Succinate dehydrogenase (Complex II) plays a dual role in respiration by catalyzing the oxidation of succinate to fumarate in the mitochondrial Krebs cycle and transferring electrons from succinate to ubiquinone in the mitochondrial electron transport chain (ETC). Mutations in Complex II are associated with a number of pathologies. SDHD, one of the four subunits of Complex II, serves by anchoring the complex to the inner-membrane and transferring electrons from the complex to ubiquinone. Thus, modeling SDHD dysfunction could be a valuable tool for understanding its importance in metabolism and developing novel therapeutics, however no suitable models exist. Via CRISPR/Cas9, we mutated SDHD in HEK293 cells and investigated the in vitro role of SDHD in metabolism. Compared to the parent HEK293, the knockout mutant HEK293ΔSDHD produced significantly less number of cells in culture. The mutant cells predictably had suppressed Complex II-mediated mitochondrial respiration, but also Complex I-mediated respiration. SDHD mutation also adversely affected glycolytic capacity and ATP synthesis. Mutant cells were more apoptotic and susceptible to necrosis. Treatment with the mitochondrial therapeutic idebenone partially improved oxygen consumption and growth of mutant cells. Overall, our results suggest that SDHD is vital for growth and metabolism of mammalian cells, and that respiratory and growth defects can be partially restored with treatment of a ubiquinone analog. This is the first report to use CRISPR/Cas9 approach to construct a knockout SDHD cell line and evaluate the efficacy of an established mitochondrial therapeutic candidate to improve bioenergetic capacity.

中文翻译：

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复合物 II 亚基 SDHD 对细胞生长和代谢至关重要，可通过合成泛醌类似物部分恢复

琥珀酸脱氢酶（复合物 II）通过在线粒体三羧酸循环中催化琥珀酸氧化为延胡索酸并将电子从琥珀酸转移到线粒体电子传递链 (ETC) 中的泛醌，在呼吸中发挥双重作用。复合体 II 中的突变与许多病理相关。SDHD 是复合物 II 的四个亚基之一，通过将复合物锚定在内膜并将电子从复合物转移到泛醌来发挥作用。因此，对 SDHD 功能障碍进行建模可能是了解其在代谢和开发新疗法中的重要性的宝贵工具，但尚无合适的模型存在。通过 CRISPR/Cas9，我们对 HEK293 细胞中的 SDHD 进行了突变，并研究了 SDHD 在代谢中的体外作用。与母体 HEK293 相比，敲除突变体 HEK293ΔSDHD 产生的培养细胞数量明显减少。可以预见，突变细胞抑制了复合物 II 介导的线粒体呼吸，也抑制了复合物 I 介导的呼吸。SDHD 突变也对糖酵解能力和 ATP 合成产生不利影响。突变细胞更易凋亡，更容易坏死。用线粒体治疗艾地苯醌治疗部分改善了突变细胞的耗氧量和生长。总体而言，我们的结果表明 SDHD 对哺乳动物细胞的生长和代谢至关重要，并且通过泛醌类似物的治疗可以部分恢复呼吸和生长缺陷。