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: To test whether the new compounds promote frataxin accumulation by preventing its UPS-dependent degradation, we evaluated their impact on frataxin ubiquitination. To this aim, we performed an in vivo ubiquitination assay. HEK-293 cells were transiently co-transfected with hemagglutinin-tagged ubiquitin (HA-Ub) and frataxin, in the presence of proteasome inhibitor and deubiquitinase inhibitor to allow the accumulation of ubiquitinated species, in the presence of the selected compounds. The ubiquitination status of frataxin was evaluated by SDS–PAGE of total cell lysates and anti-frataxin immunoblotting. As previously described, in this experimental setting, frataxin monoubiquitinated forms can be detected by anti-frataxin antibody as a slower migrating band above frataxin precursor (Rufini et al., 2011). Ubiquitination level was measured as the ratio between the levels of ubiquitinated frataxin and frataxin precursor. As shown in Fig. 3, UCM53 and UCM71 but not the control non-effective molecule UCM57, can significantly abrogate frataxin ubiquitination. These data suggest that the selected UCM interfere with frataxin ubiquitination in living cells.
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.