A novel alkyne cholesterol to trace cellular cholesterol metabolism and localization

Abstract

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. 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. Cholesterol is the major sterol of mammalian cell membranes (1Brown M.S. Goldstein J.L. Cholesterol feedback: from Schoenheimer's bottle to Scap's MELADL.J. Lipid Res. 2009; 50: S15-S27Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Beyond its role for discrete membrane structures such as caveolae (2Parton R.G. Simons K. The multiple faces of caveolae.Nat. Rev. Mol. 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Thiele C. cell imaging of lipid with a new 2009; 10: PubMed Scopus Google Scholar). and used. for the of by the acids of a and and reticulum are L. K. A. Anderson Thiele C. a new tool to Methods. 2: PubMed Scopus Google Scholar). and to the The of alkyne cholesterol and the are in the from of alkyne cholesterol by and are A acid A. C. S. Y. J. of of in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the from with the A. A. C. C. P. and reporter for in 1997; 13: PubMed Scopus Google Scholar) containing the of a using the and The of the by cells in containing to The at for and cells in containing and for to that and cells cells on an at for J. of and cell from Cell Biol. PubMed Scopus Google Scholar). and in and for from the and in containing for To to containing for from the at for for and and in for cells at and To an of of human cells by Y. M. G. 2008; Google Scholar) the cellular sterol an with with of and used. to and with the for in containing acid and cells to containing to cellular and for A. C. S. Y. J. of of in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This and the supplemented with and sterol or with at and using at for cells in in containing or B.R. A for of plasma or protein Lipid Res. Full Text PDF PubMed Google Scholar). Lipids by the with sterol from an to a concentration of with sterols with containing fatty and with in and lipid as A of lipid and J. Biochem. PubMed Scopus Google Scholar), but the of the The lipid by of by of click of of in Click and imaging as C. Papan C. Hoelper D. Kusserow K. Gaebler A. Schoene M. Piotrowitz K. Lohmann D. Spandl J. Stevanovic A. et al.Tracing fatty acid metabolism by click chemistry.ACS Chem. Biol. 2012; 7: 2004-2011Crossref PubMed Scopus (85) Google Scholar). Lipid by and using a or respectively. are in the cholesterol from in and in at and the of in the to of the in the Alkyne cholesterol at with in containing a of supplemented with for The with the and the to a The with and the Lipids in and as but using a click of of in and of the from as as the of the as A. Milan R. Straub L. Hoelper D. Kuerschner L. Thiele C. Alkyne lipids as substrates for click chemistry-based in vitro enzymatic assays.J. Lipid Res. 2013; 54: 2282-2290Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). for in containing and of alkyne cholesterol and in lipids for to a distribution of the for using to cellular lipid transport or living or with cholesterol in and at for Y. H. Cholesterol of the cell Biophys. Acta. PubMed Scopus Google Scholar) lipid sterols by and alkyne sterols by and also by with containing and alkyne cholesterol for in and using in A for at with and with A. the of the in A and to the The click by of in and at for using A. with fluorescent or and from by the using and The of in containing the and of sterol or to the using a cell and to a The a and at using a for Y. Thiele C. Huttner W.B. Cholesterol is required for the of and from the 2000; PubMed Scopus Google Scholar). the of and lipids and proteins lipid by as C. Papan C. Hoelper D. Kusserow K. Gaebler A. Schoene M. Piotrowitz K. Lohmann D. Spandl J. Stevanovic A. et al.Tracing fatty acid metabolism by click chemistry.ACS Chem. Biol. 2012; 7: 2004-2011Crossref PubMed Scopus (85) Google Scholar), to the as The protein by for the distribution of proteins of various cell using a with a and and a using the The a employing J. I. M. T. S. C. S. B. et an for Methods. 