Alkyne lipids as substrates for click chemistry-based in vitro enzymatic assays

Click chemistry is evolving as a powerful tool in biological applications because it allows the sensitive and specific 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 fluorogenic dye 3-azido-7-hydroxycoumarin, followed by the separation of the lipid mix by thin-layer chromatography and fluorescence 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.     

Canine distemper virus infection requires cholesterol in the viral envelope

Cholesterol is known to play an important role in stabilizing particular cellular membrane structures, so-called lipid or membrane rafts. For several viruses, a dependence on cholesterol for virus entry and/or morphogenesis has been shown. Using flow cytometry and fluorescence microscopy, we demonstrate that infection of cells by canine distemper virus (CDV) was not impaired after cellular cholesterol had been depleted by the drug methyl-beta-cyclodextrin. This effect was independent of the multiplicity of infection and the cellular receptor used for infection. However, cholesterol depletion of the viral envelope significantly reduced CDV infectivity. Replenishment by addition of exogenous cholesterol restored infectivity up to 80%. Thus, we conclude that CDV entry is dependent on cholesterol in the viral envelope. Furthermore, reduced syncytium formation was observed when the cells were cholesterol depleted during the course of the infection. This may be related to the observation that CDV envelope proteins H and F partitioned into cellular detergent-resistant membranes. Therefore, a role for lipid rafts during virus assembly and release as well is suggested.     

The influence of cholesterol and lipid metabolism on host cell structure and hepatitis C virus replication.

The hepatitis C virus (HCV) replicates on a membrane protein complex composed of viral proteins, replicating RNA, and altered cellular membranes. Small-molecule inhibitors of cellular lipid-cholesterol metabolism such as 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 all show a negative effect on HCV replication. Perturbation of host cell lipid and cholesterol metabolism can disrupt replication complexes by altering membranous structures where replication occurs. Changes in cholesterol and (or) lipid composition can have a general effect on membrane structure. Alternatively, metabolic changes can exert a more subtle influence over replication complexes by altering localization of host proteins through alterations in lipid anchoring. Here, we use Huh-7 cells harboring subgenomic HCV replicons to demonstrate that 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 do not disrupt the membranous web where replication occurs, whereas cholesterol-depleting agents such as beta-cyclodextrin do. Cellular imaging suggests that the HCV RNA can remain associated with subcellular compartments connected with replication complexes in the presence of metabolic inhibitors. Therefore, at least 2 different molecular mechanisms are possible for the inhibition of HCV replication through the modulation of cellular lipid and cholesterol metabolism.     

Fluorescent sterols as tools in membrane biophysics and cell biology

Cholesterol is an important constituent of cellular membranes playing a fundamental role in many biological processes. This sterol affects membrane permeability, lateral lipid organization, signal transduction and membrane trafficking. Intracellular sterol transport modes and pathways as well as the regulation of sterol metabolism and disposition in various tissues are areas of intense research. Progress is intimately linked to development and use of appropriate analogs, which closely mimic the properties of cholesterol while allowing to be detected by spectroscopic or microscopic methods. This review provides an overview of various fluorescent sterols used in membrane biophysics and cell biology including analogs of cholesterol and cholesteryl esters. Attention is paid to the natural fluorescent sterol dehydroergosterol (DHE). A survey of the many applications of DHE in biological research is presented. Special emphasis is on recent developments in fluorescence microscopy instrumentation to visualize DHE as an intrinsically fluorescent analog of cholesterol in living cells.     

Desmosterol may replace cholesterol in lipid membranes

Recently, knockout mice entirely lacking cholesterol have been described as showing only a mild phenotype. For these animals, synthesis of cholesterol was interrupted at the level of its immediate precursor, desmosterol. Since cholesterol is a major and essential constituent of mammalian cellular membranes, we asked whether cholesterol with its specific impact on membrane properties might be replaced by desmosterol. By employing various approaches of NMR, fluorescence, and EPR spectroscopy, we found that the properties of phospholipid membranes like lipid packing in the presence of cholesterol or desmosterol are very similar. However, for lanosterol, a more distant precursor of cholesterol synthesis, we found significant differences in comparison with cholesterol and desmosterol. Our results show that, from the point of view of membrane biophysics, cholesterol and desmosterol behave identically and, therefore, replacement of cholesterol by desmosterol may not impact organism homeostasis.     

