Highly specific ubiquitin-competing molecules effectively promote frataxin accumulation and partially rescue the aconitase defect in Friedreich ataxia cells.
Bottom Line: The underlying genetic defect leads to reduced expression of the mitochondrial protein frataxin.By extending our search for effective UCM, we identified a set of new and more potent compounds that more efficiently promote frataxin accumulation.Interestingly, these UCM are not effective on the ubiquitin-resistant frataxin mutant, indicating their specific action on preventing frataxin ubiquitination.
Affiliation: Laboratory of Signal Transduction, Department of Biomedicine and Prevention, University of Rome "Tor Vergata," Via Montpellier 1, Rome 00133, Italy; Fratagene Therapeutics Ltd., 22 Northumberland Rd., Dublin, Ireland.Show MeSH
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Mentions: We had previously shown that K147 is the crucial ubiquitination site on frataxin. Since we showed that these compounds are able to efficiently abrogate frataxin ubiquitination, we could anticipate that they act by interfering with ubiquitination on K147. Thus, to validate this hypothesis, we evaluated their effect on the frataxin mutant that lacks K147 (K147R). This mutant cannot be ubiquitinated and is therefore resistant to UPS-mediated degradation. Small molecules that act by preventing ubiquitination on K147 are expected to be ineffective on this mutant. We therefore tested their effect on HEK-293 cells stably expressing the ubiquitin-refractory frataxinK147R mutant (293-frataxinK147R). Cells were treated for 24 h with the indicated compounds and frataxin precursor levels analyzed by western blot on total cell extract. Indeed, we could show that when 293-frataxinK147R are treated with the selected UCM, no significant increase in frataxin precursor levels can be detected (Fig. 4), compared to what observed in 293-frataxin, expressing wild-type frataxin. These data suggest that UCM act on frataxin by interfering with the K147-dependent degradation pathway.
Affiliation: Laboratory of Signal Transduction, Department of Biomedicine and Prevention, University of Rome "Tor Vergata," Via Montpellier 1, Rome 00133, Italy; Fratagene Therapeutics Ltd., 22 Northumberland Rd., Dublin, Ireland.