NEURL4

NEURL4 is a multifunctional centrosomal and mitochondrial protein that acts as both an adaptor in ubiquitin-dependent signaling and an intrinsic ADP-ribosyltransferase. At the centrosome, NEURL4 localizes preferentially to daughter/procentrioles via its Neuralized homology repeat, where it bridges CP110 to the HERC2 ubiquitin ligase to promote CP110 ubiquitylation and degradation; this prevents ectopic microtubule organizing centers, maintains centrosome integrity, and licenses primary cilia formation (PMID:22261722, PMID:22441691, PMID:37681926). Beyond centrosome biology, NEURL4 participates in HERC2-dependent regulation of p53 oligomerization and transcriptional activity (PMID:28977907, PMID:31665549), links LRRK2 to HERC2 for endosomal recycling of the Notch ligand Dll1 (PMID:26355680), and functions with RNF8 and HERC2 in a cytoplasmic ubiquitin-signaling network that restricts synapse formation in cerebellar neurons (PMID:29097665). In mitochondria, NEURL4 is the principal ADP-ribosyltransferase, poly-ADP-ribosylating mtLIG3 to regulate mitochondrial DNA base excision repair and genome integrity (PMID:35157000).

1. 2012 High

   Establishing that NEURL4 is a centrosomal protein that bridges CP110 and HERC2 resolved how CP110 levels are controlled at centrioles and linked NEURL4 to centrosome homeostasis.

   Evidence Interaction proteomics (AP-MS), high-resolution imaging, RNAi with rescue, ubiquitylation assays in human cells; replicated across two independent labs

   PMID:22261722 PMID:22441691

   Open questions at the time

   • The ubiquitin chain type deposited on CP110 was not determined
   • Whether NEURL4 has catalytic activity independent of its adaptor function was unknown
   • Structural basis of NEURL4–CP110 and NEURL4–HERC2 interactions unresolved
2. 2012 Medium

   Identification of a high-molecular-weight complex containing HERC2, NEURL4, MAPK6, and E6AP expanded the HERC2–NEURL4 interactome beyond centriolar substrates.

   Evidence AP-MS with co-IP validation in human cells

   PMID:22645313

   Open questions at the time

   • Functional significance of MAPK6 and E6AP in the complex was not tested
   • Stoichiometry and assembly hierarchy of the complex remain undefined
3. 2015 Medium

   Discovery that LRRK2 binds NEURL4 to regulate endosomal recycling of the Notch ligand Dll1 revealed a non-centrosomal signaling role for the NEURL4–HERC2 axis in Notch pathway modulation.

   Evidence Reciprocal co-IP, domain mapping, RNAi epistasis with Notch readouts, endosomal trafficking assays

   PMID:26355680

   Open questions at the time

   • Whether HERC2 ubiquitin ligase activity is required for Dll1 recycling was not tested
   • In vivo relevance in mammalian tissues not demonstrated
   • Mechanism by which NEURL4 promotes Dll1 recycling versus degradation unclear
4. 2015 Medium

   Genetic epistasis in Drosophila confirmed evolutionary conservation of NEURL4 function upstream of CP110 at centrioles and extended the phenotype to primordial germ cell morphology.

   Evidence Drosophila loss-of-function mutant analysis, double-mutant suppression, immunofluorescence

   PMID:26116656

   Open questions at the time

   • Direct ubiquitylation of Drosophila CP110 by the Neurl4-dependent pathway was not shown
   • Whether the germ cell phenotype reflects a general centriole defect or a germ cell–specific role is unclear
5. 2017 Medium

   Demonstrating that NEURL4 modulates p53 oligomerization and transcriptional activity in concert with HERC2 established a nuclear signaling function for NEURL4 independent of centrosome maintenance.

   Evidence Co-IP with domain mapping, p53 reporter assays, RNAi knockdown and overexpression, oligomerization and clonogenic assays

   PMID:28977907

   Open questions at the time

   • Whether NEURL4 affects p53 oligomerization directly or through a cofactor is unclear
   • No in vivo tumor suppression or cancer model data presented
   • Structural details of the NEURL4–p53 interface unresolved
6. 2017 Medium

   Identification of NEURL4 as a component of the cytoplasmic RNF8–HERC2 ubiquitin-signaling complex that restrains synaptogenesis revealed a neuronal function for NEURL4.

   Evidence Proteomics, in vivo RNAi in cerebellar granule neurons, synapse counting, conditional knockout

   PMID:29097665

   Open questions at the time

   • The direct ubiquitylation substrate in this synapse-regulatory pathway was not identified
   • Whether NEURL4 depletion phenotype in neurons is HERC2 ligase-dependent was not formally tested
7. 2019 Medium

   Elucidation of how DNA damage dissociates MDM2 from the p53/HERC2/NEURL4 complex refined the mechanistic model of NEURL4-dependent p53 regulation by linking it to the DNA damage response.

   Evidence Co-IP of endogenous complexes, promoter reporter assays, p53 post-translational modification analysis

   PMID:31665549

   Open questions at the time

   • Whether NEURL4 is required for the dissociation event or is a passive scaffold is unclear
   • Relevance of this mechanism to physiological DNA damage in vivo not tested
8. 2022 High

   Discovery that NEURL4 is the principal mitochondrial ADP-ribosyltransferase, poly-ADP-ribosylating mtLIG3 to regulate mtDNA integrity, fundamentally redefined NEURL4 as a catalytic enzyme beyond its adaptor roles.

   Evidence ART activity assays in mitochondrial extracts from NEURL4 knockout cells, ADP-ribosylome mass spectrometry, mtDNA integrity assays

   PMID:35157000

   Open questions at the time

   • Catalytic domain and active site residues responsible for ART activity were not structurally defined
   • Relationship between centrosomal adaptor function and mitochondrial ART activity is unclear
   • Whether mtLIG3 poly-ADP-ribosylation activates or inhibits its repair function requires kinetic analysis
9. 2023 Medium

   Showing that NEURL4 overexpression alone suffices to drive primary cilia formation via CP110 removal, with ODF2 acting as the centriolar recruitment scaffold, completed the mechanistic pathway from NEURL4 recruitment to cilia biogenesis.

   Evidence Rapamycin-induced protein dimerization, RNAi of ODF2, CP110 quantification, cilia formation assay

   PMID:37681926

   Open questions at the time

   • Direct physical interaction between ODF2 and NEURL4 was not demonstrated biochemically
   • Whether this pathway operates in multiciliated cells or is restricted to primary cilia is unknown

• How NEURL4 partitions between centrosomal adaptor and mitochondrial enzymatic functions — whether through distinct pools, isoforms, or cell-cycle regulation — remains unresolved.
• No structural model of NEURL4 ADP-ribosyltransferase domain exists
• Mechanism directing NEURL4 to mitochondria versus centrosomes is unknown
• No Mendelian disease or in vivo mammalian knockout phenotype has been reported