Among the five enzyme complexes in the oxidative phosphorylation system, NADH-coenzyme Q oxidoreductase (also called complex I) is the largest, most intricate, and least understood. This enzyme complex spans the inner mitochondrial membrane and catalyzes the first step of electron transfer by the oxidation of NADH, and thereby provides two electrons for the reduction of quinone to quinol. Complex I deficiency is associated with many severe mitochondrial diseases, including Leber hereditary optic neuropathy and Leigh syndrome. However, to date, conventional treatments for the majority of genetic mitochondrial diseases are only palliative. Developing a reliable and convenient therapeutic approach is therefore considered to be an urgent need. Targeted proteins fused with the protein transduction domain of human immunodeficiency virus 1 transactivator of transcription (TAT) have been shown to enter cells by crossing plasma membranes while retaining their biological activities. Recent developments show that, in fusion with mitochondrial targeting sequences (MTSs), TAT-MTS-bound cargo can be correctly transported into mitochondria and restore the missing function of the cargo protein in patients' cells. The available evidence suggests that the TAT-mediated protein transduction system holds great promise as a potential therapeutic approach to treat complex I deficiency, as well as other mitochondrial diseases.

NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8) is a nuclear-encoded core subunit of human mitochondrial complex I. Defects in NDUFS8 are associated with Leigh syndrome and encephalomyopathy. Cell-penetrating peptide derived from the HIV-1 transactivator of transcription protein (TAT) has been successfully applied as a carrier to bring fusion proteins into cells without compromising the biological function of the cargoes. In this study, we developed a TAT-mediated protein transduction system to rescue complex I deficiency caused by NDUFS8 defects. Two fusion proteins (TAT-NDUFS8 and NDUFS8-TAT) were exogenously expressed and purified from Escherichia coli for transduction of human cells. In addition, similar constructs were generated and used in transfection studies for comparison. The results showed that both exogenous TAT-NDUFS8 and NDUFS8-TAT were delivered into mitochondria and correctly processed. Interestingly, the mitochondrial import of TAT-containing NDUFS8 was independent of mitochondrial membrane potential. Treatment with TAT-NDUFS8 not only significantly improved the assembly of complex I in an NDUFS8-deficient cell line, but also partially rescued complex I functions both in the in-gel activity assay and the oxygen consumption assay. Our current findings suggest the considerable potential of applying the TAT-mediated protein transduction system for treatment of complex I deficiency.