High-content screening for compounds that affect mtDNA-encoded protein levels in eukaryotic cells

Journal of Biomolecular Screening
Sashi NadanacivaYvonne Will

Abstract

Compounds that interfere with the synthesis of either mitochondrial DNA or mtDNA-encoded proteins reduce the levels of 13 proteins essential for oxidative phosphorylation, leading to a decrease in mitochondrial adenosine triphosphate (ATP) production. Toxicity caused by these compounds is seldom identified in 24- to 72-h cytotoxicity assays due to the low turnover rates of both mtDNA and mtDNA-encoded proteins. To address this problem, the authors developed a 96-well format, high-content screening (HCS) assay that measures, in eukaryotic cells, the level of Complex IV-subunit 1, an mtDNA-encoded protein synthesized on mitochondrial ribosomes, and the level of Complex V-alpha subunit, a nuclear DNA-encoded protein synthesized on cytosolic ribosomes. The effect of several antibiotics and antiretrovirals on these 2 proteins was assessed, in transformed human liver epithelial cells, 6 days after compound treatment. The results confirmed effects of drugs known to reduce mtDNA-encoded protein levels and also revealed novel information showing that several fluoroquinolones and a macrolide, josamycin, impaired expression of mtDNA-encoded proteins. The HCS assay was robust with an average Z' factor of 0.62. The assay enables large-scale...Continue Reading

References

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Citations

Dec 15, 2011·Toxicological Sciences : an Official Journal of the Society of Toxicology·Zhiwu Lin, Yvonne Will
Jul 16, 2014·Expert Opinion on Drug Metabolism & Toxicology·Yvonne Will, James Dykens
Feb 11, 2015·The International Journal of Biochemistry & Cell Biology·Eligio F IannettiWerner J H Koopman
Sep 23, 2014·Regulatory Toxicology and Pharmacology : RTP·Christine M HuntAyako Suzuki
Apr 25, 2013·Journal of Medicinal Chemistry·Tatiana StoilovaMario Salmona
Apr 9, 2014·Journal of Biomolecular Screening·Shantanu SinghAuguste Genovesio
Dec 8, 2017·Frontiers in Molecular Biosciences·Matthew J Young
Mar 23, 2018·Nature·Jo Marchant
Jan 10, 2020·The Journal of Biological Chemistry·Amita GuptaChaitan Khosla
Apr 28, 2019·International Journal of Molecular Sciences·Steffi GoffartJaakko L O Pohjoismäki
Jul 30, 2020·BMJ Open Ophthalmology·Nasim SalimiaghdamM Cristina Kenney

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Methods Mentioned

BETA
electrophoresis
Infrared Imaging
Fluorescence

Software Mentioned

GraphPad Prism
Compartmental Analysis Bioapplication

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