Abstract: FR-PO155
Re-Routing of Mutant Protein from Mitochondria to Peroxisome: A Therapeutic Approach for Primary Hyperoxaluria Type I
Session Information
- Mitochondriacs and More
November 03, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Genetic Diseases of the Kidney
- 802 Non-Cystic Mendelian Diseases
Authors
- Belostotsky, Ruth, Shaarey Zedek Medical Center, Jerusalem, Israel
- Lyakhovetsky, Roman, Teva Pharmaceuticals, Maale Adumim, Israel
- Beck, Bodo B., University of Cologne Medical Center, Cologne, Germany
- Reusch, Björn, University of Cologne, Cologne, Germany
- Shkedy, Fanny, Shaare Zedek Medical Center, Jerusalem, Israel
- Frishberg, Yaacov, Shaare Zedek Medical Center, Jerusalem, Israel
Background
Primary hyperoxaluria type I (PH1) is caused by deficiency of the liver specific peroxisomal (Px) alanine-glyoxylate aminotransferase (AGT). Due to low solubility of calcium oxalate, PH1 results in progressive nephrocalcinosis and decline in kidney function to ESRD.
The most frequent AGT mutation G170R results in aberrant mitochondrial localization with preserved catalytic activity. Thus, molecules that stimulate Px localization of the mutated protein may prevent oxalate production. To identify such molecules we developed the split GFP system in which only peroxisomal AGT sub-population produces a fluorescent signal. This sensitive and specific tool allows precise monitoring of Px sub-population of AGT: confocal microscopy confirms that GFP signal of both WT and G170R AGT is localized exclusively in Px and is substantially stronger in the WT-AGT.
Using split GFP, we demonstrated that localization of G170R-AGT can be corrected by mild translation inhibition using the inhibitors emetine (a known antiprotozoal agent) and GC7. 7 days incubation of G170R-AGT-GFP transfected CHO cells with 40 nM emetine resulted in 70-80% increase in the number of GFP positive cells as quantified by FACS analysis. Under these conditions, total protein synthesis decreased by less than 25%. We also measured the effect of mitochondrial transport inhibitors: DECA and monensin, recently indicated to correct G170R-AGT localization. Both compounds, but not GC7, presented synergistic effect with emetine. We assume that DECA and monensin assist AGT relocation by mitochondrial transport interference while emetine and GC7 interfere with protein synthesis. The functional competence of peroxisomal retargeted AGT-LRM was confirmed in cultured human hepatocytes bearing G170R mutation. Augmented oxalate level in culture media was substantially reduced.
In summary, we demonstrate that mild translation inhibition can re-route nascent AGT molecules into the Px. We suggest that this approach may be applicable not only for treating PH1 but also for other diseases caused by protein misfolding. The split-AGT system can be a useful tool in developing new treatments for PH1.
Funding
- Government Support - Non-U.S.