2012; PubMed Scopus Google Scholar) and of by To trace cellular we have an analog of alkyne cholesterol The alkyne moiety the terminal of the and in an analog of structural to To the of alkyne we a sterol the A. C. S. Y. J. of of in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these cells are to sterols and on to the various and alkyne or the of acid to these cells and their Alkyne as as the sterols and the an analog a in the for cell In cells by cell M.G. A. D. T. P. of sterol cell and cell Natl. Acad. Sci. USA. 2010; PubMed Scopus Google Scholar), and alkyne cholesterol to the These data that alkyne cholesterol can sterol in these cells. we the of alkyne cholesterol to the by cholesterol using detection for alkyne lipids C. Papan C. Hoelper D. Kusserow K. Gaebler A. Schoene M. Piotrowitz K. Lohmann D. Spandl J. Stevanovic A. et al.Tracing fatty acid metabolism by click chemistry.ACS Chem. Biol. 2012; 7: 2004-2011Crossref PubMed Scopus (85) Google Scholar), for in vitro A. Milan R. Straub L. Hoelper D. Kuerschner L. Thiele C. Alkyne lipids as substrates for click chemistry-based in vitro enzymatic assays.J. Lipid Res. 2013; 54: 2282-2290Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Cholesterol from accepted alkyne cholesterol as a and the enzymatic The as by These enzymatic by studies in living mammalian cells the of alkyne cholesterol to alkyne cholesterol human cells with alkyne cholesterol and for cellular alkyne metabolites by click and in the alkyne cholesterol and its cellular levels of cells in with a in alkyne cholesterol and but the of alkyne cholesterol with cells in the of the alkyne The of alkyne and the for both The of in the different studied in in of with alkyne be in for to the alkyne cholesterol levels of a that be by the with acid to sterol ester the cells in levels of in the of acid of of alkyne cholesterol The of sterol also studied in cells of the with alkyne cholesterol in the and to generate alkyne cholesterol to different the of the levels of alkyne cholesterol and alkyne cholesterol of to alkyne by these cells (6Russell D.W. Oxysterol biosynthetic enzymes.Biochim. Biophys. Acta. 2000; 1529: 126-135Crossref PubMed Scopus (308) Google Scholar). To the of alkyne cholesterol on cellular sterol cells with alkyne or in with for and cellular sterols by with sterol cells levels of and important in cellular sterol biosynthesis, with alkyne This is as cholesterol in a of its The cellular levels of an enzymatic of the of the cholesterol concentration the different alkyne a of alkyne cholesterol to cholesterol of The of from and the supplemented with also the cellular levels of the different and cells with cells of the in a of the of both cholesterol and alkyne cholesterol that by levels of cholesterol also in a of in these of sterol metabolites in Cholesterol alkyne cells in containing and supplemented with alkyne or sterol lipids by are in a new of in Alkyne Cholesterol Cholesterol Alkyne Cholesterol Cholesterol cells in containing and supplemented with alkyne or also sterol lipids by are in a new cells in containing and supplemented with alkyne or sterol lipids by are cells in containing and supplemented with alkyne or also sterol lipids by are important of alkyne cholesterol is its to microscopy. Using the allows for on cholesterol metabolism and To alkyne cholesterol by fluorescence in we a employing different azido the use of these detection allows for a great of with different In we detected cellular alkyne cholesterol click with an to a fluorescent or an to and with a fluorescent With both we alkyne cholesterol at the plasma membrane and for sterol using the in a is and be with cells in sterol the with alkyne cholesterol the of the cellular and membranes a labeling This to the binding of to alkyne cholesterol using an in vitro the of the sterol interaction J. J. P. M.A. of the with and membranes containing Biophys. Acta. PubMed Scopus Google Scholar). that binds to both cholesterol and alkyne but an