Biology subjects

Armao, D.

Publications and source records attributed to Armao, D..

4 recordsLinked to original sources

bioRxiv2025

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, 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 ({Delta}K1-{Delta}K6) promoted the degradation of soluble vimentin. The {Delta}K3 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, {Delta}K3 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 {Delta}K3-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.

cell biology↗

bioRxiv2024

Gigaxonin, mutated in Giant Axonal Neuropathy, interacts with TDP-43 and other RNA binding proteins

Giant Axonal Neuropathy (GAN) is a neurodegenerative disease caused by loss-of-function mutations in the KLHL16 gene, encoding the cytoskeleton regulator gigaxonin. In the absence of functional gigaxonin, intermediate filament (IF) proteins accumulate in neurons and other cell types due to impaired turnover and transport. GAN neurons exhibit distended, swollen axons and distal axonal degeneration, but the mechanisms behind this selective neuronal vulnerability are unknown. Our objective was to identify novel gigaxonin interactors pertinent to GAN neurons. Unbiased proteomics revealed a statistically significant predominance of RNA-binding proteins (RBPs) within the soluble gigaxonin interactome and among differentially-expressed proteins in iPSC-neuron progenitors from a patient with classic GAN. Among the identified RBPs was TAR DNA-binding protein 43 (TDP-43), which associated with the gigaxonin protein and its mRNA transcript. TDP-43 co-localized within large axonal neurofilament IFs aggregates in iPSC-motor neurons derived from a GAN patient with the axonal CMT-plus disease phenotype. Our results implicate RBP dysfunction as a potential underappreciated contributor to GAN-related neurodegeneration. SummaryThis work reveals that the neurodegeneration-associated protein and cytoskeleton regulator gigaxonin and its mRNA associate with numerous RNA binding proteins. These findings shift understanding of normal gigaxonin function and provide insights into how disease-causing mutations in the gigaxonin-encoding gene (KLHL16) may ignite a pathogenic cascade in neurons.

cell biology↗

bioRxiv2023

Intermediate filament dysregulation and astrocytopathy in the human disease model of KLHL16 mutation in giant axonal neuropathy (GAN)

Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by KLHL16 mutations. KLHL16 encodes gigaxonin, a regulator of intermediate filament (IF) protein turnover. Previous neuropathological studies and our own examination of postmortem GAN brain tissue in the current study revealed astrocyte involvement in GAN. To study the underlying mechanisms, we reprogrammed skin fibroblasts from seven GAN patients carrying different KLHL16 mutations to iPSCs. Isogenic controls with restored IF phenotypes were derived via CRISPR/Cas9 editing of one patient carrying a homozygous missense mutation (G332R). Neural progenitor cells (NPCs), astrocytes, and brain organoids were generated through directed differentiation. All GAN iPSC lines were deficient for gigaxonin, which was restored in the isogenic control. GAN iPSCs displayed patient-specific increased vimentin expression, while GAN NPCs had decreased nestin expression compared to isogenic control. The most striking phenotypes were observed in GAN iPSC-astrocytes and brain organoids, which exhibited dense perinuclear IF accumulations and abnormal nuclear morphology. GAN patient cells with large perinuclear vimentin aggregates accumulated nuclear KLHL16 mRNA. In over-expression studies, GFAP oligomerization and perinuclear aggregation were potentiated in the presence of vimentin. As an early effector of KLHL16 mutations, vimentin may serve as a potential therapeutic target in GAN.

cell biology↗

bioRxiv2022

Calpain-mediated proteolysis of vimentin filaments is augmented in Giant Axonal Neuropathy (GAN) fibroblasts exposed to hypotonic stress

Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by loss-of-function mutations in the E3 ubiquitin ligase adaptor gigaxonin, which is encoded by the GAN (KLHL16) gene. Gigaxonin regulates the degradation of multiple intermediate filament (IF) proteins, including neurofilaments, GFAP, and vimentin. In the absence of functional gigaxonin, GAN patients display abnormal cytoplasmic IF aggregates in multiple cell types. Understanding how normal IF networks and abnormal IF aggregates respond and are processed under physiologic stress can reveal new GAN disease mechanisms and potential targets for therapy. Here we tested the hypothesis that hypotonic stress-induced vimentin proteolysis is impaired in GAN. In both GAN and control fibroblasts exposed to hypotonic stress, we observed time-dependent vimentin cleavage, resulting in two prominent ~40-45 kDa fragments readily detectable by immunoblot in the total cell lysates and detergent-insoluble fractions. However, vimentin proteolysis occurred more rapidly and extensively in GAN cells compared to unaffected controls. Both fragments were generated earlier in GAN cells and at 4-6-fold higher levels (p<0.0001) compared to control fibroblasts. To test enzymatic involvement, we determined the expression levels and localization of the calcium-sensitive calpain proteases-1 and -2 and their endogenous inhibitor calpastatin. While the latter was not affected, the expression of both calpains was 2-fold higher in GAN cells compared to control cells (p<0.01). Moreover, pharmacologic inhibition of calpains with MDL-28170 or MG-132 attenuated vimentin cleavage, the latter resulting in >95% reduced cleavage (p<0.0001). Imaging analysis revealed striking colocalization between large perinuclear vimentin aggregates and calpain-2 in GAN fibroblasts. This colocalization was dramatically altered by hypotonic stress, where selective breakdown of IF networks with relative sparing of IF aggregates occurred rapidly in GAN cells and coincided with cytoplasmic redistribution of calpain-2. Finally, mass spectrometry-based proteomics revealed that phosphorylation at Ser-412, located at the junction between the central "rod" domain and C-terminal "tail" domain on vimentin, is involved in this stress response. Over-expression studies using phospho-deficient (S412A) and phospho-mimic (S412D) mutants revealed that Ser-412 is important for filament organization, solubility dynamics, and cleavage of vimentin upon hypotonic stress exposure. Collectively, our work reveals that osmotic stress induces calpain- and proteasome-mediated vimentin degradation and IF network breakdown. These effects are significantly augmented in the presence of disease-causing KLHL16 mutations that alter IF spatial distribution and intermediate filament organization. While the specific roles of calpain-generated vimentin IF fragments in GAN cells remain to be defined, this proteolytic pathway is translationally-relevant to GAN because maintaining osmotic homeostasis is critical for nervous system function.

cell biology↗