| Summary: | 碩士 === 國立清華大學 === 分子醫學研究所 === 106 === NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) is a nuclear-encoded subunit of human mitochondrial complex I. It contains a binuclear iron sulfur cluster N1a, and may play a role in temporary storage of excess electrons to prevent free radical production. Defects of NDUFV2 have been associated with Alzheimer's disease and Parkinson's disease. Cell-penetrating peptide derived from HIV-1’s transactivator of transcription (TAT) has been successfully applied as a carrier to bring fusion proteins into cells by crossing plasma membranes without compromising the biological function of proteins. In this study, we tried to develop a TAT-mediated protein transduction system to rescue complex I deficiency caused by NDUFV2 defects. Two fusion proteins (TAT-NDUFV2 and NDUFV2-TAT) were exogenously expressed and purified from E. coli for transduction of human cells. In addition, similar constructs were also generated and used in transfection studies for comparison. The results showed that both exogenous TAT-NDUFV2 and NDUFV2-TAT could be delivered into mitochondria and correctly assembled in complex I. Interestingly, the mitochondrial import of TAT-containing NDUFV2 was independent of mitochondrial membrane potential. To explore the therapeutic application of the developed system, a NDUFV2 knockdown cell line (IF4) generated in previous studies was applied for rescuing studies. Treating with TAT-NDUFV2 not only significantly improve the assembly of complex I in IF4 cells, but also partially rescue complex I functions both in the in-gel activity assay and the complex I enzymatic activity assay. In addition, the oxygen consumption rate and mitochondrial membrane potential of IF4 cells were also greatly increased. Similar results were also observed while IF4 cells were treated with NDUFV2-TAT. Our current findings suggest a great potential of applying the TAT-mediated protein transduction system for treatment of complex I deficiency and other mitochondrial disease.
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