Abstract
Defects in subunits of mitochondrial complex I are associated with severe diseases, including Leber hereditary optic neuropathy and Leigh syndrome. However, to date, conventional treatment for the majority of genetic-based mitochondrial diseases can only be palliative. Therefore, developing a reliable and convenient treatment approach is in an urgent need. Fusion of the protein transduction domain (PTD) of HIV-1 transactivator of transcription (TAT) with proteins has been demonstrated to bring proteins into cells by crossing plasma membranes while retaining the biological activity of proteins. In this study, we tried to apply the protein transduction concept of TAT with the mitochondrial-targeting capability of the specific leader sequence to generate a therapeutic protein delivery system which can specifically carry target proteins into mitochondria. Here, NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8), the first complex I subunit linked to Leigh syndrome, was used as the model subunit to test our specific aims, with a hope that this newly developed method could become a novel treatment for complex I deficiency. Currently, our findings showed that both exogenously produced TAT-NDUFS8 and NDUFS8-TAT could be delivered into mitochondria and processed into the mature forms of NDUFS8. We also showed that the mechanism of TAT-NDUFS8 and NDUFS8-TAT entering mitochondria is not through the well-recognized translocase of the outer membrane (Tom) /translocase of the inner membrane (Tim) mitochondrial import pathway. Furthermore, in order to mimic the rescue of complex I deficiency, a NDUFS8 expression knockdown cell line (shRNA-C3) was used in functional analyses as the therapeutic model. Treating with TAT-NDUFS8 could completely restore the assembly of complex I in shRNA-C3 cells, and the respiratory rate of these NDUFS8 knockdown cells was also increased about 31% and 79% in the in-gel activity assay and oxygen consumption assay, respectively. Moreover, we demonstrated that when cells were cultured with TAT-NDUFS8, endosomes were found to be retrieved in close proximity to mitochondria, indicating that TAT-NDUFS8 may enter mitochondria via the endosomes-mitochondria juxtaposition. In conclusion, our findings provide both the possible mechanism of TAT-NDUFS8 entering mitochondria and the model for therapeutic treatment of mitochondrial disorders.