Trypanosoma cruzi epimastigotes are able to store and mobilize high amounts of cholesterol in reservosome lipid inclusions

Background

Reservosomes are lysosome-related organelles found in Trypanosoma cruzi epimastigotes. They represent the last step in epimastigote endocytic route, accumulating a set of proteins and enzymes related to protein digestion and lipid metabolism. The reservosome matrix contains planar membranes, vesicles and lipid inclusions. Some of the latter may assume rectangular or sword-shaped crystalloid forms surrounded by a phospholipid monolayer, resembling the cholesterol crystals in foam cells.

Methodology/Principal Findings

Using Nile Red fluorimetry and fluorescence microscopy, as well as electron microscopy, we have established a direct correlation between serum concentration in culture medium and the presence of crystalloid lipid inclusions. Starting from a reservosome purified fraction, we have developed a fractionation protocol to isolate lipid inclusions. Gas-chromatography mass-spectrometry (GC-MS) analysis revealed that lipid inclusions are composed mainly by cholesterol and cholesterol esters. Moreover, when the parasites with crystalloid lipid-loaded reservosomes were maintained in serum free medium for 48 hours the inclusions disappeared almost completely, including the sword shaped ones.

Conclusions/Significance

Taken together, our results suggest that epimastigote forms of T. cruzi store high amounts of neutral lipids from extracellular medium, mostly cholesterol or cholesterol esters inside reservosomes. Interestingly, the parasites are able to disassemble the reservosome cholesterol crystalloid inclusions when submitted to serum starvation.     

Hepatic cholesterol transport and its role in non-alcoholic fatty liver disease and atherosclerosis

Non-alcoholic fatty liver disease (NAFLD) is a quickly emerging global health problem representing the most common chronic liver disease in the world. Atherosclerotic cardiovascular disease represents the leading cause of mortality in NAFLD patients. Cholesterol metabolism has a crucial role in the pathogenesis of both NAFLD and atherosclerosis. The liver is the major organ for cholesterol metabolism. Abnormal hepatic cholesterol metabolism not only leads to NAFLD but also drives the development of atherosclerotic dyslipidemia. The cholesterol level in hepatocytes reflects the dynamic balance between endogenous synthesis, uptake, esterification, and export, a process in which cholesterol is converted to neutral cholesteryl esters either for storage in cytosolic lipid droplets or for secretion as a major constituent of plasma lipoproteins, including very-low-density lipoproteins, chylomicrons, high-density lipoproteins, and low-density lipoproteins. In this review, we describe decades of research aimed at identifying key molecules and cellular players involved in each main aspect of hepatic cholesterol metabolism. Furthermore, we summarize the recent advances regarding the biological processes of hepatic cholesterol transport and its role in NAFLD and atherosclerosis.     

Ultrastructural localization of cholesterol by enzyme histochemistry

Summary The histochemical localization of cholesterol using oxidized diaminobenzidine as the final reaction product was studied at the electron microscopical level and compared with the digitonin method of cholesterol localization based on cholesterol digitonide as the final reaction product. Tissue chopper sections of fixed rat adrenal glands were incubated at 37° C in a medium consisting of 0.8 units/ml cholesterol oxidase, 1.4 units/ml cholesterol ester hydrolase, 50 units/ml horseradish peroxidase, 0.5 mg/ml diaminobenzidine, 0.1% v/v Triton X-100 (or Surfal) and an endogenous peroxidase inhibitor in 0.1m phosphate buffer, pH 7.0. An electron-dense osmiophilic reaction product was observed in many lipid droplets, intracellular vesicles and focally around mitochondria. Appropriate control experiments indicated that deposition of reaction product depended on the presence of cholesterol and the necessary enzymes. Comparison studies using digitonin confirmed the presence of cholesterol in the lipid droplets, but ultrastructural distortion limited the resolution of the more discrete deposits of cholesterol such as around mitochondria. The enzyme method permits finer resolution of these discrete deposits of cholesterol than the digitonin method because it does not cause distortion of cellular ultrastructure attributed to the formation of cholesterol digitonide. The enzyme method or a combination of enzyme and digitonin enables localization of free, esterified or total cholesterol.     

