Biology subjects

Snider, N. T.

Publications and source records attributed to Snider, N. T..

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↗

bioRxiv2022

Disassembly of embryonic keratin filaments promotes pancreatic cancer

Keratin 17 (K17), an oncofetal intermediate filament protein, is one of the most abundantly expressed proteins in pancreatic ductal adenocarcinomas (PDACs) of the most aggressive molecular subtype. The mechanistic roles of this protein in malignancy, however, are largely unexplored. Here we show that K17 expression and disassembly enhances tumor growth and metastatic potential and shortens survival. Using mass spectrometry in K17 isolated from patients tumors, we identified a hotspot phosphorylation site in serines 10-13. Site-mutagenesis revealed that phosphorylation of this hotspot is sufficient to disassemble K17 and promote its nuclear translocation. In silico and pharmacologic inhibition studies uncovered the role of the PKC/MEK/RSK pathway in the phosphorylation and disassembly of K17. Murine models bearing tumors expressing phosphomimetic mutations at the serine hotspot displayed enhanced metastases, compared to mice bearing tumors expressing wild-type K17 or phosphorylation-resistant K17. Lastly, we found that detergent-soluble nuclear K17 promotes the expression of metastasis promoting genes in both patient and murine tumors. These results suggest that phosphorylation at specific serines is sufficient to promote pancreatic cancer metastasis and shorter survival, and that these sites could provide novel, druggable therapeutic domains to enhance PDAC patient survival.

cancer biology↗

bioRxiv2020

CD73 maintains hepatocyte metabolic integrity and mouse liver homeostasis in a sex-dependentmanner

Background & AimsMetabolic imbalance and inflammation are common features of chronic liver diseases. Molecular factors controlling these mechanisms represent potential therapeutic targets. One promising target is CD73, the major enzyme that dephosphorylates extracellular adenosine monophosphate (AMP) to form the anti-inflammatory adenosine. In normal liver, CD73 is expressed on pericentral hepatocytes, which are important for long-term liver homeostasis. The aim of this study was to determine if CD73 has non-redundant hepatoprotective functions. Approach & ResultsWe generated mice with a targeted deletion of the CD73-encoding gene (Nt5e) in hepatocytes (CD73-LKO). Deletion of hepatocyte Nt5e resulted in approximately 70% reduction in total liver CD73 protein (p<0.0001). Male and female CD73-LKO mice developed normally during the first 21 weeks, without significant liver phenotypes. Between 21-42 weeks, the CD73-LKO mice developed spontaneous onset liver disease with significant severity in male mice. Notably, middle-aged male CD73-LKO mice displayed hepatocyte swelling and ballooning (p<0.05), inflammation (p<0.01) and variable steatosis. Female CD73-LKO mice had lower serum albumin (p<0.05) and elevated inflammatory markers (p<0.01), but did not exhibit the spectrum of histopathologic changes characteristic of the male mice, potentially due to compensatory induction of adenosine receptors. Serum analysis and proteomic profiling of hepatocytes from male CD73-LKO mice revealed significant metabolic imbalance, with elevated blood urea nitrogen (p<0.0001) and impairments in major metabolic pathways, including oxidative phosphorylation and AMP-activated protein kinase (AMPK) signaling. There was significant hypo-phosphorylation in AMPK substrate in CD73-LKO livers (p<0.0001), while in isolated hepatocytes treated with AMP, soluble CD73 induced AMPK activation (p<0.001). ConclusionsHepatocyte CD73 supports long-term metabolic liver homeostasis through AMPK in a sex-dependent manner. These findings have implications for human liver diseases marked by CD73 dysregulation.

physiology↗