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

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 by affecting IF protein degradation and transport. This leads to focal abnormal accumulations of IFs and compromised cellular function, with neurons being most severely impacted. 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-K6. All six gigaxonin deletion mutants (ΔK1-ΔK6) promoted the degradation of soluble vimentin. Compared to WT-gigaxonin, ΔK3-gigaxonin exhibited increased soluble vimentin degradation and increased presence of thick bundles of vimentin IFs. The ΔK4 mutant showed similar, but milder phenotypes compared to ΔK3. Using mass spectrometry proteomics we found that, relative to WT gigaxonin, ΔK3 gigaxonin had increased associations with ubiquitination-associated and mitochondrial proteins but lost the association with the NudC domain-containing protein 3 (NUDCD3), a molecular chaperone enriched in the nervous system. AlphaFold modeling revealed loss of gigaxonin-NUDCD3 binding with ΔK3 and altered binding with ΔK4. Collectively, our cell biological data show the induction of an abnormal GAN-like IF phenotype in cells expressing ΔK3- and, to a lesser extent, ΔK4-gigaxonin, while our proteomic profiling links the loss of gigaxonin-NUDCD3 interactions with defective IF proteostasis.