The Kelch 3 motif on gigaxonin mediates the interaction with NUDCD3 and regulates vimentin filament morphology

Abstract

Gigaxonin is an intermediate filament (IF)-interacting partner belonging to the Kelch-like (KLHL) protein family. Gigaxonin is encoded by the KLHL16 gene, which is mutated in Giant Axonal Neuropathy (GAN). The lack of functional gigaxonin in GAN patient cells impairs IF proteostasis, leading to focal abnormal accumulations of IFs and compromised neuronal function. We hypothesized that gigaxonin forms molecular interactions via specific sequence motifs to regulate IF proteostasis. The goal of this study was to examine how distinct Kelch motifs on gigaxonin regulate IF protein degradation and filament morphology. We analyzed vimentin IFs in HEK293 cells overexpressing wild type (WT) gigaxonin, or gigaxonin lacking each of the six individual Kelch motifs: K1 (aa274-326), K2 (aa327-374), K3 (aa376-421), K4 (aa422-468), K5 (aa470-522), and K6 (aa528-574). All six gigaxonin deletion mutants (ΔK1-ΔK6) promoted the degradation of soluble vimentin. The ΔK3 gigaxonin mutant exhibited soluble vimentin degradation and promoted the bundling of vimentin IFs relative to WT gigaxonin. Using mass spectrometry proteomic analysis we found that, relative to WT gigaxonin, ΔK3 gigaxonin had increased associations with ubiquitination-associated and mitochondrial proteins and lost the association with the NudC domain-containing protein 3 (NUDCD3), a molecular chaperone enriched in the nervous system. Collectively, our cell biological data show the induction of an abnormal GAN-like IF phenotype in cells expressing ΔK3-gigaxonin, while our mass spectrometry profiling links the loss of gigaxonin-NUDCD3 interactions with defective IF proteostasis, revealing NUDCD3 as a potential new target in GAN.

Figure 1.. Pathogenicity of GAN missense variants across the gigaxonin protein domains.

A. Missense mutations are the most common GAN variants; based on a genetic analysis of the GAN natural history study cohort in Bharucha-Goebel et al., Brain 2021. B. AlphaMissense scores on known disease-causing GAN variants from the natural history study, as represented by box-and-whisker plot (n=22; median score=0.9850). C. Missense GAN variants from ClinVar plotted by their respective cDNA location (x-axis) and AlphaMissense pathogenicity score (y-axis). Black dots represent known disease-causing GAN variants; from Bharucha-Goebel et al., Brain 2021 and Lescouzeres & Bomont, Front Physiol 2020. Blue and red dots represent variants of unknown significance (VUS), where blue is predicted benign and red is predicted pathogenic. Yellow boxes represent the gigaxonin functional domains BTB, BACK, and Kelch, as defined in UniProt (ID: Q9H2C0). D. Frequency distribution plot of AlphaMissense pathogenicity scores of gigaxonin VUS, according to the protein domain affected: BTB (green), BACK (blue), KELCH (magenta).

Figure 2.. Functional validation of GFP-tagged wild type gigaxonin.

A. Schematic representation of the gigaxonin domains and the location of the turbo-GFP tag at the C-terminus. B. Immunoblot for wild type GFP-tagged and untagged gigaxonin and the effect of their overexpression on TritonX-soluble vimentin levels in HEK293 cells; actin serves as a loading control. C. Quantification of the vimentin immunoblots from panel B. *p<0.05; one-way ANOVA. D. Overexpression of wild type GFP-gigaxonin (green) in U251 glioma cells and the effect on GFAP (red) clearance. ***p<0.001; unpaired t-test.

Figure 3.. Effect of WT and Kelch deletion mutant gigaxonin expression on soluble vimentin.

A. AlphaFold model of gigaxonin showing the predicted beta propeller structure and location of each Kelch motif that compose the gigaxonin Kelch domain. B. Immunoblot of GFP-gigaxonin and endogenous gigaxonin in cells expressing WT or Kelch deletion mutants. C. Immunofluorescence analysis of GFP-tagged WT and Kelch deletion mutants of gigaxonin. D. Immunoblot of TritonX-soluble vimentin in HEK293 cells transfected with WT and Kelch deletion mutants of gigaxonin. E. Quantitative vimentin ELISA of soluble vimentin. **p<0.01; one-way ANOVA compared to WT-Gig group.

Figure 4.. Vimentin filament morphology in HEK293 cells expressing WT and Kelch deletion mutants of gigaxonin.

Immunofluorescence analysis of GFP-gigaxonin (green), vimentin (magenta), and DAPI (cyan) in HEK293 cells transfected with GFP-tagged WT gigaxonin (Gig) and Kelch motif deletion mutants (ΔK1-K6); Lipofectamine-only condition serves as transfection control. Scale bars=20μm.

Figure 5.. Effect of gigaxonin Kelch 3 deletion on vimentin filament morphology and gigaxonin protein interactions.

A-C. Immunofluorescence analysis of GFP-gigaxonin (green), vimentin (magenta), and DAPI (cyan) in HEK293 cells treated with (A) Lipofectamine only or transfected with (B) WT or (C) Kelch 3 deletion mutant (ΔK3) GFP-gigaxonin. Yellow arrows point to vimentin bundles in ΔK3 condition. Scale bars=20μm. D. ImageJ analysis of number and area size of vimentin particles in Lipofectamine control (blue), WT-Gig (green), and ΔK3-Gig (magenta) conditions. **p<0.01; *p<0.05; one-way ANOV compared to WT-Gig group. E. Volcano plot of protein interactors of ΔK3-gigaxonin that were significantly changed from WT-gigaxonin, based on mass spectrometry analysis on GFP-Gig pulldowns. Cyan dots represent increased interactions; magenta dot represents decreased interaction. F. Heat maps of the two major categories of differentially interacting gigaxonin partners in the absence of the Kelch 3 motif. Left map shows ubiquitin ligases/deubiquitinases and right map shows mitochondrial proteins (selected out of 71 statistically significant proteins detected in the ΔK3-Gig pulldown relative to WT-Gig).

Figure 6.. The Kelch 3 motif is required for gigaxonin interaction with the chaperone NUDCD3.

A. Immunoblot analysis of NUDCD3 and GFP-Gig in GFP pulldowns on cell lysates from Lipofectamine control, GFP empty vector, and GFP-gigaxonin conditions (WT and ΔK3 mutant). Input lanes show total levels of NUDCD3 and actin (loading control) in the lysates. B. AlphaFold 3 predicted model of gigaxonin (green) interacting with NUDCD3 (cyan). Kelch repeat motifs are colored by domain: Kelch 1 (aa274–326, yellow), Kelch 2 (aa327–374, blue), Kelch 3 (aa376–421, red), Kelch 4 (aa422–468, black), Kelch 5 (470–522, purple), Kelch 6 (528–574, orange). C. AlphaFold 3 predicted model of gigaxonin ΔK3 (aa376–421) (green) interacting with NUDCD3 (cyan), with the remaining Kelch motifs colored as in B. With the removal of Kelch 3, NUDCD3 is no longer predicted to interact with the Kelch domain.