Replacement of the C6ORF66 assembly factor (NDUFAF4) restores complex I activity in patient cells

Abstract

Disorders of the oxidative phosphorylation (OXPHOS) system frequently result in a severe multisystem disease with the consequence of early childhood death. Among these disorders, isolated complex I deficiency is the most frequently diagnosed, accounting for one-third of all cases of respiratory chain deficiency. We chose to focus on complex I deficiency, caused by mutation in the assembly factor chromosome 6, open reading frame 66 (C6ORF66; NADH dehydrogenase [ubiquinone] complex I assembly factor 4 [NDUFAF4]) protein. We used the approach of cell- and organelle-directed protein/enzyme replacement therapy, with the transactivator of transcription (TAT) peptide as the moiety delivery system. This step will enable us to deliver the wild-type assembly factor C6ORF66 into patient cells and their mitochondria, leading to the proper assembly and function of complex I and, as a result, to a functional OXPHOS system. We designed and constructed the TAT-ORF fusion protein by gene fusion techniques, expressed the protein in an Escherichia coli expression system and highly purified it. Our results indicate that TAT-ORF enters patients' cells and their mitochondria rapidly and efficiently. TAT-ORF is biologically active and led to an increase in complex I activity. TAT-ORF also increased the number of patient cells and improved the activity of their mitochondria. Moreover, we observed an increase in ATP production, a decrease in the content of mitochondria and a decrease in the level of reactive oxygen species. Our results suggest that this approach of protein replacement therapy for the treatment of mitochondrial disorders is a promising one.

Figure 1

Schematic representation of TAT-ORF and its purification. (A) Schematic representation of TAT-ORF fusion protein. SDS-PAGE (B) and Western blots using anti-His antibodies (C) or anti-C6ORF66 antibodies (D) to analyze the purification steps of TAT-ORF by affinity chromatography as described in Materials and Methods are shown. Arrows indicate the TAT-ORF fusion protein. FT1, flow through 1; FT2, flow through 2; His, 6xhis tag; M., marker; MTS, mitochondrial targeting sequence; Sol., soluble fraction; TAT, Tat peptide.

Figure 2

Internalization of TAT-ORF into isolated mitochondria and cells of F528 patients. (A, B) Internalization of TAT-ORF into mitochondria isolated from cells of F528 patients. Isolated mitochondria were added with TAT-ORF (0.11 μg/μL; final concentration) for 2 h, washed and submitted to Western blot analysis by using anti-His (A) (1:30,000 dilution) or anti-ORF (B) (1:10,000 dilution) antibodies. (C, D) Internalization of TAT-ORF into cells from F528 patients. F528 cells were treated with TAT-ORF (0.02 μg/μL; final concentration) for various time periods. The cells were washed and mitochondria were isolated and submitted to Western blot analysis using anti-ORF (C1 1:50,000 dilution), anti-E1α (C2; 1:1,000 dilution) or anti-His (C3; 1:10,000 dilution) antibodies. (D) Repeating the experiment with a higher concentration of the fusion protein added to the F528 cells (0.1 μg/μL, final concentration) and loading twice the amount of purified mitochondria (25 μg protein per lane), from both F528 treated cells and normal fibroblast. Proteins were identified by using anti-ORF antibodies. Arrows indicate the TAT-ORF fusion protein (A–C1, 3; D, right image), the E1α (C2) or the endogenous C6ORF66 protein (D, left image). (E) After internalization of FITC-labeled TAT-ORF into F528 cells for 1 h (E1), 2 h (E2) and 3 h (E3), by using confocal microscopy. Magnification 60×.

Figure 3

Complex I activity in various cells. (A) Complex I activity in healthy fibroblasts as a function of amount (in micrograms protein added) of mitochondria added. (B) Complex I activity in mitochondria isolated from patient cells mutated in C6ORF66; data for F528, F334 and F511 cells, as compared with activity in mitochondria isolated from healthy fibroblasts, are shown. The effect on complex I activity is shown for TAT-ORF added to isolated mitochondria from F528 cells (C). TAT-ORF was added to patients’ cells: F528 (D), F334 (E), F511 (F) and nonrelevant NDUFS2 cells (G); mitochondria was isolated and complex I activity was measured. TAT-ORF was added at a final concentration of 0.022 μg/μL (C–F) or 0.032 μg/μL (G), for 3 h (C) or 24 h (D–G). Complex I activity was measured as described in Material and Methods. Activity values are expressed as nmol/min/mg protein and presented as average of triplicates ± standard deviation (SD).

Figure 4

Effect of TAT-ORF fusion protein on patients’ cell viability. F528 cells were incubated with TAT-ORF (0.02 μg/μL final concentration; however, at different start concentrations) for 24 h (A) or 48 h (B), and cell viability was measured. Results are expressed as percentage of proliferation (untreated cells being 100%) and are the mean of triplicates ± SD. *p < 0.05.

Figure 5

Complex I activity in TAT-ORF–treated cells: calibration studies. (A, B) Complex I activity in F528 cells treated with TAT-ORF fusion protein (0.022 μg/μL, final concentration) as a function of the amount of mitochondria (A): 1.67 μg protein (1), 3.35 μg (2), 5 μg (3) of isolated mitochondria and incubation time (B). (C,D) Complex I activity in F528 cells treated with TAT-ORF fusion protein as a function of the start concentration of the TAT-ORF fusion protein (C) and final concentration in the assay (D). Incubation was performed for 24 h. Complex I activity was measured as described in Material and Methods. Activity values are expressed as nmol/min/mg protein and presented as average of triplicates ± SD.

Figure 6

Treatment with TAT-ORF improves functionality of patients’ mitochondria mutated in the C6ORF66 (NDUFAF4) assembly factor. The effect of TAT-ORF treatment was tested on the following: amount of the cells (A); mitochondrial content (B); production of total ATP in the cells, normalized to the amount of cell ATP/methylene blue (MB) (C) and normalized to mitochondrial content ATP/MitoTracker Green (MTG) (D); mitochondrial membrane potential TMRE/MTG (E); and production of free radicals DCF/MTG (F). The experiment was performed with cells from patient F511 treated with TAT-ORF (0.022 μg/μL, final concentration) for 24 h. All measures were performed as described in Materials and Methods. Data are presented as the average of four to five repeats ± SD. *p < 0.5.