Yen Hsiu-Chuan, Liu Yi-Chun, Kan Chia-Chi, Wei Hsing-Ju, Lee Szu-Hsien, Wei Yau-Huei, Feng Yu-Hsiu, Chen Chih-Wei, Huang Chin-Chang
Graduate Institute and Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Department of Nephrology, Chang Gung Memorial Hospital, Taoyuan, Taiwan.
Graduate Institute and Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
Biochim Biophys Acta. 2016 Sep;1860(9):1864-76. doi: 10.1016/j.bbagen.2016.05.005. Epub 2016 May 4.
The Coq protein complex assembled from several Coq proteins is critical for coenzyme Q6 (CoQ6) biosynthesis in yeast. Secondary CoQ10 deficiency is associated with mitochondrial DNA (mtDNA) mutations in patients. We previously demonstrated that carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) suppressed CoQ10 levels and COQ5 protein maturation in human 143B cells.
This study explored the putative COQ protein complex in human cells through two-dimensional blue native-polyacrylamide gel electrophoresis and Western blotting to investigate its status in 143B cells after FCCP treatment and in cybrids harboring the mtDNA mutation that caused myoclonic epilepsy with ragged-red fibers (MERRF) syndrome. Ubiquinol-10 and ubiquinone-10 levels were detected by high-performance liquid chromatography. Mitochondrial energy status, mRNA levels of various PDSS and COQ genes, and protein levels of COQ5 and COQ9 in cybrids were examined.
A high-molecular-weight protein complex containing COQ5, but not COQ9, in the mitochondria was identified and its level was suppressed by FCCP and in cybrids with MERRF mutation. That was associated with decreased mitochondrial membrane potential and mitochondrial ATP production. Total CoQ10 levels were decreased under both conditions, but the ubiquinol-10:ubiquinone-10 ratio was increased in mutant cybrids. The expression of COQ5 was increased but COQ5 protein maturation was suppressed in the mutant cybrids.
A novel COQ5-containing protein complex was discovered in human cells. Its destabilization was associated with reduced CoQ10 levels and mitochondrial energy deficiency in human cells treated with FCCP or exhibiting MERRF mutation.
The findings elucidate a possible mechanism for mitochondrial dysfunction-induced CoQ10 deficiency in human cells.
由几种Coq蛋白组装而成的Coq蛋白复合物对酵母中辅酶Q6（CoQ6）的生物合成至关重要。继发性辅酶Q10缺乏与患者的线粒体DNA（mtDNA）突变有关。我们之前证明，羰基氰化物 - 对 - 三氟甲氧基苯腙（FCCP）可抑制人143B细胞中辅酶Q10水平和COQ5蛋白成熟。
本研究通过二维蓝色非变性聚丙烯酰胺凝胶电泳和蛋白质印迹法探索人细胞中假定的COQ蛋白复合物，以研究其在FCCP处理后的143B细胞以及携带导致肌阵挛性癫痫伴破碎红纤维（MERRF）综合征的mtDNA突变的细胞杂交体中的状态。通过高效液相色谱法检测泛醇 - 10和泛醌 - 10水平。检测细胞杂交体中的线粒体能量状态、各种PDSS和COQ基因的mRNA水平以及COQ5和COQ9的蛋白质水平。
在mitochondria中鉴定出一种含有COQ5但不含COQ9的高分子量蛋白复合物，其水平在FCCP处理的细胞以及具有MERRF突变的细胞杂交体中受到抑制。这与线粒体膜电位降低和线粒体ATP产生减少有关。在这两种情况下，总辅酶Q10水平均降低，但突变细胞杂交体中泛醇 - 10：泛醌 - 10的比率增加。突变细胞杂交体中COQ5的表达增加，但COQ5蛋白成熟受到抑制。
在人细胞中发现了一种新型的含COQ5蛋白复合物。在用FCCP处理或表现出MERRF突变的人细胞中，其不稳定与辅酶Q10水平降低和线粒体能量缺乏有关。
这些发现阐明了人细胞中线粒体功能障碍诱导的辅酶Q10缺乏的一种可能机制。
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