Sterol Interactions Research Articles

Sterol Migration during Rotational Frying of Food Products in Modified Rapeseed and Soybean Oils.

This study explores the impact of rotational frying of three different food products on degradation of sterols, as well as their migration between frying oils and food. The research addresses a gap in the existing literature, which primarily focuses on changes in fat during the frying of single food items, providing limited information on the interaction of sterols from the frying medium with those from the food product. The frying was conducted at 185 ± 5 °C for up to 10 days where French fries, battered chicken, and fish sticks were fried in succession. The sterol content was determined by Gas Chromatography. This research is the first to highlight the influence of the type of oil on sterol degradation in both oils and food. Notably, sterols were found to be most stable when food products were fried in high-oleic low-linolenic rapeseed oil (HOLLRO). High-oleic soybean oil (HOSO) exhibited higher sterol degradation than high-oleic rapeseed oil (HORO). It was proven that cholesterol from fried chicken and fish sticks did not transfer to the fried oils or French fries. Despite initially having the highest sterol content in fish, the lowest sterol amount was recorded in fried fish, suggesting rapid degradation, possibly due to prefrying in oil with a high sterol content, regardless of the medium used.

Measuring the Interaction of Sterols and Steroids with Proteins by Microscale Thermophoresis.

Determining the affinity and specificity of protein-lipid interactions is crucial for understanding the physiological function and mode of action of signaling lipids, including steroids. Here we describe a method that relies on microscale thermophoresis (MST) to monitor the binding of sterols and steroids to proteins. The protein of interest is expressed as a polyhistidine-tagged fusion in E. coli and purified by affinity chromatography on a nickel-based resin. The purified protein is then labeled with a fluorescent dye and incubated with a serial dilution of the lipid ligand. The protein-ligand interaction is monitored by MST, which detects the fraction of the protein bound to the ligand based on its altered mobility in a thermal gradient. A binding curve fitted to the measured data points is then used to calculate the corresponding dissociation constant.

The Effects of Cholesterol Oxidation on Erythrocyte Plasma Membranes: A Monolayer Study.

Cholesterol plays a key role in the molecular and mesoscopic organisation of lipid membranes and it is expected that changes in its molecular structure (e.g., through environmental factors such as oxidative stress) may affect adversely membrane properties and function. In this study, we present evidence that oxidation of cholesterol has significant effects on the mechanical properties, molecular and mesoscopic organisation and lipid–sterol interactions in condensed monolayers composed of the main species found in the inner leaflet of the erythrocyte membrane. Using a combination of experimental methods (static area compressibility, surface dilatational rheology, fluorescence microscopy, and surface sensitive X-ray techniques) and atomistic molecular dynamics simulations, we show that oxidation of cholesterol to 7-ketocholesterol leads to stiffening of the monolayer (under both static and dynamic conditions), significant changes in the monolayer microdomain organisation, disruption in the van der Waals, electrostatic and hydrophobic interactions between the sterol and the other lipid species, and the lipid membrane hydration. Surface sensitive X-ray techniques reveal that, whilst the molecular packing mode is not significantly affected by cholesterol oxidation in these condensed phases, there are subtle changes in membrane thickness and a significant decrease in the coherence length in monolayers containing 7-ketocholesterol.

S-Palmitoylation and Sterol Interactions Mediate Antiviral Specificity of IFITMs.

Interferon-induced transmembrane proteins (IFITM1, 2, and 3) are important antiviral proteins that are active against many viruses, including influenza A virus (IAV), dengue virus (DENV), Ebola virus (EBOV), Zika virus (ZIKV), and severe acute respiratory syndrome coronavirus (SARS-CoV). IFITM proteins exhibit specificity in activity, but their distinct mechanisms of action and regulation are unclear. Since S-palmitoylation and cholesterol homeostasis are crucial for viral infections, we investigated IFITM interactions with cholesterol by photoaffinity cross-linking in mammalian cells along with molecular dynamic simulations and nuclear magnetic resonance analysis in vitro. These studies suggest that cholesterol can directly interact with S-palmitoylated IFITMs in cells and alter the conformation of IFITMs in membrane bilayers. Notably, we discovered that the S-palmitoylation levels regulate differential IFITM protein interactions with cholesterol in mammalian cells and specificity of antiviral activity toward IAV, SARS-CoV-2, and EBOV. Our studies suggest that modulation of IFITM S-palmitoylation levels and cholesterol interaction influence host susceptibility to different viruses.

