High-Throughput Assays for Assessing Mitochondrial Dysfunction Caused by Compounds that Impair mtDNA-Encoded Protein Levels in Eukaryotic Cells

Sashi Nadanaciva, James Murray, Casey Wilson, David F. Gebhard, Yvonne Will
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引用次数: 6

Abstract

Compounds that impair the synthesis of either mitochondrial DNA (mtNDA) or mtDNA-encoded proteins reduce the levels of 13 proteins essential for oxidative phosphorylation, leading to a decrease in mitochondrial ATP production. Toxicity caused by these compounds is seldom identified in 24 to 72 hr cytotoxicity assays due to the low turnover rates of both mtDNA and mtDNA-encoded proteins. Here, we describe three high-throughput screening assays that detect compounds that affect mtDNA-encoded protein levels. All three assays measure the levels of two proteins, one a mtDNA-encoded protein synthesized on mitochondrial ribosomes and the other, a nuclear DNA-encoded protein synthesized on cytosolic ribosomes. The first assay measures the levels of these two proteins by quantitative image analysis and requires a high-content imaging system. The second assay is an in-cell immunoassay that utilizes infrared dyes for detection of the two proteins and, thus, requires a LI-COR Odyssey system. The third assay is an in-cell immunoassay that utilizes colorimetric detection of the two proteins and requires an absorbance microplate reader. Curr. Protoc. Toxicol. 48:3.11.1-3.11.17. © 2011 by John Wiley & Sons, Inc.

评估真核细胞中损害mtdna编码蛋白水平的化合物引起的线粒体功能障碍的高通量测定
损害线粒体DNA (mtNDA)或mtdna编码蛋白质合成的化合物会降低氧化磷酸化所需的13种蛋白质的水平,导致线粒体ATP生成减少。由于mtDNA和mtDNA编码蛋白的低周转率,这些化合物引起的毒性很少在24至72小时的细胞毒性测定中被鉴定出来。在这里,我们描述了三种检测影响mtdna编码蛋白水平的化合物的高通量筛选试验。这三种检测方法测量两种蛋白质的水平,一种是在线粒体核糖体上合成的mtdna编码蛋白质,另一种是在细胞质核糖体上合成的核dna编码蛋白质。第一种方法通过定量图像分析来测量这两种蛋白质的水平,需要高含量的成像系统。第二种分析是细胞内免疫分析,利用红外染料检测两种蛋白质,因此需要LI-COR Odyssey系统。第三种分析是细胞内免疫分析,利用比色法检测两种蛋白质,需要吸光度微孔板读取器。咕咕叫。Protoc。Toxicol 48:3.11.1-3.11.17。©2011 by John Wiley &儿子,Inc。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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