A novel alkyne cholesterol to trace cellular cholesterol metabolism and localization[S]

Cholesterol is an important lipid of mammalian cells and plays a fundamental role in many biological processes. Its concentration in the various cellular membranes differs and is tightly regulated. Here, we present a novel alkyne cholesterol analog suitable for tracing both cholesterol metabolism an...

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Bibliographic Details
Published in:	Journal of lipid research Vol. 55; no. 3; pp. 583 - 591
Main Authors:	Hofmann, Kristina, Thiele, Christoph, Schött, Hans-Frieder, Gaebler, Anne, Schoene, Mario, Kiver, Yuriy, Friedrichs, Silvia, Litjohann, Dieter, Kuerschner, Lars
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 01-03-2014 The American Society for Biochemistry and Molecular Biology Elsevier
Subjects:	5-Aminolevulinate Synthetase - genetics Acyltransferases - metabolism alkyne lipid Alkynes - chemistry Alkynes - metabolism analog Animals Cell Line, Tumor Cell Membrane - metabolism Cell Tracking - methods Cholesterol - analogs & derivatives Cholesterol - chemistry Cholesterol - metabolism Cholesterol Esters - metabolism Cholesterol Oxidase - metabolism click reaction Endoplasmic Reticulum - metabolism Golgi Apparatus - metabolism Humans Kinetics Methods Microscopy, Fluorescence Mitochondria - metabolism Molecular Structure Mutation oxysterols probe Rats Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae - growth & development Saccharomyces cerevisiae - metabolism Saccharomyces cerevisiae Proteins - genetics Sterols - metabolism analog alkyne lipid click reaction oxysterols probe
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Summary:	Cholesterol is an important lipid of mammalian cells and plays a fundamental role in many biological processes. Its concentration in the various cellular membranes differs and is tightly regulated. Here, we present a novel alkyne cholesterol analog suitable for tracing both cholesterol metabolism and localization. This probe can be detected by click chemistry employing various reporter azides. Alkyne cholesterol is accepted by cellular enzymes from different biological species (Brevibacterium, yeast, rat, human) and these enzymes include cholesterol oxidases, hydroxylases, and acyl transferases that generate the expected metabolites in in vitro and in vivo assays. Using fluorescence microscopy, we studied the distribution of cholesterol at subcellular resolution, detecting the lipid in the Golgi and at the plasma membrane, but also in the endoplasmic reticulum and mitochondria. In summary, alkyne cholesterol represents a versatile, sensitive, and easy-to-use tool for tracking cellular cholesterol metabolism and localization as it allows for manifold detection methods including mass spectrometry, thin-layer chromatography/fluorography, and fluorescence microscopy.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0022-2275 1539-7262
DOI:	10.1194/jlr.D044727