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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 increase frataxin levels.(A) To detect frataxin precursor accumulation, 293 Flp-In cells stably expressing frataxin1 -210 were treated for 24 hrs with 10 μM of the indicated UCM or 10 μM MG132 (MG). Total cell extracts were resolved on SDS–PAGE and analyzed with anti-frataxin antibody, or anti-tubulin, as a loading control. Pre: precursor; tub: tubulin. (B) To detect mature frataxin accumulation, 293 Flp-In cells stably expressing frataxin1 -210 were treated for 72 hrs with 10 μM of the indicated UCM. Total cell extracts were resolved on SDS–PAGE and analyzed with anti-frataxin antibody, or anti-tubulin, as a loading control. Pre: precursor, int: intermediate, mat: mature; tub: tubulin.
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f0010: UCM increase frataxin levels.(A) To detect frataxin precursor accumulation, 293 Flp-In cells stably expressing frataxin1 -210 were treated for 24 hrs with 10 μM of the indicated UCM or 10 μM MG132 (MG). Total cell extracts were resolved on SDS–PAGE and analyzed with anti-frataxin antibody, or anti-tubulin, as a loading control. Pre: precursor; tub: tubulin. (B) To detect mature frataxin accumulation, 293 Flp-In cells stably expressing frataxin1 -210 were treated for 72 hrs with 10 μM of the indicated UCM. Total cell extracts were resolved on SDS–PAGE and analyzed with anti-frataxin antibody, or anti-tubulin, as a loading control. Pre: precursor, int: intermediate, mat: mature; tub: tubulin.

Mentions: To validate UCM activity, we tested their effect in human HEK-293 cells stably expressing single copy frataxin (293-frataxin). These cells allow the detection of all forms of frataxin, including the frataxin precursor. Compounds that were able to enhance frataxin precursor levels were further chemically modified to better fit the docking model, synthesized and tested again in 293-frataxin. This process was repeated in an iterative cycle with the aim to improve the efficacy of the UCM. Approximately 200 new candidate UCM were tested in functional assays. Through this process, we were able to identify new UCM that promote frataxin precursor accumulation more efficiently than the previously described compounds. Structures of the compounds described in this study are shown in Table 1. Indeed, the treatment of 293-frataxin cells with 10 μM UCM53, UCM108 and UCM71 is able to induce frataxin precursor accumulation (Fig. 2A) more efficiently than with the previously described UCM2 (referred to as NSC620301 in (Rufini et al., 2011)) or with the proteasome inihibitor MG132. Importantly, an accumulation of mature frataxin can also be observed in these cells when treatment is prolonged for 3 days (Fig. 2B).


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 increase frataxin levels.(A) To detect frataxin precursor accumulation, 293 Flp-In cells stably expressing frataxin1 -210 were treated for 24 hrs with 10 μM of the indicated UCM or 10 μM MG132 (MG). Total cell extracts were resolved on SDS–PAGE and analyzed with anti-frataxin antibody, or anti-tubulin, as a loading control. Pre: precursor; tub: tubulin. (B) To detect mature frataxin accumulation, 293 Flp-In cells stably expressing frataxin1 -210 were treated for 72 hrs with 10 μM of the indicated UCM. Total cell extracts were resolved on SDS–PAGE and analyzed with anti-frataxin antibody, or anti-tubulin, as a loading control. Pre: precursor, int: intermediate, mat: mature; tub: tubulin.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0010: UCM increase frataxin levels.(A) To detect frataxin precursor accumulation, 293 Flp-In cells stably expressing frataxin1 -210 were treated for 24 hrs with 10 μM of the indicated UCM or 10 μM MG132 (MG). Total cell extracts were resolved on SDS–PAGE and analyzed with anti-frataxin antibody, or anti-tubulin, as a loading control. Pre: precursor; tub: tubulin. (B) To detect mature frataxin accumulation, 293 Flp-In cells stably expressing frataxin1 -210 were treated for 72 hrs with 10 μM of the indicated UCM. Total cell extracts were resolved on SDS–PAGE and analyzed with anti-frataxin antibody, or anti-tubulin, as a loading control. Pre: precursor, int: intermediate, mat: mature; tub: tubulin.
Mentions: To validate UCM activity, we tested their effect in human HEK-293 cells stably expressing single copy frataxin (293-frataxin). These cells allow the detection of all forms of frataxin, including the frataxin precursor. Compounds that were able to enhance frataxin precursor levels were further chemically modified to better fit the docking model, synthesized and tested again in 293-frataxin. This process was repeated in an iterative cycle with the aim to improve the efficacy of the UCM. Approximately 200 new candidate UCM were tested in functional assays. Through this process, we were able to identify new UCM that promote frataxin precursor accumulation more efficiently than the previously described compounds. Structures of the compounds described in this study are shown in Table 1. Indeed, the treatment of 293-frataxin cells with 10 μM UCM53, UCM108 and UCM71 is able to induce frataxin precursor accumulation (Fig. 2A) more efficiently than with the previously described UCM2 (referred to as NSC620301 in (Rufini et al., 2011)) or with the proteasome inihibitor MG132. Importantly, an accumulation of mature frataxin can also be observed in these cells when treatment is prolonged for 3 days (Fig. 2B).

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