Molecular Medicine

, Volume 19, Issue 1, pp 124–134 | Cite as

Replacement of the C6ORF66 Assembly Factor (NDUFAF4) Restores Complex I Activity in Patient Cells

  • Dana Marcus
  • Michal Lichtenstein
  • Ann Saada
  • Haya Lorberboum-Galski
Research Article


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.



This work was supported by a grant from the Israel Science Foundation (grant 322/09 to H Lorberboum-Galski) and in part by the Israel Science Foundation (grant 1462/09 to A Saada).


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Authors and Affiliations

  • Dana Marcus
    • 1
  • Michal Lichtenstein
    • 1
  • Ann Saada
    • 2
  • Haya Lorberboum-Galski
    • 1
  1. 1.Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada (IMRIC)Hebrew University-Hadassah Medical SchoolJerusalemIsrael
  2. 2.Monique and Jacques Roboh Department of Genetic Research, Department of Genetics and Metabolic Diseases, HadassahHebrew University Medical CenterJerusalemIsrael

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