Complex I protein NDUFS2 is vital for growth, ROS generation, membrane integrity, apoptosis, and mitochondrial energetics.
Complex I protein NDUFS2 is vital for growth, ROS generation, membrane integrity, apoptosis, and mitochondrial energetics.
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DOI：
10.1016/j.mito.2021.03.003
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发表时间：
2021-05
期刊：
影响因子：
4.4
通讯作者：
Brown DA
中科院分区：
文献类型：
--
作者：
Bandara AB;Drake JC;James CC;Smyth JW;Brown DA
复合体I是线粒体电子传递链(ETC)中最大、最复杂的蛋白质复合体。这种L形状的酶由一个外围亲水的基质结构域和一个膜结合的正交疏水结构域组成。已知这两个臂之间的界面区域对于泛醌部分的结合是关键的，也被证明是络合物I抑制剂的结合部位。由于缺乏基因敲除的细胞系和动物模型，对ETC界面区蛋白的具体作用知之甚少。在这里，我们使用CRISPR/Cas9程序，在人胚胎肾细胞系293中突变了核编码的NADH脱氢酶[泛醌]铁硫蛋白2(NDUFS2)，它是界面区的三个蛋白质亚基之一。NDUFS2的干扰显著降低了细胞在培养液中的生长、复合体I的比呼吸、糖酵解能力、ATP库和细胞膜完整性，但显著增加复合体II的呼吸、ROS的产生、细胞的凋亡和坏死。艾地苯农是一种临床苯醌，目前正在研究其他适应症，治疗部分恢复了突变株的生长、ATP池和氧气消耗。总体而言，我们的结果表明，NDUFS2对哺乳动物细胞的生长和代谢至关重要，NDUFS2功能障碍的呼吸缺陷可以通过对已建立的线粒体治疗候选药物的治疗部分纠正。这是第一次使用CRISPR/Cas9方法构建敲除NDUFS2细胞系，并使用构建的突变体来评估已知的线粒体疗法增强生物能量能力的疗效。
Complex I is the largest and most intricate of the protein complexes of mitochondrial electron transport chain (ETC). This L-shaped enzyme consists of a peripheral hydrophilic matrix domain and a membrane-bound orthogonal hydrophobic domain. The interfacial region between these two arms is known to be critical for binding of ubiquinone moieties and has also been shown to be the binding site of Complex I inhibitors. Knowledge on specific roles of the ETC interfacial region proteins is scarce due to lack of knockout cell lines and animal models. Here we mutated nuclear encoded NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2), one of three protein subunits of the interfacial region, in a human embryonic kidney cell line 293 using a CRISPR/Cas9 procedure. Disruption of NDUFS2 significantly decreased cell growth in medium, Complex I specific respiration, glycolytic capacity, ATP pool and cell-membrane integrity, but significantly increased Complex II respiration, ROS generation, apoptosis, and necrosis. Treatment with idebenone, a clinical benzoquinone currently being investigated in other indications, partially restored growth, ATP pool, and oxygen consumption of the mutant. Overall, our results suggest that NDUFS2 is vital for growth and metabolism of mammalian cells, and respiratory defects of NDUFS2 dysfunction can be partially corrected with treatment of an established mitochondrial therapeutic candidate. This is the first report to use CRISPR/Cas9 approach to construct a knockout NDUFS2 cell line and use the constructed mutant to evaluate the efficacy of a known mitochondrial therapeutic to enhance bioenergetic capacity.
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