Institute of Molecular Medicine, National Tsing Hua University, Hsinchu 30013, Taiwan.
Department of Life Science, College of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan.
Int J Mol Sci. 2021 Jun 17;22(12):6524. doi: 10.3390/ijms22126524.
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 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.
NADH 脱氢酶（泛醌）Fe-S 蛋白 8（NDUFS8）是人类线粒体复合物 I 的核编码核心亚基。NDUFS8 的缺陷与 Leigh 综合征和脑肌病有关。来自 HIV-1 转录激活蛋白（TAT）的穿膜肽已成功应用于作为载体将融合蛋白带入细胞而不影响货物的生物功能。在这项研究中，我们开发了一种 TAT 介导的蛋白转导系统来挽救由 NDUFS8 缺陷引起的复合物 I 缺乏。两种融合蛋白（TAT-NDUFS8 和 NDUFS8-TAT）从大肠杆菌中表达和纯化，用于转导人细胞。此外，还生成了类似的构建体并用于转染研究进行比较。结果表明，外源性 TAT-NDUFS8 和 NDUFS8-TAT 均被递送至线粒体并正确加工。有趣的是，含 TAT 的 NDUFS8 的线粒体导入不依赖于线粒体膜电位。TAT-NDUFS8 的处理不仅显著改善了 NDUFS8 缺陷细胞系中复合物 I 的组装，而且在凝胶活性测定和耗氧量测定中部分挽救了复合物 I 的功能。我们目前的发现表明，应用 TAT 介导的蛋白转导系统治疗复合物 I 缺乏症具有相当大的潜力。
