Fatty acids are abundant constituents of all biological systems, and their metabolism is important for normal function at all levels of an organism. Aberrations in fatty acid metabolism are associated with pathological states and have become a focus of current research, particularly due to the interest in metabolic overload diseases. Here we present a click-chemistry-based method that allows tracing of fatty acid metabolism in virtually any biological system. It combines high sensitivity with excellent linearity and fast sample turnover. Since it is free of radioactivity, it can be combined with any other modern analysis technology and can be used in high-throughput applications. Using the new method, we provide for the first time an analysis of cellular fatty metabolism with high time resolution and a comprehensive comparison of utilization of a broad spectrum of fatty acids in hepatoma and adipose cell lines.
1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids that are elevated in the plasma of patients with type 2 diabetes and hereditary sensory and autonomic neuropathy type 1 (HSAN1). Clinically, diabetic neuropathy and HSAN1 are very similar, suggesting the involvement of deoxySLs in the pathology of both diseases. However, very little is known about the biology of these lipids and the underlying pathomechanism. We synthesized an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, to trace the metabolism and localization of deoxySLs. Our results indicate that the metabolism of these lipids is restricted to only some lipid species and that they are not converted to canonical sphingolipids or fatty acids. Furthermore, exogenously added alkyne-doxSA [(2S,3R)-2-aminooctadec-17-yn-3-ol] localized to mitochondria, causing mitochondrial fragmentation and dysfunction. The induced mitochondrial toxicity was also shown for natural doxSA, but not for sphinganine, and was rescued by inhibition of ceramide synthase activity. Our findings therefore indicate that mitochondrial enrichment of an N-acylated doxSA metabolite may contribute to the neurotoxicity seen in diabetic neuropathy and HSAN1. Hence, we provide a potential explanation for the characteristic vulnerability of peripheral nerves to elevated levels of deoxySLs.
the cellular fate of cholesterol by fl uorescence microscopy, various fl uorescent analogs ( 13-16 ), sterol binding toxins ( 17 ), and anti-cholesterol antibodies ( 18 ) have been used. Isotope-labeled cholesterol ( 1 ) and isotope-labeled toxins ( 19 ) have been employed to follow metabolism or organization of cholesterol in membranes, respectively. A probe suitable for conveniently tracing both cholesterol metabolism and localization would be of great value.With the advent of bioorthogonal chemistry ( 20 ), including click chemistry ( 21 ), the sensitive and specifi c detection of compounds containing azido groups or terminal alkynes has become possible. Lipids containing an alkyne moiety have been used to trace their metabolism and distribution ( 22-26 ) and to monitor protein lipidation ( 27-30 ) or protein-lipid interaction ( 31 ). Thus far, no alkyne-bearing analog of cholesterol has been used to trace cholesterol in mammalian cells.Here we report on the synthesis and use of alkyne cholesterol to study cholesterol metabolism by in vitro assays, and in vivo by using various cells. These data are supplemented by cellular localization studies of the probe. MATERIALS AND METHODSFilipin and azide-PEG3-biotin conjugate were obtained from Sigma. Borondifl uorodipyrromethene (Bodipy)-cholesterol [23-(dipyrrometheneboron-difl uoride)-24-norcholesterol] was from 26,26,27,27, H 6 , and 4-cholesten-3-one were from C/D/N Isotopes and Steraloids, respectively. LD540 was described before ( 32 ). Antibodies against calnexin (Stressgen, SPA-860), EGFR (Epitomics, 2235-1), GM130 (Epitomics, 1837-1), and HSP60 (Stressgen, SPA-806) were used.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 fl uorescence 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/fl uorography, and fl uorescence microscopy. Cholesterol is the major sterol of mammalian cell membranes ( 1 ). Beyond its role for discrete membrane structures such as caveolae ( 2 ) or microdomains ( 3 ), cholesterol also specifi cally interacts with numerous...
This article is available online at http://www.jlr.org to radioactive labeling. They are convenient and sensitive reporters, but in general contain spacious tags of high impact on the structure, likely to infl uence specifi city and kinetic parameters of enzymatic reactions. Although there are reports of assays with unchanged affi nity of the enzyme to fl uorescently labeled substrates, notably nitrobenzoxadiazole (NBD)-sphingolipids [e.g., NBD-sphinganine ( 2 ), NBDsphingosine-1-phosphate ( 3 )] the applicability of a substrate has to be validated thoroughly. For instance, when the boron dipyrromethene (BDP) analog, BDP-sphingosine-1-phosphate, was developed as a more photostable and hydrophobic alternative to NBD-sphingosine-1-phosphate, the BDP-, but not the NBD-derivate displayed a higher K m value than the natural substrate ( 4 ). A smaller nonradioactive universally applicable tag with less impact on the substrate structure would hence be benefi cial.Click chemistry ( 5 ), and especially the copper-catalyzed Huisgen 1,3-dipolar cycloaddition between a terminal alkyne group and an azide, provide new possibilities for the bioorthogonal labeling of molecules like amino acids ( 6 ), sugars ( 7 ), and nucleotides ( 8 ), which have already triggered many applications in life sciences ( 9, 10 ). In lipid biology, alkyne and azide analogs and precursors are already applied broadly, e.g., in protein lipidation analyses [reviewed in ( 11 )], receptor binding studies ( 12 ), tracing of fatty acid metabolism ( 13 ), and the labeling of lipids in Abstract Click chemistry is evolving as a powerful tool in biological applications because it allows the sensitive and specifi c detection of compounds with alkyne or azido groups. Here we describe the use of alkyne lipids as substrates for in vitro enzymatic assays of lipid modifying enzymes. The small alkyne moiety is introduced synthetically at the terminus of the hydrocarbon chain of various substrate lipids. After the assay, the label is click-reacted with the azide-bearing fl uorogenic dye 3-azido-7-hydroxycoumarin, followed by the separation of the lipid mix by thinlayer chromatography and fl uorescence detection, resulting in high sensitivity and wide-range linearity. Kinetic analyses using alkyne-labeled substrates for lysophosphatidic acid acyltransferases, lysophosphatidylcholine acyltransferases, and ceramide synthases resulted in Michaelis-Menten constants similar to those for radiolabeled or natural substrates. We tested additional alkyne substrates for several hydrolases and acyltransferases in lipid metabolism. In this pilot study we establish alkyne lipids as a new class of convenient substrates for in vitro enzymatic assays. In vitro enzymatic assays are a major source of information about the characteristics of enzymes. In particular the determination of functional parameters relies on data in well-defi ned in vitro systems. Enzymes of lipid metabolism have been investigated with radiolabeled substrates for decades ( 1 ). They represent ideal tracers that di...
Raman imaging has become an attractive technology in molecular biology because of its ability to detect multiple molecular components simultaneously without labeling. Two major limitations in accurately accounting for spectral features, viz., background removal and spectral unmixing, have been overcome by employing a modified and effective routine in multivariate curve resolution (MCR). With our improved strategy, we have spectrally isolated seven structurally specific biomolecules without any post-acquisition spectral treatments. Consequently, the isolated intensity profiles reflected concentrations of corresponding biomolecules with high statistical accuracy. Our study reveals the changes in the molecular composition of lipid droplets (LDs) inside HuH7 cells and its relation to the physiological state of the cell. Further, we show that the accurate separation of spectral components permits analysis of structural modification of molecules after cellular uptake. A detailed discussion is presented to highlight the potential of Raman spectroscopy with MCR in semiquantitative molecular profiling of living cells.
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