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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 interact with frataxin.Fluorescence studies of the interaction of 2 μM recombinant frataxin precursor with different concentration of the indicated UCM (ligand). The graphs represent the fractional loss of the protein fluorescence intensity (ΔF/F0), in the presence of different concentration of ligand, versus the ratio between ligand and frataxin. The interaction with UCM53 was also analyzed in the case of frataxin precursor previously denaturated in 3 M guanidinium hydrochloride (upper left panel, black square symbols). The half-saturation binding constant, L1/2 (μM), of frataxin precursor with UCM53, UCM108 and UCM72 is indicated in each corresponding panel.
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f0025: UCM interact with frataxin.Fluorescence studies of the interaction of 2 μM recombinant frataxin precursor with different concentration of the indicated UCM (ligand). The graphs represent the fractional loss of the protein fluorescence intensity (ΔF/F0), in the presence of different concentration of ligand, versus the ratio between ligand and frataxin. The interaction with UCM53 was also analyzed in the case of frataxin precursor previously denaturated in 3 M guanidinium hydrochloride (upper left panel, black square symbols). The half-saturation binding constant, L1/2 (μM), of frataxin precursor with UCM53, UCM108 and UCM72 is indicated in each corresponding panel.

Mentions: Ubiquitin-competing molecules were selected through structure-based virtual screening for their potential ability to interact with frataxin on its ubiquitination site. Since they are in fact able to prevent frataxin ubiquitination and to interfere with its K147-dependent degradation, we wanted to confirm their ability to physically interact with frataxin. Therefore, the interaction propensities of frataxin with these molecules were investigated by fluorescence spectroscopy through the analysis of the changes of the signal of the protein tryptophan residues in the presence of the different compounds. In Fig. 5, we have reported the binding isotherm of the protein to four different compounds, UCM53 and UCM108 that promote frataxin accumulation, and UCM72 and UCM57 that are unable to promote frataxin accumulation (see below), as a control. One of the effective compounds (UCM53) was also exposed to the denatured protein. The half-saturation binding constant, L1/2, for UCM53, UCM108 and UCM72 are reported in each corresponding panel. UCM53 and UCM108 showed strong fluorescence changes (left panels) and the lowest binding constants, while the control UCM72 showed the highest binding constant (upper right panel). Fluorescent changes induced by UCM53 were completely lost using the denatured protein (upper left panel, black square symbols). The interaction of frataxin with the control molecule UMC57 was also studied by fluorescence (lower right panel). In this case, the spectroscopic properties of the protein do not significantly change in the presence of this compound obtaining similar results to those of denatured protein + UCM53 (upper left panel, black squares). These results indicate that UCM53 and UCM108 strongly interact with tryptophans likely due to a quite efficient binding.


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 interact with frataxin.Fluorescence studies of the interaction of 2 μM recombinant frataxin precursor with different concentration of the indicated UCM (ligand). The graphs represent the fractional loss of the protein fluorescence intensity (ΔF/F0), in the presence of different concentration of ligand, versus the ratio between ligand and frataxin. The interaction with UCM53 was also analyzed in the case of frataxin precursor previously denaturated in 3 M guanidinium hydrochloride (upper left panel, black square symbols). The half-saturation binding constant, L1/2 (μM), of frataxin precursor with UCM53, UCM108 and UCM72 is indicated in each corresponding panel.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4358773&req=5

f0025: UCM interact with frataxin.Fluorescence studies of the interaction of 2 μM recombinant frataxin precursor with different concentration of the indicated UCM (ligand). The graphs represent the fractional loss of the protein fluorescence intensity (ΔF/F0), in the presence of different concentration of ligand, versus the ratio between ligand and frataxin. The interaction with UCM53 was also analyzed in the case of frataxin precursor previously denaturated in 3 M guanidinium hydrochloride (upper left panel, black square symbols). The half-saturation binding constant, L1/2 (μM), of frataxin precursor with UCM53, UCM108 and UCM72 is indicated in each corresponding panel.
Mentions: Ubiquitin-competing molecules were selected through structure-based virtual screening for their potential ability to interact with frataxin on its ubiquitination site. Since they are in fact able to prevent frataxin ubiquitination and to interfere with its K147-dependent degradation, we wanted to confirm their ability to physically interact with frataxin. Therefore, the interaction propensities of frataxin with these molecules were investigated by fluorescence spectroscopy through the analysis of the changes of the signal of the protein tryptophan residues in the presence of the different compounds. In Fig. 5, we have reported the binding isotherm of the protein to four different compounds, UCM53 and UCM108 that promote frataxin accumulation, and UCM72 and UCM57 that are unable to promote frataxin accumulation (see below), as a control. One of the effective compounds (UCM53) was also exposed to the denatured protein. The half-saturation binding constant, L1/2, for UCM53, UCM108 and UCM72 are reported in each corresponding panel. UCM53 and UCM108 showed strong fluorescence changes (left panels) and the lowest binding constants, while the control UCM72 showed the highest binding constant (upper right panel). Fluorescent changes induced by UCM53 were completely lost using the denatured protein (upper left panel, black square symbols). The interaction of frataxin with the control molecule UMC57 was also studied by fluorescence (lower right panel). In this case, the spectroscopic properties of the protein do not significantly change in the presence of this compound obtaining similar results to those of denatured protein + UCM53 (upper left panel, black squares). These results indicate that UCM53 and UCM108 strongly interact with tryptophans likely due to a quite efficient binding.

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