Localization of mammalian NAD(P)H steroid dehydrogenase-like protein on lipid droplets

Mammalian enzymes in late cholesterol biosynthesis have been localized uniformly over the endoplasmic reticulum by enzymatic methods. We report here the first mammalian cholesterol biosynthetic enzyme unequivocally localized at the surface of intracellular lipid storage droplets. NAD(P)H steroid dehydrogenase-like protein (Nsdhl), a mammalian C-3 sterol dehydrogenase involved in the conversion of lanosterol into cholesterol, was localized on lipid droplets by immunofluorescence microscopy and subcellular fractionation. Nsdhl was localized on lipid droplets even when cell growth exclusively depended on cholesterol biosynthesis mediated by this enzyme. Depletion of fatty acids in culture medium reduced the development of lipid droplets and caused Nsdhl redistribution to the endoplasmic reticulum. Elevating oleic acid in medium induced well developed, Nsdhl-positive lipid droplets, and simultaneously caused a reduction in cellular conversion of lanosterol into cholesterol. Manipulated human NSDHL with a missense mutation (G205S) causing a human embryonic developmental disorder, congenital hemidysplasia with ichthyosiform nevus and limb defects (CHILD) syndrome, could no longer be localized on lipid droplets. Although the expression of wild-type NSDHL could restore the defective growth of a CHO cholesterol auxotroph, LEX2 in cholesterol-deficient medium, the expression of NSDHL(G205S) failed to do so. These results point to functional significance of the localization of Nsdhl on lipid droplets. Functional significance was also suggested by the colocalization of Nsdhl on lipid droplets with TIP47, a cargo selection protein for mannose 6-phosphate receptors from late endosomes to the trans-Golgi network. These results add to the growing notion that the lipid droplet is an organelle endowed with more complex roles in various biological phenomena.     

Intracellular localization of organized lipid domains of C16-ceramide/cholesterol

Lipid microdomains, also called lipid rafts, consisting of sphingolipids and cholesterol, play important roles in membrane trafficking and in signaling. Despite years of study of the composition, size, half-life and dynamic organization of these domains, many open questions remain about their precise characteristics. To address some of these issues, we have developed a new experimental approach involving the use of specific monoclonal antibodies as recognition tools. One such antibody was raised against a homogeneous, mixed, ordered monolayer phase comprised of 60:40 mol% cholesterol:C16-ceramide, and has been used previously to demonstrate the existence of C16-ceramide/cholesterol domains in the membranes of cultured cells. We now use a combination of quantitative fluorescence microscopy, immuno-transmission electron microscopy and immuno-scanning cryo-electron microscopy, optimized for the study of intracellular lipid antigens. In a variety of cultured cells, C16-ceramide/cholesterol structural domains were found at high levels in late endosomes and in the trans-Golgi network, but were not found at statistically significant levels in early endosomes, lysosomes or the endoplasmic reticulum. We discuss the relevance of these results to understanding the role of lipid lateral organization in biological membranes.     

The fluorescent cholesterol analog dehydroergosterol induces liquid-ordered domains in model membranes

The fluorescent sterol dehydroergosterol (DHE) is often used as a marker for cholesterol in cellular studies. We show by vesicle fluctuation analysis that DHE has a lower ability than cholesterol to stiffen lipid bilayers suggesting less efficient packing with phospholipid acyl chains. Despite this difference, we found by fluorescence and atomic force microscopy, that DHE induces liquid-ordered/-disordered coexistent domains in giant unilamellar vesicles (GUVs) and supported bilayers made of dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC) and DHE or cholesterol. DHE-induced phases have a height difference of 0.9-1 nm similar as known for cholesterol-containing domains. DHE not only promotes formation of liquid-liquid immiscibility but also shows strong partition preference for the liquid-ordered phase further supporting its suitability as cholesterol probe.     

Tripterygium regelii decreases the biosynthesis of triacylglycerol and cholesterol in HepG2 cells

In the course of screening to find a plant material decreasing the activity of triacylglycerol and cholesterol, we identified Tripterygium regelii (TR). The methanol extract of TR leaves (TR-LM) was shown to reduce the intracellular lipid contents consisting of triacylglycerol (TG) and cholesterol in HepG2 cells. TR-LM also downregulated the mRNA and protein expression of the lipogenic genes such as SREBP-1 and its target enzymes. Consequently, TR-LM reduced the TG biosynthesis in HepG2 cells. In addition, TR-LM decreased SREBP2 and its target enzyme HMG-CoA reductase, which is involved in cholesterol synthesis. In this study, we evaluated that TR-LM attenuated cellular lipid contents through the suppression of de novo TG and cholesterol biosynthesis in HepG2 cells. All these taken together, TR-LM could be beneficial in regulating lipid metabolism and useful preventing the hyperlipidemia and its complications, in that liver is a crucial tissue for the secretion of serum lipids.     

Effects of cholesterol surface transfer on cholesterol and phosphatidylcholine synthesis in cultured rat arterial smooth muscle cells

The purpose of this study was to examine the effects of cholesterol surface transfer between lipid vesicles and rat arterial smooth muscle cells on endogenous synthesis of cholesterol and phosphatidylcholine. Lipid vesicles containing cholesterol and egg phosphatidylcholine in different proportions were used as the extracellular lipid source. The rate of cellular cholesterol and phosphatidylcholine synthesis was determined from the [14C]acetate incorporation into these lipid classes. [3H]Cholesterol in lipid vesicles, with a cholesterol/phospholipid (C/P) mole ratio of 1:1, was rapidly transferred into rat smooth muscle cells, with a half-time of about 3.6 hours in the absence of serum proteins. Incubation of cells for 5 hours with vesicles of a high C/P mole ratio (i.e. 1.5:1) at vesicle-cholesterol concentrations above 100 micrograms/ml resulted in a marked reduction of cellular cholesterol synthesis, whereas the rate of phosphatidylcholine synthesis was increased. Cells incubated with lipid vesicles of C/P 1:2 did not show any change in cellular cholesterol or phosphatidylcholine synthesis. Incubation of cells with egg phosphatidylcholine vesicles at concentrations above 300 micrograms/ml, on the other hand, stimulated endogenous synthesis of cholesterol without affecting cellular phosphatidylcholine synthesis. The main conclusion is that cholesterol surface transfer may influence cellular lipid metabolism in the absence of mediating serum lipoproteins in a model system with cultured cells and lipid vesicles.     

The cholesterol‐binding motif of the HIV‐1 glycoprotein gp41 regulates lateral sorting and oligomerization

Enveloped viruses often use membrane lipid rafts to assemble and bud, augment infection and spread efficiently. However, the molecular bases and functional consequences of the partitioning of viral glycoproteins into microdomains remain intriguing questions in virus biology. Here, we measured Foerster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM‐FRET) to study the role of distinct membrane proximal regions of the human immunodeficiency virus glycoprotein gp41 for lipid raft partitioning in living Chinese hamster ovary cells (CHO‐K1). Gp41 was labelled with a fluorescent protein at the exoplasmic face of the membrane, preventing any interference of the fluorophore with the proposed role of the transmembrane and cytoplasmic domains in lateral organization of gp41. Raft localization was deduced from interaction with an established raft marker, a fluorescently tagged glycophosphatidylinositol anchor and the cholesterol recognition amino acid consensus (CRAC) was identified as the crucial lateral sorting determinant in CHO‐K1 cells. Interestingly, the raft association of gp41 indicates a substantial cell‐to‐cell heterogeneity of the plasma membrane microdomains. In complementary fluorescence polarization microscopy, a distinct CRAC requirement was found for the oligomerization of the gp41 variants. Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral sorting of viral glycoproteins for virus assembly at cellular membranes.     

Activity based subcellular resolution imaging of lipases

Lipases play a key role in whole body energy homeostasis. Dysregulation of lipolytic activities affects lipid absorption, mobilization, and transport, and is causative for lipid-related diseases. Regulation of enzymes involved in lipid metabolism is governed by a complex network of protein-protein and protein-small molecule interactions. Thus these enzymes have to be studied under the physiologically most relevant conditions, that is, in vivo. Our latest generation of activity based probes designed for capturing of lipases employs bioorthogonal chemical linker groups, which are membrane permeable and thus allow studying protein activity in living cells. Another advantage is the virtually unlimited choice of reporter tags. Here we report on a novel method combining in vivo activity based labeling of lipases with in situ detection of lipolytic activities by on slide click chemistry and imaging by fluorescence microscopy. We demonstrate that cytosolic as well as organelle resident lipases are specifically labeled in intact living cells. This method will shed light on the (sub)cellular localization of lipolytic proteomes of cells and tissues in health and disease directly at enzymatic activity level without the need of prior knowledge of the identities of the responsible enzymes or dependence on the availability of specific antibodies.     

The effect of non-receptor-mediated uptake of cholesterol on intracellular cholesterol metabolism in human skin fibroblasts.

Unilamellar lipid vesicles of various cholesterol:phosphatidylcholine molar ratios were used to alter, via passive exchange at the plasma membrane, the cellular free cholesterol content of cultured human skin fibroblasts which had been preincubated in lipoprotein-deficient serum. The effects of these net surface transfers of cholesterol on cellular cholesterol biosynthesis, cholesterol esterification and low density lipoprotein (LDL) binding were determined and were compared with the effects of cholesterol delivered to the cell interior via the receptor-mediated endocytosis of LDL. Both LDL and cholesterol-rich lipid vesicles increased cell cholesterol within 6 h. Cells exposed to LDL also showed, within 6 h, decreased cholesterol synthesis, decreased LDL binding and increased cholesterol esterification. Cells incubated with the cholesterol-rich vesicles showed similar changes but these were delayed and did not occur until 24 h. Fibroblasts incubated with cholesterol-free phosphatidylcholine vesicles had decreased cell cholesterol, increased cholesterol synthesis, increased LDL binding, and decreased esterification, but only after 24 h of incubation. These results suggest that passive net transfers of cholesterol occurring at the cell surface can with time modulate intracellular cholesterol metabolism. These findings are consistent with the idea that the movement of cholesterol from the cell surface to the cell interior is a limited and relatively slow process.     

Cellular cholesterol trafficking and compartmentalization

Cholesterol is an essential structural component in the cell membranes of most vertebrates. The biophysical properties of cholesterol and the enzymology of cholesterol metabolism provide the basis for how cells handle cholesterol and exchange it with one another. A tightly controlled--but only partially characterized--network of cellular signalling and lipid transfer systems orchestrates the functional compartmentalization of this lipid within and between organellar membranes. This largely dictates the exchange of cholesterol between tissues at the whole body level. Increased understanding of these processes and their integration at the organ systems level provides fundamental insights into the physiology of cholesterol trafficking.     

Diet-induced elevations in serum cholesterol are associated with alterations in hippocampal lipid metabolism and increased oxidative stress

The structure and function of the hippocampus, a brain region critical for learning and memory, is impaired by obesity and hyperlipidemia. Peripheral cholesterol and sphingolipids increase progressively with aging and are associated with a range of age-related diseases. However, the mechanisms linking peripheral cholesterol metabolism to hippocampal neuroplasticity remain poorly understood. To determine whether diets that elevate serum cholesterol influence lipid metabolism in the hippocampus, we maintained rats on a diet with high amounts of saturated fat and simple sugars for 3 months and then analyzed hippocampal lipid species using tandem mass spectrometry. The high fat diet was associated with increased serum and liver cholesterol and triglyceride levels, and also promoted cholesterol accumulation in the hippocampus. Increases in hippocampal cholesterol were associated with elevated galactosyl ceramide and sphingomyelin. To determine whether changes in lipid composition exerted biological effects, we measured levels of the lipid peroxidation products 4-hydroxynonenal-lysine and 4-hydroxynonenal-histidine; both were increased locally in the hippocampus, indicative of cell membrane-associated oxidative stress. Taken together, these observations support the existence of a potentially pathogenic relationship between dietary fat intake, peripheral cholesterol and triglyceride levels, brain cell sphingolipid metabolism, and oxidative stress.     

Lateral distribution of cholesterol in dioleoylphosphatidylcholine lipid bilayers: cholesterol-phospholipid interactions at high cholesterol limit

Lateral organization of cholesterol in dioleoyl-phosphatidylcholine (DOPC) lipid bilayers at high cholesterol concentration (>45 mol%) was investigated using steady-state fluorescence anisotropy and fluorescent resonance energy transfer techniques. The recently devised Low Temperature Trap method was used to prepare compositionally uniform cholesterol/DOPC liposomes to avoid the problem of lipid demixing. The fluorescence anisotropy of diphenylhexatrience chain-labeled phosphatidylcholine (DPH-PC) in these liposomes exhibited local maxima at cholesterol mol fractions of 0.50 and 0.57, and a sharp drop at 0.67. For the liposomes labeled with both dehydroergosterol and DPH-PC, the fluorescent resonance energy transfer efficiency from dehydroergosterol to DPH-PC displayed a steep jump at cholesterol mol fraction of 0.5, and dips at 0.57 and 0.68. These results indicate the presence of highly ordered cholesterol regular distribution domains at those observed critical compositions. The observed critical mol fraction at 0.67 agreed favorably with the solubility limit of cholesterol in DOPC bilayers as independently measured by light scattering and optical microscopy. The regular distribution at 0.57 was previously predicted from a Monte Carlo simulation based on the Umbrella model. The results strongly support the hypothesis that the primary requirement for cholesterol-phospholipid mixing is that the polar phospholipid headgroups need to cover the nonpolar body of cholesterol to avoid the exposure of cholesterol to water.