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A Live-cell High-throughput Screening Assay for Identification of Fatty Acid Uptake Inhibitors by Hong Li; Paul N. Black; Concetta C. DiRusso is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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We developed a live-cell high-throughput assay system using the bakerÕs yeast Saccharomyces cerevisiae to screen for chemical compounds that will inhibit fatty acid uptake. The target for the inhibitors is a mammalian fatty acid transport protein (mmFATP2), which is involved in the fatty acid transport and activation pathway. The mmFATP2 was expressed in a S. cerevisiae mutant strain deficient in Fat1p-dependent fatty acid uptake and reduced in long-chain fatty acid activation, fat1Dfaa1D. To detect fatty acid import, a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C 1 -BODIPY-C 12 ), was incubated with cells expressing FATP2 in a 96-well plate. The mmFATP2-dependent C 1 -BODIPY-C 12 uptake was monitored by measuring intracellular C 1 -BODIPY-C 12 fluorescence on a microtiter plate reader, whereas extracellular fluorescence was quenched by a cell viability dye, trypan blue. Using this high-throughput screening method, we demonstrate that the uptake of the fluorescent fatty acid ligand was effectively competed by the natural fatty acid oleate. Inhibition of uptake was also demonstrated to occur when cells were pretreated with s

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Author

Hong Li; Paul N. Black; Concetta C. DiRusso

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

2005

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)