Lipid-sensing high-throughput ApoA-I assays

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Journal titleJournal of Biomolecular Screening
Pages10501061; # of pages: 12
SubjectABC transporter A1; apolipoprotein A1; biotin; high density lipoprotein cholesterol; lipid; streptavidin; terbium; amino terminal sequence; artery wall; article; atherosclerosis; biotinylation; cholesterol metabolism; cholesterol transport; controlled study; fluorescence resonance energy transfer; foam cell; high throughput screening; human; human cell; lipid analysis; phospholipid transfer; priority journal; protein determination; radioactivity; risk assessment; Apolipoprotein A-I; Atherosclerosis; ATP-Binding Cassette Transporters; Biotin; Cells, Cultured; Cholesterol; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; High-Throughput Screening Assays; Humans; Lipid Metabolism; Lipids; Lipoproteins, HDL; Macrophages; Streptavidin
AbstractApolipoprotein A-I (ApoA-I), a primary protein component of high-density lipoprotein (HDL), plays an important role in cholesterol metabolism mediating the formation of HDL and the efflux of cellular cholesterol from macrophage foam cells in arterial walls. Lipidation of ApoA-I is mediated by adenosine triphosphate (ATP) binding cassette A1 (ABCA1). Insufficient ABCA1 activity may lead to increased risk of atherosclerosis due to reduced HDL formation and cholesterol efflux. The standard radioactive assay for measuring cholesterol transport to ApoA-I has low throughput and poor dynamic range, and it fails to measure phospholipid transfer. We describe the development of two sensitive, nonradioactive high-throughput assays that report on the lipidation of ApoA-I: a homogeneous assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) and a discontinuous assay that uses the label-free Epic platform. The TR-FRET assay employs HiLyte Fluor 647-labeled ApoA-I with N-terminal biotin bound to streptavidin-terbium. When fluorescent ApoA-I was incorporated into HDL, TR-FRET decreased proportionally to the increase in the ratio of lipids to ApoA-I, demonstrating that the assay was sensitive to the amount of lipid bound to ApoA-I. In the Epic assay, biotinylated ApoA-I was captured on a streptavidin-coated biosensor. Measured resonant wavelength shift was proportional to the amount of lipids associated with ApoA-I, indicating that the assay senses ApoA-I lipidation. © 2012 Society for Laboratory Automation and Screening.
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AffiliationNational Research Council Canada (NRC-CNRC); NRC Institute for Nutrisciences and Health (INH-ISNS)
Peer reviewedYes
NPARC number21269417
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Record identifier0c65e909-444a-4fc9-8e51-edaf0d8fc109
Record created2013-12-12
Record modified2016-05-09
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