Figure 2

Mitochondrial disulfide relay system dysfunction and effect of MIA40 overexpression in fibroblasts from premutation carriers. (A) Nuclearly encoded proteins are imported into the inner membrane space and translocated in their reduced form into mitochondria. The oxidized form of MIA40 gives rise to a transient intermolecular disulfide bridge with the reduced precursor protein, resulting in oxidation of the precursor protein that now contains an intramolecular disulfide bond and is able to undergo folding in the IMS. Reduced MIA40 is then reoxidized by GFER via a disulfide relay system. Reduced GFER is reoxidized, transferring its electrons to O₂ via cytochrome c and cytochrome c oxidase, linking MDRS to respiratory chain activity (38). (B) Representative Western blot images and densitometry of the protein levels of substrates (COX17 and NDUFB7) and elements of the MDRS system (MIA40, GFER, cytochrome c) in cell extracts from controls, PA and PS carriers. Band intensity of the proteins of interest was normalized to actin and data are shown as mean ± SEM (n = 4 for control, n = 5 for PA, n = 3 for PS). Data were analyzed by ANOVA followed by Bonferroni’s post-hoc test. *p < 0.05; **p < 0.005; ***p < 0.001 versus controls. (C) Activities of Complex I, IV and citrate synthase were evaluated in control, PA and PS. Complex I and IV activities were normalized to citrate synthase. The 95% CI for citrate synthase activity was 48-68 nmol × (min x mg protein)⁻¹. Statistical analysis was performed with the Student t test between PS and control. *p < 0.05. (D) Two cell lines (one control and one premutation) were chosen to over-express MIA40. Cells were cultured in regular media and transfected at 90% confluency with either empty vector (E.V.) or MIA40 coding or overexpression vector (O.E.). At 48 h, MIA40 protein expression was evaluated by immunoblots in E.V. or O.E. using β-actin as the loading control, Average of 4 independent experiments. (E) Confocal images of control fibroblasts transfected with either E.V. or MIA40 O.E. vector showing mitochondria staining and MIA40 subcellular distribution. Red = mitochondria; Green = MIA40; Yellow = overlap. (F) Cell viability (evaluated by Trypan blue exclusion) and Complex I activity (NADH-quinone reductase normalized to citrate synthase) were assessed in E.V. and O.V. Values represent the average of 4 independent experiments normalized to control values. *p < 0.05,