Human xenomitochondrial cybrids - Cellular models of mitochondrial complex I deficiency
Human xenomitochondrial cybrids - Cellular models of mitochondrial complex I deficiency
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DOI：
10.1074/jbc.273.23.14210
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发表时间：
1998-06-05
影响因子：
4.8
通讯作者：
Moraes, CT
中科院分区：
文献类型：
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作者：
Barrientos, A;Kenyon, L;Moraes, CT
线粒体氧化磷酸化系统的亚基由核基因和线粒体基因共同编码。最近，我们试图将非人类类人猿的mtDNA导入缺乏mtDNA(Rho度)的人类细胞系，并成功地产生了人-普通黑猩猩、人-侏儒黑猩猩和人-大猩猩异种线粒体胞质杂交体(HXC)，在这里，我们对这些细胞的氧化磷酸化功能进行了全面的表征。线粒体复合体TC、III、IV和V的活性与亲代人或非人灵长类细胞没有区别。相反，在所有HXC中都观察到复杂的I缺乏。以低分子鱼藤酮或NADH为底物的络合物I的动力学研究表明，在HXC中，Vmax降低了约40%，NADH的Km显著增加(3倍，p<；0.001)，对完整细胞呼吸和通透性细胞中丙酮酸-苹果酸氧化的鱼藤酮抑制研究表明，3 NM鱼藤酮对对照细胞产生轻微的抑制作用(抑制0-10%)，但对HXC的呼吸有明显的抑制(50%-75%)。免疫印迹分析表明，在HXC细胞中，它们的相对含量没有明显变化，这些结果建立了EMC作为人类复合体I缺乏的细胞模型，并强调了核和线粒体基因组共同进化在优化氧化磷酸化功能方面的重要性。
The subunits forming the mitochondrial oxidative phosphorylation system are coded by both nuclear and mitochondrial genes. Recently, we attempted to introduce mtDNA from non-human apes into a human cell line lacking mtDNA (rho degrees), and succeeded in producing human-common chimpanzee, human-pigmy chimpanzee, and human-gorilla xenomitochondrial cybrids (HXC), Here, we present a comprehensive characterization of oxidative phosphorylation function in these cells. Mitochondrial complexes TC, III, IV, and V had activities indistinguishable from parental human or non-human primate cells. In contrast, a complex I deficiency was observed in all HXC. Kinetic studies of complex I using decylubiquinone or NADH as Limiting substrates showed that the V-max was decreased in HXC by approximately 40%, and the K-m for the NADH was significantly increased (3-fold, p < 0.001), Rotenone inhibition studies of intact cell respiration and pyruvate-malate oxidation in permeabilized cells showed that 3 nM rotenone produced a mild effect in control cells (0-10% inhibition) but produced a marked inhibition of HXC respiration (50-75%). Immunoblotting analyses of three subunits of complex I (ND1, 75 and 49 kDa) showed that their relative amounts were not significantly altered in HXC cells, These results establish EMC as cellular models of complex I deficiency in humans and underscore the importance of nuclear and mitochondrial genomes co-evolution in optimizing oxidative phosphorylation function.