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Highly specific ubiquitin-competing molecules effectively promote frataxin accumulation and partially rescue the aconitase defect in Friedreich ataxia cells.

Rufini A, Cavallo F, Condò I, Fortuni S, De Martino G, Incani O, Di Venere A, Benini M, Massaro DS, Arcuri G, Serio D, Malisan F, Testi R - Neurobiol. Dis. (2014)

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.

View Article: PubMed Central - PubMed

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.

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UCM prevent frataxin ubiquitination.(A) 293 cells were transiently co-transfected with HA-Ub and frataxin1 -210. Twenty-four hours after transfection, cells were treated with 10 μM of the indicated UCM or with DMSO alone (contr). UCM57 was used as a non-effective control molecule. Cells were harvested 48 h after transfection. Where indicated (MG), cells were also treated with 10 μM MG132 and 50 ng/ml ubiquitin-aldehyde for the last 16 h. Total cell extracts were resolved on SDS–PAGE and revealed with anti-frataxin antibody. The arrows indicate the bands corresponding to frataxin precursor (Fxn-pre) and ubiquitin-conjugated frataxin (Ub-Fxn). (B) The graph represents the relative ubiquitination levels, quantified as the densitometric ratio between ubiquitinated frataxin bands and frataxin precursor bands for each MG132-treated lanes. Data represent the mean ± SEM from five different independent experiments. P-values were calculated with Student's t-test and were statistically significant (**P < 0.01) compared to non-treated control.
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f0015: UCM prevent frataxin ubiquitination.(A) 293 cells were transiently co-transfected with HA-Ub and frataxin1 -210. Twenty-four hours after transfection, cells were treated with 10 μM of the indicated UCM or with DMSO alone (contr). UCM57 was used as a non-effective control molecule. Cells were harvested 48 h after transfection. Where indicated (MG), cells were also treated with 10 μM MG132 and 50 ng/ml ubiquitin-aldehyde for the last 16 h. Total cell extracts were resolved on SDS–PAGE and revealed with anti-frataxin antibody. The arrows indicate the bands corresponding to frataxin precursor (Fxn-pre) and ubiquitin-conjugated frataxin (Ub-Fxn). (B) The graph represents the relative ubiquitination levels, quantified as the densitometric ratio between ubiquitinated frataxin bands and frataxin precursor bands for each MG132-treated lanes. Data represent the mean ± SEM from five different independent experiments. P-values were calculated with Student's t-test and were statistically significant (**P < 0.01) compared to non-treated control.

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.


Highly specific ubiquitin-competing molecules effectively promote frataxin accumulation and partially rescue the aconitase defect in Friedreich ataxia cells.

Rufini A, Cavallo F, Condò I, Fortuni S, De Martino G, Incani O, Di Venere A, Benini M, Massaro DS, Arcuri G, Serio D, Malisan F, Testi R - Neurobiol. Dis. (2014)

UCM prevent frataxin ubiquitination.(A) 293 cells were transiently co-transfected with HA-Ub and frataxin1 -210. Twenty-four hours after transfection, cells were treated with 10 μM of the indicated UCM or with DMSO alone (contr). UCM57 was used as a non-effective control molecule. Cells were harvested 48 h after transfection. Where indicated (MG), cells were also treated with 10 μM MG132 and 50 ng/ml ubiquitin-aldehyde for the last 16 h. Total cell extracts were resolved on SDS–PAGE and revealed with anti-frataxin antibody. The arrows indicate the bands corresponding to frataxin precursor (Fxn-pre) and ubiquitin-conjugated frataxin (Ub-Fxn). (B) The graph represents the relative ubiquitination levels, quantified as the densitometric ratio between ubiquitinated frataxin bands and frataxin precursor bands for each MG132-treated lanes. Data represent the mean ± SEM from five different independent experiments. P-values were calculated with Student's t-test and were statistically significant (**P < 0.01) compared to non-treated control.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4358773&req=5

f0015: UCM prevent frataxin ubiquitination.(A) 293 cells were transiently co-transfected with HA-Ub and frataxin1 -210. Twenty-four hours after transfection, cells were treated with 10 μM of the indicated UCM or with DMSO alone (contr). UCM57 was used as a non-effective control molecule. Cells were harvested 48 h after transfection. Where indicated (MG), cells were also treated with 10 μM MG132 and 50 ng/ml ubiquitin-aldehyde for the last 16 h. Total cell extracts were resolved on SDS–PAGE and revealed with anti-frataxin antibody. The arrows indicate the bands corresponding to frataxin precursor (Fxn-pre) and ubiquitin-conjugated frataxin (Ub-Fxn). (B) The graph represents the relative ubiquitination levels, quantified as the densitometric ratio between ubiquitinated frataxin bands and frataxin precursor bands for each MG132-treated lanes. Data represent the mean ± SEM from five different independent experiments. P-values were calculated with Student's t-test and were statistically significant (**P < 0.01) compared to non-treated control.
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.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus