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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 promote frataxin accumulation in FRDA cells and rescue the aconitase defect.FRDA patients-derived lymphoblasts cell lines FRDA 798 (A) and FRDA 214 (B) or lymphoblasts derived from the corresponding unaffected carrier siblings, FRDA 241 or FRDA 215, respectively, were cultured in the presence of 10 μM of the indicated UCM, or DMSO alone (contr) for 5 days. Total cell extracts were resolved on SDS–PAGE and analyzed with anti-frataxin antibody, or anti-tubulin, as a loading control. The graphs represent the relative frataxin abundance as quantified by densitometric analysis and normalized with tubulin levels. Data represent the mean ± SEM from four different independent experiments. P-values were calculated with Student's t-test and were statistically significant (*P < 0.05; **P < 0.01) compared to non-treated conditions. Tub: tubulin; mat: mature frataxin. (C) Patients-derived lymphoblasts FRDA 214 were cultured with DMSO alone (contr) or in the presence of 10 μM of UCM108 for 5 days. The unaffected carrier siblings FRDA 215 lymphoblasts were cultured with DMSO alone. Total aconitase activities were measured and normalized as described in the Methods section. Data represent the mean ± SEM from four different independent experiments. P-values was calculated with Student's t-test and was statistically significant (**P < 0.01) compared to non-treated condition.
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f0030: UCM promote frataxin accumulation in FRDA cells and rescue the aconitase defect.FRDA patients-derived lymphoblasts cell lines FRDA 798 (A) and FRDA 214 (B) or lymphoblasts derived from the corresponding unaffected carrier siblings, FRDA 241 or FRDA 215, respectively, were cultured in the presence of 10 μM of the indicated UCM, or DMSO alone (contr) for 5 days. Total cell extracts were resolved on SDS–PAGE and analyzed with anti-frataxin antibody, or anti-tubulin, as a loading control. The graphs represent the relative frataxin abundance as quantified by densitometric analysis and normalized with tubulin levels. Data represent the mean ± SEM from four different independent experiments. P-values were calculated with Student's t-test and were statistically significant (*P < 0.05; **P < 0.01) compared to non-treated conditions. Tub: tubulin; mat: mature frataxin. (C) Patients-derived lymphoblasts FRDA 214 were cultured with DMSO alone (contr) or in the presence of 10 μM of UCM108 for 5 days. The unaffected carrier siblings FRDA 215 lymphoblasts were cultured with DMSO alone. Total aconitase activities were measured and normalized as described in the Methods section. Data represent the mean ± SEM from four different independent experiments. P-values was calculated with Student's t-test and was statistically significant (**P < 0.01) compared to non-treated condition.

Mentions: We finally analyzed the efficacy of our compounds in cells derived from FRDA patients. Lymphoblast cell lines derived from two different patients, FRDA 798 and FRDA 214 were cultured for 5 days in the presence of 10 μM of UCM53 or UCM108, the two most promising compounds. UCM71 could not be evaluated because of its toxicity over a long-term treatment (Fig. S1). Frataxin levels were quantified by western blot analysis on whole cell extracts. Importantly, we could observe a significant accumulation of mature frataxin when cells are cultured in the presence of UCM53 or UCM108, compared to cells treated with vehicle alone (control), or UCM72 (Figs. 6A and B). Frataxin levels in treated cells derived from patients are also compared to the levels observed in cells derived from unaffected carrier siblings (FRDA 241 and FRDA 215). These UCM are also more effective than the previously described compounds (Rufini et al., 2011). Indeed, at the concentration of 10 μM that is efficacious for the new UCM, the previously described compound does not show a significant effect (Fig. S2). Moreover, to validate a functional recovery of frataxin levels, rescue of cellular aconitases activity was evaluated in FRDA cells upon treatment with UCM. A significant increase in aconitases activity was observed in patient-derived lymphoblast cell line FRDA 214 after treatment with UCM108 for 5 days (Fig. 6C). Aconitases activity in cells derived from an unaffected carrier sibling (FRDA 215) is also shown for comparison. Thus, importantly, these data indicate that treatment with UCM allows accumulation of a functional form of mature frataxin with consequent reactivation of ISC biogenesis.


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 promote frataxin accumulation in FRDA cells and rescue the aconitase defect.FRDA patients-derived lymphoblasts cell lines FRDA 798 (A) and FRDA 214 (B) or lymphoblasts derived from the corresponding unaffected carrier siblings, FRDA 241 or FRDA 215, respectively, were cultured in the presence of 10 μM of the indicated UCM, or DMSO alone (contr) for 5 days. Total cell extracts were resolved on SDS–PAGE and analyzed with anti-frataxin antibody, or anti-tubulin, as a loading control. The graphs represent the relative frataxin abundance as quantified by densitometric analysis and normalized with tubulin levels. Data represent the mean ± SEM from four different independent experiments. P-values were calculated with Student's t-test and were statistically significant (*P < 0.05; **P < 0.01) compared to non-treated conditions. Tub: tubulin; mat: mature frataxin. (C) Patients-derived lymphoblasts FRDA 214 were cultured with DMSO alone (contr) or in the presence of 10 μM of UCM108 for 5 days. The unaffected carrier siblings FRDA 215 lymphoblasts were cultured with DMSO alone. Total aconitase activities were measured and normalized as described in the Methods section. Data represent the mean ± SEM from four different independent experiments. P-values was calculated with Student's t-test and was statistically significant (**P < 0.01) compared to non-treated condition.
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Related In: Results  -  Collection

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f0030: UCM promote frataxin accumulation in FRDA cells and rescue the aconitase defect.FRDA patients-derived lymphoblasts cell lines FRDA 798 (A) and FRDA 214 (B) or lymphoblasts derived from the corresponding unaffected carrier siblings, FRDA 241 or FRDA 215, respectively, were cultured in the presence of 10 μM of the indicated UCM, or DMSO alone (contr) for 5 days. Total cell extracts were resolved on SDS–PAGE and analyzed with anti-frataxin antibody, or anti-tubulin, as a loading control. The graphs represent the relative frataxin abundance as quantified by densitometric analysis and normalized with tubulin levels. Data represent the mean ± SEM from four different independent experiments. P-values were calculated with Student's t-test and were statistically significant (*P < 0.05; **P < 0.01) compared to non-treated conditions. Tub: tubulin; mat: mature frataxin. (C) Patients-derived lymphoblasts FRDA 214 were cultured with DMSO alone (contr) or in the presence of 10 μM of UCM108 for 5 days. The unaffected carrier siblings FRDA 215 lymphoblasts were cultured with DMSO alone. Total aconitase activities were measured and normalized as described in the Methods section. Data represent the mean ± SEM from four different independent experiments. P-values was calculated with Student's t-test and was statistically significant (**P < 0.01) compared to non-treated condition.
Mentions: We finally analyzed the efficacy of our compounds in cells derived from FRDA patients. Lymphoblast cell lines derived from two different patients, FRDA 798 and FRDA 214 were cultured for 5 days in the presence of 10 μM of UCM53 or UCM108, the two most promising compounds. UCM71 could not be evaluated because of its toxicity over a long-term treatment (Fig. S1). Frataxin levels were quantified by western blot analysis on whole cell extracts. Importantly, we could observe a significant accumulation of mature frataxin when cells are cultured in the presence of UCM53 or UCM108, compared to cells treated with vehicle alone (control), or UCM72 (Figs. 6A and B). Frataxin levels in treated cells derived from patients are also compared to the levels observed in cells derived from unaffected carrier siblings (FRDA 241 and FRDA 215). These UCM are also more effective than the previously described compounds (Rufini et al., 2011). Indeed, at the concentration of 10 μM that is efficacious for the new UCM, the previously described compound does not show a significant effect (Fig. S2). Moreover, to validate a functional recovery of frataxin levels, rescue of cellular aconitases activity was evaluated in FRDA cells upon treatment with UCM. A significant increase in aconitases activity was observed in patient-derived lymphoblast cell line FRDA 214 after treatment with UCM108 for 5 days (Fig. 6C). Aconitases activity in cells derived from an unaffected carrier sibling (FRDA 215) is also shown for comparison. Thus, importantly, these data indicate that treatment with UCM allows accumulation of a functional form of mature frataxin with consequent reactivation of ISC biogenesis.

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