immunoprecipitation (ZNRF1-LCN2 interaction), TFAM knockout mice, ferroptosis assays, ZNRF1 overexpression in CKD mouse model European journal of pharmacology Low 39349116
2025 ZNRF1-dependent AKT degradation in neurons induces axon initial segment (AIS) shift and increases cell surface localization of voltage-gated sodium channel Nav1.2. ZNRF1 knockout mice exhibit enhanced short-term and contextual fear memory, linking ZNRF1-mediated AKT degradation to AIS plasticity and behavior. ZNRF1 knockout mice, AIS morphology analysis, Nav1.2 surface localization (fractionation/imaging), fear conditioning behavioral assays Journal of biochemistry Medium 40331626
2025 ZNRF1 in peripheral myeloid cells suppresses MHC class II surface expression on macrophages following EAE induction, thereby limiting antigen-specific T cell proliferation, Th1/Th17 polarization, and neuroinflammation. Myeloid-specific ZNRF1 knockout mice, EAE model, flow cytometry for MHC-II surface expression and T cell polarization, CNS immune cell infiltration analysis Journal of neuroinflammation Medium 41126242
2026 ZNRF1 E3 ligase activity (requiring catalytically active C184) is required for terminal trafficking and surface exposure of FasL in macrophages. ZNRF1 deficiency weakens the Munc18-2 (Stxbp2)-Syntaxin-3 (Stx3) interaction; reconstitution with wild-type but not catalytically inactive ZNRF1 restores surface FasL, defining a ZNRF1-Munc18-2-Stx3 axis for lysosome-related organelle fusion. Myeloid-specific ZNRF1 knockout mice, confocal imaging of LAMP1 and FasL localization, biochemical co-immunoprecipitation (Stxbp2-Stx3 interaction), catalytic mutant (C184A) reconstitution, FasL surface flow cytometry, T cell killing assay Cell death & disease High 41896526

Source papers

Stage 0 corpus · 17 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2011 ZNRF1 promotes Wallerian degeneration by degrading AKT to induce GSK3B-dependent CRMP2 phosphorylation. Nature cell biology 132 22057101
2017 The ubiquitin ligase ZNRF1 promotes caveolin-1 ubiquitination and degradation to modulate inflammation. Nature communications 53 28593998
2015 Oxidative stress-dependent phosphorylation activates ZNRF1 to induce neuronal/axonal degeneration. The Journal of cell biology 46 26572622
2012 ZNRF2 is released from membranes by growth factors and, together with ZNRF1, regulates the Na+/K+ATPase. Journal of cell science 26 22797923
2021 ZNRF1 Mediates Epidermal Growth Factor Receptor Ubiquitination to Control Receptor Lysosomal Trafficking and Degradation. Frontiers in cell and developmental biology 24 33996800
2009 ZNRF1 interacts with tubulin and regulates cell morphogenesis. Biochemical and biophysical research communications 14 19737534
2016 NADPH oxidases promote apoptosis by activating ZNRF1 ubiquitin ligase in neurons treated with an exogenously applied oxidant. Communicative & integrative biology 13 27195063
2018 Structural insights into the nanomolar affinity of RING E3 ligase ZNRF1 for Ube2N and its functional implications. The Biochemical journal 11 29626159
2018 Regulation of neuronal/axonal degeneration by ZNRF1 ubiquitin ligase. Neuroscience research 11 30118738
2024 The Drosophila ZNRF1/2 homologue, detour, interacts with HOPS complex and regulates autophagy. Communications biology 6 38360932
2023 The Src-ZNRF1 axis controls TLR3 trafficking and interferon responses to limit lung barrier damage. The Journal of experimental medicine 6 37158982
2024 Myricanol represses renal fibrosis by activating TFAM and ZNRF1 to inhibit tubular epithelial cells ferroptosis. European journal of pharmacology 4 39349116
2023 LZTFL1 inhibits kidney tumor cell growth by destabilizing AKT through ZNRF1-mediated ubiquitin proteosome pathway. Oncogene 4 36966254
2025 The essential role of E3 ubiquitin ligases in the pathogenesis of neurodevelopmental and psychiatric disorders: Cul3, Cul4, Ube3a, and ZNRF1. Biochemical and biophysical research communications 1 40233431
2025 Myeloid ZNRF1 suppresses autoimmune demyelination and neuroinflammation by regulating MHC-II-mediated T cell activation. Journal of neuroinflammation 1 41126242
2026 ZNRF1 deficiency disrupts Fas ligand trafficking and immune balance. Cell death & disease 0 41896526
2025 ZNRF1-dependent regulation of AKT activity modulates Nav subcellular localization and AIS position in neurons to regulate fear-related behaviour. Journal of biochemistry 0 40331626

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