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 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.

[1]  D. Russell,et al.  Oxysterol biosynthetic enzymes. , 2000, Biochimica et biophysica acta.

[2]  Heinrich J.G. Matthies,et al.  Cholesterol oxidase susceptibility of the red cell membrane. , 1984, Biochimica et biophysica acta.

[3]  P. Walter,et al.  Structure of sterol aliphatic chains affects yeast cell shape and cell fusion during mating , 2010, Proceedings of the National Academy of Sciences.

[4]  P. Philippsen,et al.  Heterologous HIS3 Marker and GFP Reporter Modules for PCR‐Targeting in Saccharomyces cerevisiae , 1997, Yeast.

[5]  F. Schroeder,et al.  Fluorescent sterols: probe molecules of membrane structure and function. , 1984, Progress in lipid research.

[6]  Brent R. Martin,et al.  Large-scale profiling of protein palmitoylation in mammalian cells , 2009, Nature Methods.

[7]  W. Huttner,et al.  Cholesterol is Required for the Formation of Regulated and Constitutive Secretory Vesicles from the trans‐Golgi Network , 2000, Traffic.

[8]  J. Goldstein,et al.  Use of mutant 125I-Perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells , 2013, Proceedings of the National Academy of Sciences.

[9]  Y. Yamaguchi-Iwai,et al.  Inhibition of Heme Biosynthesis Prevents Transcription of Iron Uptake Genes in Yeast* , 2003, Journal of Biological Chemistry.

[10]  T. Fujimoto,et al.  Crosslinked Plasmalemmal Cholesterol Is Sequestered to Caveolae: Analysis with a New Cytochemical Probe , 1997, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[11]  J. Peychl,et al.  Live Cell Multicolor Imaging of Lipid Droplets with a New Dye, LD540 , 2009, Traffic.

[12]  J. Slot,et al.  Immunoelectron Microscopic Localization of Cholesterol Using Biotinylated and Non-cytolytic Perfringolysin O , 2002, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[13]  Jennifer A. Prescher,et al.  Rapid and selective detection of fatty acylated proteins using ω-alkynyl-fatty acids and click chemistry[S] , 2010, Journal of Lipid Research.

[14]  J. Vance.  Dysregulation of cholesterol balance in the brain: contribution to neurodegenerative diseases , 2012, Disease Models & Mechanisms.

[15]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[16]  Shobha Ghosh,et al.  Macrophage cholesteryl ester mobilization and atherosclerosis. , 2010, Vascular pharmacology.

[17]  Daniel Wüstner,et al.  Analysis of cholesterol trafficking with fluorescent probes. , 2012, Methods in cell biology.

[18]  Edward W. Tate,et al.  Bioorthogonal chemical tagging of protein cholesterylation in living cells. , 2011, Chemical communications.

[19]  E. Leitner,et al.  Lipid analysis of mitochondrial membranes from the yeast Pichia pastoris. , 2009, Biochimica et biophysica acta.

[20]  F. Maxfield,et al.  Cholesterol distribution in living cells: fluorescence imaging using dehydroergosterol as a fluorescent cholesterol analog. , 1998, Biophysical journal.

[21]  A. Gavin,et al.  In vivo profiling and visualization of cellular protein-lipid interactions using bifunctional fatty acids. , 2013, Angewandte Chemie.

[22]  A. Shevchenko,et al.  Tracing fatty acid metabolism by click chemistry. , 2012, ACS chemical biology.

[23]  Y. Shimada,et al.  Cholesterol-binding toxins and anti-cholesterol antibodies as structural probes for cholesterol localization. , 2010, Sub-cellular biochemistry.

[24]  K. Moore,et al.  Macrophages in atherosclerosis: a dynamic balance , 2013, Nature Reviews Immunology.

[25]  C. Schultz,et al.  Selective fluorescence labeling of lipids in living cells. , 2009, Angewandte Chemie.

[26]  R. Hannoush,et al.  The chemical toolbox for monitoring protein fatty acylation and prenylation. , 2010, Nature chemical biology.

[27]  A. Radhakrishnan,et al.  Accessibility of Cholesterol in Endoplasmic Reticulum Membranes and Activation of SREBP-2 Switch Abruptly at a Common Cholesterol Threshold , 2010, The Journal of Biological Chemistry.

[28]  Xianlin Han,et al.  Examination of the brain mitochondrial lipidome using shotgun lipidomics. , 2009, Methods in molecular biology.

[29]  J. Bolard,et al.  Interaction of the polyene antibiotic filipin with model and natural membranes containing plant sterols. , 1988, Biochimica et biophysica acta.

[30]  C. Thiele,et al.  Alkyne lipids as substrates for click chemistry-based in vitro enzymatic assays[S] , 2013, Journal of Lipid Research.

[31]  W. J. Dyer,et al.  A rapid method of total lipid extraction and purification. , 1959, Canadian journal of biochemistry and physiology.

[32]  A. McIntosh,et al.  Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking , 2008, Lipids.

[33]  K. McCarthy,et al.  Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue , 1980, The Journal of cell biology.

[34]  D. Wüstner.  Fluorescent sterols as tools in membrane biophysics and cell biology. , 2007, Chemistry and physics of lipids.

[35]  Andrew J. Brown,et al.  Sterol regulators of cholesterol homeostasis and beyond: the oxysterol hypothesis revisited and revised. , 2008, Progress in lipid research.

[36]  H. Bostic,et al.  Exploiting bioorthogonal chemistry to elucidate protein-lipid binding interactions and other biological roles of phospholipids. , 2011, Accounts of chemical research.

[37]  J. McDonald,et al.  Switch-like control of SREBP-2 transport triggered by small changes in ER cholesterol: a delicate balance. , 2008, Cell metabolism.

[38]  A. Salic,et al.  Metabolic labeling and direct imaging of choline phospholipids in vivo , 2009, Proceedings of the National Academy of Sciences.

[39]  M. Brown,et al.  A receptor-mediated pathway for cholesterol homeostasis. , 1986, Science.

[40]  B. Cravatt,et al.  Proteome-wide Mapping of Cholesterol-Interacting Proteins in Mammalian Cells , 2013, Nature Methods.

[41]  B. Cham,et al.  A solvent system for delipidation of plasma or serum without protein precipitation. , 1976, Journal of lipid research.

[42]  R. Bittman,et al.  Use of Bodipy-labeled sphingolipid and cholesterol analogs to examine membrane microdomains in cells , 2008, Histochemistry and Cell Biology.

[43]  G. Charron,et al.  Chemical tools for understanding protein lipidation in eukaryotes. , 2009, Current opinion in chemical biology.

[44]  R. Parton,et al.  The multiple faces of caveolae , 2007, Nature Reviews Molecular Cell Biology.

[45]  F. Fahrenholz,et al.  Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles , 1999, Nature Cell Biology.

[46]  M. G. Finn,et al.  Click Chemistry: Diverse Chemical Function from a Few Good Reactions. , 2001, Angewandte Chemie.

[47]  G. Gimpl,et al.  Probes for studying cholesterol binding and cell biology , 2011, Steroids.

[48]  M. Swaisgood,et al.  Plasma membranes contain half the phospholipid and 90% of the cholesterol and sphingomyelin in cultured human fibroblasts. , 1989, The Journal of biological chemistry.

[49]  C. Bertozzi.  A decade of bioorthogonal chemistry. , 2011, Accounts of chemical research.

[50]  Kai Simons,et al.  Lipid Rafts As a Membrane-Organizing Principle , 2010, Science.

[51]  Christer S. Ejsing,et al.  Polyene-lipids: A new tool to image lipids , 2005, Nature Methods.