Molecular and Functional Effects of Loss of Cytochrome c Oxidase Subunit 8A
Molecular and Functional Effects of Loss of Cytochrome c Oxidase Subunit 8A
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DOI：
10.1134/s0006297921010041
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发表时间：
2021-01
期刊：
影响因子：
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通讯作者：
Daria Rotko;A. Kudin;G. Zsurka;Bogusz Kulawiak;A. Szewczyk;W. Kunz
中科院分区：
文献类型：
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作者：
Daria Rotko;A. Kudin;G. Zsurka;Bogusz Kulawiak;A. Szewczyk;W. Kunz
在这项工作中，我们研究了细胞色素氧化酶COX8A最小核编码亚基缺失在纯合剪接位点突变患者的成纤维细胞和CRISPR/Cas9基因组编辑的HEK293T细胞中的分子和功能影响。在两种细胞模型系统中，可检测到细胞色素氧化酶（COX）残留酶活性的20%至30%。在两种细胞模型的BN-PAGE分离线粒体的免疫印迹中，几乎看不到完全组装的COX的单体和二聚体。有趣的是，与复合物III形成的COX超复合物以及与复合物I和III形成的COX超复合物保留了相当大的免疫反应性，而几乎没有发现可归因于亚组装的免疫反应性。这表明缺乏亚基8A的COX在超配合物中是稳定的，而单体和二聚体则被迅速降解。通过3 ' -RNA测序转录组分析，我们未能在我们的COX8A缺乏的细胞模型中检测到线粒体未折叠蛋白反应（mtUPR）和综合应激反应（ISR）相关基因的转录变化。因此，我们的数据强烈表明，细胞色素氧化酶COX8A的最小亚基对于维持COX单体和二聚体的结构稳定性是必需的。
AbstractIn this work we studied molecular and functional effects of the loss of the smallest nuclear encoded subunit of cytochromecoxidase COX8A in fibroblasts from a patient with a homozygous splice site mutation and in CRISPR/Cas9 genome-edited HEK293T cells. In both cellular model systems, between 20 to 30% of the residual enzymatic activity of cytochromecoxidase (COX) was detectable. In immunoblots of BN-PAGE separated mitochondria from both cellular models almost no monomers and dimers of the fully assembled COX could be visualized. Interestingly, supercomplexes of COX formed with complex III and also with complexes I and III retained considerable immunoreactivity, while nearly no immunoreactivity attributable to subassemblies was found. That indicates that COX lacking subunit 8A is stabilized in supercomplexes, while monomers and dimers are rapidly degraded. With transcriptome analysis by 3′-RNA sequencing we failed to detect in our cellular models of COX8A deficiency transcriptional changes of genes involved in the mitochondrial unfolded protein response (mtUPR) and the integrated stress response (ISR). Thus, our data strongly suggest that the smallest subunit of cytochromecoxidase COX8A is required for maintenance of the structural stability of COX monomers and dimers.