interaction with cholesteryl To the subcellular distribution of alkyne cholesterol in we using fluorescent proteins for different cellular detected in the Golgi and and in but alkyne fluorescence in lipid The plasma membrane of alkyne cholesterol in by and by an in vivo employing cholesterol of alkyne cholesterol and we the by and the of enzymatic to A of both supplemented sterols in the plasma membrane to by the cells a the that the sterol at the plasma membrane with by cellular for the enzymatic from alkyne cholesterol by analysis and The the from the also cellular and have the distribution of alkyne and sterols in the different of a a distribution for alkyne cholesterol and cholesterol on and their sterol on the other by for proteins of various cell the to and from and from the plasma lipid the both sterols that the cells alkyne cholesterol from analysis of alkyne cholesterol and fluorescent proteins with alkyne cholesterol in for and for alkyne cholesterol by a click using an to used for the of click reporter and proteins in with in a The are as To trace the cellular metabolism and localization of the alkyne analog cholesterol Its is as and are alkyne cholesterol are of are with analysis Alkyne cholesterol and other alkyne lipids can be detected from lipid by as we for alkyne fatty by and with click with a reporter C. Papan C. Hoelper D. Kusserow K. Gaebler A. Schoene M. Piotrowitz K. Lohmann D. Spandl J. Stevanovic A. et al.Tracing fatty acid metabolism by click chemistry.ACS Chem. Biol. 2012; 7: 2004-2011Crossref PubMed Scopus (85) Google Scholar). the cholesterol we to the alkyne moiety at the of the This allows for a on the and biological as important of the cholesterol are F. Fluorescent probe of membrane and Lipid Res. PubMed Scopus Google Scholar) and of B. S. chemical of protein in living 2011; 47: PubMed Scopus Google Scholar) is the structural alkyne cholesterol its to cholesterol with the fluorescent dehydroergosterol and (13Mukherjee S. Zha X. Tabas I. Maxfield F.R. Cholesterol distribution in living cells: fluorescence imaging using dehydroergosterol as a fluorescent cholesterol analog.Biophys. J. 1998; 75: 1915-1925Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 14Wistner D. Fluorescent sterols as tools in membrane biophysics and cell biology.Chem. Phys. Lipids. 2007; 146: 1-25Crossref PubMed Scopus (121) Google Scholar, 15McIntosh A.L. Atshaves B.P. Huang H. Gallegos A.M. Kier A.B. Schroeder F. Fluorescence techniques using dehydroergosterol to study cholesterol trafficking.Lipids. 2008; 43: 1185-1208Crossref PubMed Scopus (58) Google Scholar, F.R. D. of cholesterol with fluorescent Cell Biol. 2012; PubMed Scopus Google Scholar). these probes in the sterol and in but the of cholesterol is for many alkyne cholesterol a probe with an from as a To alkyne cholesterol we employed various biological Alkyne cholesterol is accepted by enzymes from different biological species (Brevibacterium, yeast, rat, and these enzymes include cholesterol oxidases, hydroxylases, and acyl have the use of alkyne cholesterol in in vitro and in vivo and its enzymatic to various alkyne cholesterol for the and of a on of A that also is for sterol has been used for of (13Mukherjee S. Zha X. Tabas I. Maxfield F.R. Cholesterol distribution in living cells: fluorescence imaging using dehydroergosterol as a fluorescent cholesterol analog.Biophys. J. 1998; 75: 1915-1925Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). A major of the alkyne cholesterol analog is its to microscopy. a click employing a of reporter and alkyne cholesterol conveniently at subcellular alkyne cholesterol binds to a cholesterol an detection is In a localization using or both and click we detected sterol labeling of the plasma membrane in with the that the of cellular cholesterol is Y. membranes the and of the cholesterol and in human Biol. Chem. Full Text PDF PubMed Google Scholar). a in the the of the at alkyne cholesterol we that also interacts with alkyne cholesterol in in with in vitro binding These cellular of cholesterol have been in many studies employing (13Mukherjee S. Zha X. Tabas I. Maxfield F.R. Cholesterol distribution in living cells: fluorescence imaging using dehydroergosterol as a fluorescent cholesterol analog.Biophys. J. 1998; 75: 1915-1925Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 14Wistner D. Fluorescent sterols as tools in membrane biophysics and cell biology.Chem. Phys. Lipids. 