Fig. 6: Cellular respiration of FRDA fibroblasts expressing the frataxin-like protein Nqo15 of T. thermophilus complex I.

A On the top, cartoon representation of human mature frataxin (PDB ID: 1EKG) and the subunit Nqo15 from T. thermophilus respiratory complex I (PDB ID: 6Y11). In both structures, α helices and β strands are highlighted in dark and light green, respectively. On the bottom, structural representation of respiratory complex I from human mitochondria (PDB ID: 5XTD) and T. thermophilus (PDB ID: 6Y11). Iron and sulfur atoms of FeS clusters are shown as brown and yellow spheres, respectively. For sake of clarity, the domain of T. thermophilus complex I containing Nqo15 (in green) and the corresponding region in the human structure have been zoomed. In the bacterial complex, Nqo15 faces the subunit Nqo3 which corresponds to the homologue subunit NDUFS1 in human complex I (both subunits are shown in light blue). B Western blotting analysis of the whole protein extract obtained 72 h after lentiviral transduction of FRDA fibroblast with Nqo15^COQ6 vector or FXN^1-210 vector. As negative control, cells were infected with the empty vector. The expression of the exogenous proteins was detected immunodecorating the blot with an anti-FLAG primary antibody. Equal volumes of sample in each lane (i.e., 20 µL) were loaded and β-actin was used as loading control. C Oxygen consumption rates (OCR) of cells were measured in real time under basal conditions and after injection of oligomycin (1 µM), FCCP (0.75 µM), rotenone (1 µM) and antimycin (1 µM) as indicated in the figure. Values were normalized to the total protein concentration (µg/mL) and the respiration profiles of the transduced fibroblasts are represented in dark gray for negative control, in red for FXN^1-210 vector and in light blue for Nqo15^COQ6 vector. Bioenergetic parameters were calculated as described in Materials and Methods. Reported data result from the mean of three independent experiments ± SEM. Statistical significance was determined using Two-way ANOVA with Bonferroni’s post-hoc test (*p ≤ 0.05, compared to empty vector transduction).