Manfredi Giovanni, Fu Jin, Ojaimi Joseline, Sadlock James E, Kwong Jennifer Q, Guy John, Schon Eric A
Department of Neurology and Neuroscience, Cornell University, New York, New York, USA.
Nat Genet. 2002 Apr;30(4):394-9. doi: 10.1038/ng851. Epub 2002 Feb 25.
A T-->G transversion at nt 8993 in mitochondrial DNA of MTATP6 (encoding ATPase 6 of complex V of the respiratory chain) causes impaired mitochondrial ATP synthesis in two related mitochondrial disorders: neuropathy, ataxia and retinitis pigmentosa and maternally inherited Leigh syndrome. To overcome the biochemical defect, we expressed wildtype ATPase 6 protein allotopically from nucleus-transfected constructs encoding an amino-terminal mitochondrial targeting signal appended to a recoded ATPase 6 gene (made compatible with the universal genetic code) that also contained a carboxy-terminal FLAG epitope tag. After transfection of human cells, the precursor polypeptide was expressed, imported into and processed within mitochondria, and incorporated into complex V. Allotopic expression of stably transfected constructs in cytoplasmic hybrids (cybrids) homoplasmic with respect to the 8993T-->G mutation showed a significantly improved recovery after growth in selective medium as well as a significant increase in ATP synthesis. This is the first successful demonstration of allotopic expression of an mtDNA-encoded polypeptide in mammalian cells and could form the basis of a genetic approach to treat a number of human mitochondrial disorders.
MTATP6（编码呼吸链复合体V的ATP酶6）线粒体DNA第8993位核苷酸处的T→G颠换，在两种相关的线粒体疾病中导致线粒体ATP合成受损：神经病变、共济失调和色素性视网膜炎以及母系遗传的Leigh综合征。为了克服这种生化缺陷，我们从核转染构建体异位表达野生型ATP酶6蛋白，该构建体编码一个附加到重新编码的ATP酶6基因（与通用遗传密码兼容）的氨基末端线粒体靶向信号，该基因还包含一个羧基末端FLAG表位标签。在转染人类细胞后，前体多肽被表达、导入线粒体并在其中加工，然后整合到复合体V中。在相对于8993T→G突变同质的细胞质杂种（细胞杂交体）中稳定转染构建体的异位表达显示，在选择性培养基中生长后恢复显著改善，ATP合成也显著增加。这是首次在哺乳动物细胞中成功证明mtDNA编码多肽的异位表达，可为治疗多种人类线粒体疾病的遗传方法奠定基础。