2007; 146: 1-25Crossref PubMed Scopus (121) Google Scholar, 15McIntosh A.L. Atshaves B.P. Huang H. Gallegos A.M. Kier A.B. Schroeder F. Fluorescence techniques using dehydroergosterol to study cholesterol trafficking.Lipids. 2008; 43: 1185-1208Crossref PubMed Scopus (58) Google Scholar, F.R. D. of cholesterol with fluorescent Cell Biol. 2012; PubMed Scopus Google Scholar), and a of the sterol binding O (17Fujimoto T. Hayashi M. Iwamoto M. Ohno-Iwashita Y. Crosslinked plasmalemmal cholesterol is sequestered to caveolae: analysis with a new cytochemical probe.J. Histochem. Cytochem. 1997; 45: 1197-1205Crossref PubMed Scopus (61) Google Scholar, Y. Shimada Y. Hayashi M. Iwamoto M. Iwashita S. Inomata M. Cholesterol-binding toxins and anti-cholesterol antibodies as structural probes for cholesterol localization.Subcell. Biochem. 2010; 51: 597-621Crossref PubMed Scopus (42) Google Scholar, Ohno-Iwashita Y. van Shimada Y. T. localization of cholesterol using and Histochem. Cytochem. 50: PubMed Scopus Google Scholar). Using alkyne cholesterol and we of the and mitochondria. subcellular analysis to the imaging and of alkyne and cholesterol localization. Thus far, and labeling have been in studies using or the in an analysis Ohno-Iwashita Y. van Shimada Y. T. localization of cholesterol using and Histochem. Cytochem. 50: PubMed Scopus Google Scholar). is the of alkyne cholesterol to cells the sterol at the a A. Radhakrishnan A. of cholesterol in endoplasmic reticulum membranes and of at a cholesterol Biol. Chem. 2010; Full Text Full Text PDF PubMed Scopus (76) Google Scholar, A. Goldstein J.L. Brown M.S. of transport by in a 2008; 8: Full Text Full Text PDF PubMed Scopus Google Scholar), that would for labeling by A of the interaction on the cholesterol levels has been (18Ohno-Iwashita Y. Shimada Y. Hayashi M. Iwamoto M. Iwashita S. Inomata M. Cholesterol-binding toxins and anti-cholesterol antibodies as structural probes for cholesterol localization.Subcell. Biochem. 2010; 51: 597-621Crossref PubMed Scopus (42) Google Scholar, A. Radhakrishnan A. of cholesterol in endoplasmic reticulum membranes and of at a cholesterol Biol. Chem. 2010; Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Filipin is to to membranes G. K. for cholesterol binding and cell 2011; PubMed Scopus Google Scholar), and the of and F.R. D. of cholesterol with fluorescent Cell Biol. 2012; PubMed Scopus Google Scholar) detection of levels of sterol in specific cellular the is to an important of cholesterol on cholesterol in are by that of or cholesterol in from the T. G. Lipid analysis of membranes from the Biophys. Acta. 2009; PubMed Scopus Google Scholar) and in from M.A. X. of the using Mol. Biol. 2009; PubMed Scopus Google Scholar), respectively. In summary, the novel alkyne cholesterol for tracking cellular cholesterol metabolism and localization. represents a versatile, sensitive, and easy-to-use tool for manifold of detection such as and fluorescence microscopy. important studies using alkyne cholesterol are of to great be to a probe represents a suitable tool for the specific alkyne cholesterol and metabolites by using is the for an chemical detection alkyne cholesterol is to the sterol that are in lipid the be to the click with a reporter the of these of lipid in is to microscopy, to on cholesterol ester and as cholesterol to the cellular sterol the of the cellular cholesterol by imaging the a it for lipid tracking in living cells. be in the The are to and for the for human and respectively. for with the The of their are for of the with acid dehydroergosterol endoplasmic reticulum cell