Dataset Information

Mitochondrial stress in GLA mutants Zebrafish is independent of globotriaosylceramide accumulation

ABSTRACT: Fabry disease (FD) is a hereditary lysosomal storage disorder caused by mutations in GLA gene resulting in reduction or lack of α-galactosidase A activity. In humans, enzymatic deficiency leads to accumulation of globotriaosylceramide (Gb3) and other glycosphingolipids in lysosomes. Current therapies focus on reversing Gb3 accumulation but fail to restore altered cellular signaling in the long run. Zebrafish (ZF) lacks Alpha 1,4-Galactosyltransferase (A4GALT) gene and cannot synthesize Gb3 or lysoGb3. We used previously generated GLA-mutant ZF model to investigate Gb3-accumulation-independent alterations by untargeted proteomics analysis of renal tissues. We observed lysosomal and mitochondrial-related pathways disfunction, higher oxidative stress, as indicated by GSH/GSSH and MDA levels, and higher antioxidant activity in mutant ZF. Moreover, mitochondrial morphological alterations also affecting cristae structure were evident. By immunohistochemistry, we also detected decreased lysosomal Tetraspanin (CD63), Superoxide dismutase 2 (SOD2), and Cadherin 1 (CDH1) protein expression. Thus, this ZF model Gb3-independently mirrors GLA mutation-related changes, and unravels novel FD pathogenic mechanisms, thereby representing a powerful tool for innovative drug screening, and the identification of markers of potential clinical relevance.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Danio Rerio (zebrafish) (brachydanio Rerio)

SUBMITTER: Even Birkeland

LAB HEAD: Jessica Furriol

PROVIDER: PXD035409 | Pride | 2023-10-24

REPOSITORIES: Pride

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Publications

Proteomic analysis unveils Gb3-independent alterations and mitochondrial dysfunction in a gla<sup>-/-</sup> zebrafish model of Fabry disease.

Elsaid Hassan Osman Alhassan HOA Rivedal Mariell M Skandalou Eleni E Svarstad Einar E Tøndel Camilla C Birkeland Even E Eikrem Øystein Ø Babickova Janka J Marti Hans-Peter HP Furriol Jessica J

Journal of translational medicine 20230905 1

<h4>Background</h4>Fabry disease (FD) is a rare lysosomal storage disorder caused by mutations in the GLA gene, resulting in reduced or lack of α-galactosidase A activity. This results in the accumulation of globotriaosylceramide (Gb3) and other glycosphingolipids in lysosomes causing cellular impairment and organ failures. While current therapies focus on reversing Gb3 accumulation, they do not address the altered cellular signaling in FD. Therefore, this study aims to explore Gb3-independent m ...[more]

PMID: 37670295

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Project description:Transcription factors are often regarded as having two distinct components: a DNA binding domain (DBD) to allow binding to the regulatory region of a target gene and a trans-acting functional domain (FD) to modulate gene expression. Recently, it is becoming clear that the DBDs of transcription factors alone are incapable of providing sufficient specificity to account for the highly complex genomic structures in eukaryotes. Other regions, outside of the DBD, may play a role in transcription factor DNA binding specificity in vivo. In order to test this hypothesis, we examined a model zinc finger transcription factor, KrÃ¼ppel-like factor 3 (KLF3) with a FD that recruits partner proteins for its repressive activity and a three zinc finger DBD. Previously, we showed that deletion of the entire KLF3 FD reduced DNA binding across the genome. This implied the importance of the FD for in vivo DNA binding specificity. In the current study, we fused the KLF3 FD onto an unrelated, but well characterised Artificial Zinc Finger (AZF) targeting a standard target gene, VEGF-A. We compared the genomic DNA binding profile of this chimeric KLF3 construct, termed KLF3FD-AZF, to that of the AZF alone. Chromatin Immunoprecipitation followed by next generation sequencing was used to assess genomic wide DNA-binding of these AZFs. Remarkably, in these gain-of-function experiments, we again observed that the KLF3 FD is critical for in vivo DNA binding specificity. The addition of KLF3 FD increased the number of binding sites by more than two fold compared to the AZF alone. KLF3 FD also directed more binding to the promoter regions. Further investigations of these acquired sites identified a substantial portion of these sites as the known endogenous KLF3 bound regions, whilst others contain sequences that are similar but not identical to the predicted AZF target sequence. These results clearly demonstrate that the specific localisation of this model transcription factor to target genes is not solely dependent on the DBD and that the regions outside of this domain may also play an important role. ChIP-Seq experiments were performed on four HEK293 cell lines, two each stably expressing AZF or KLF3FD.AZF or KLF3FD only (two biological replicates). Input samples were included as controls.

Dataset Information

Mitochondrial stress in GLA mutants Zebrafish is independent of globotriaosylceramide accumulation

Publications

Proteomic analysis unveils Gb3-independent alterations and mitochondrial dysfunction in a gla<sup>-/-</sup> zebrafish model of Fabry disease.

Similar Datasets

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