Dishevelled localization and function are differentially regulated by structurally distinct sterols

Aberrant cholesterol synthesis, characterized by depletion of cellular cholesterol and accumulation of sterol precursors, is directly linked to a group of rare human diseases exhibiting broad clinical phenotypes and developmental malformations. Despite decreased cholesterol content being shared across these diseases, patients exhibit distinct clinical manifestations associated with specific tissues or systems with varying degrees of phenotypic overlap. This tissue specificity may result from disease‐specific disruption of important critical signaling pathways due to the inability of sterol precursors to structurally compensate for cholesterol. We previously determined that the cholesterol precursor 7‐dehydrocholesterol (7DHC), which accumulates in Smith‐Lemli‐Opitz syndrome, perturbs Dishevelled 2 (DVL2) protein function due to reduced binding affinity of 7DHC to DVL2’s PDZ domain (Postsynaptic density 95, Disc large 1, and Zonular occludens 1 domain) in comparison to cholesterol, resulting in loss of canonical Wnt/β‐catenin signaling (1). We hypothesized that structural differences between cholesterol and alternative sterols will inhibit binding with DVL2 and other PDZ‐domain containing proteins, producing dire cellular consequences and directly affecting Wnt signaling (2). Protein homology modeling and ligand‐protein docking analysis suggested a differential binding affinity and specificity of DVL2 for cholesterol versus alternative sterols. Using PM‐mimetic vesicles incorporating cholesterol versus alternative sterols in liposome/DVL2 pulldowns, we experimentally observed a reduced binding affinity between DVL2 and alternate sterols. Surface plasmon resonance analysis to define the kinetics of protein‐sterol interaction corroborates in silicomodeling and pulldowns. Our data suggests orientation of the beta‐ and alpha‐sterol face within the DVL2‐PDZ domain binding pocket is predictive of DVL2‐sterol interaction. At the cellular level, we have also observed disruption of DVL2‐sterol binding shifts the subcellular localization of DVL2 from the plasma membrane to the nucleus. Increased nuclear DVL2 promotes non‐canonical WNT signaling and DVL2 protein‐protein binding with nuclear factors. Our work suggests that disrupted sterol homeostasis broadly impacts cellular signaling in a sterol‐specific and pathway‐specific manner. Defining the structural and biochemical requirements for sterol interactions with DVL2 and other cholesterol‐binding proteins will help define the critical role sterol homeostasis plays in human health.(1)Francis KR et al, Nature medicine. 2016;22(4):388‐96.(2) Sheng R et al, Nature communications. 2012;3:1249.

Phytosterol-rich compressed fluids extracts from Phormidium autumnale cyanobacteria with neuroprotective potential

Phormidium autumnale (P. autumnale) is a cyanobacteria with an unknown metabolic system, since only few studies have been reported. Among the produced metabolites, sterols profile is important considering the ability to this cyanobacterium in producing such compounds and their important benefits for human health. In this study, compressed fluid technologies were evaluated to obtain phytosterol-rich extracts from P. autumnale cyanobacteria for investigating their potential neuroprotective properties. A preliminary study was performed by comparing gas-expanded liquid extraction and supercritical fluid extraction using ethanol as a co-solvent. The following steps were optimized using a two-level factorial design and considering solvent composition and pressure as experimental factors. The bioactive potential of the optimized compressed fluid extract was tested using in vitro bioactivity assays, including acetylcholinesterase, lipoxygenase inhibition, and antioxidant capacity. The supercritical fluid multi-optimization response presented optimum phytosterol enrichment conditions at 266.3 bar of pressure and 7% of ethanol. Moreover, the optimized extract had higher in-vitro neuroprotective activity than the non-enriched extract, presenting the biochemical half maximal inhibitory concentration (IC50) values of 65.80 μg mL−1 for acetylcholinesterase, 58.20 μg mL−1 for lipoxygenase inhibition, and 7.40 μg mL−1 for antioxidant activity. In-silico molecular docking analyses showed the specificity of sterol interaction with acetylcholinesterase active sites. These results provide evidence for further exploring P. autumnale as a source of bioactive phytosterols, demonstrating that compressed fluid technologies are powerful tools to obtain phytosterol-rich extracts.

Physical mechanism analysis of cholesterol concentration effect on asymmetric phospholipid membrane

Cell membrane is a multi-component complex system containing large number of membrane proteins and various types of sterols, so its properties are closely relevant to the interaction of sterols with phospholipid membranes. In this paper, we use the molecular dynamics method to investigate the effect of cholesterol concentration on asymmetric phospholipid system. To describe the membrane with the complex lipid components, we employ the coarse-grained model and choose three types of saturated phospholipids, DPPC, DPPE and DPPS, as the membrane components. The different concentrations of cholesterol are represented by five systems, so different physical influences on asymmetric phospholipid membrane can be exhibited. After enough time of the molecular simulation, the structural properties of the membrane, such as lipid accumulation, lipid tail sequence, lipid diffusion and cholesterol turnover, are obtained. The analysis on the total energy of the system, the L-J and electrostatic interaction between cholesterol molecules and phospholipid membrane system shows that the effect of cholesterol concentration on asymmetric phospholipid membrane is through changing the fluidity of asymmetric phospholipid membrane. Meanwhile, the distribution of cholesterol molecules in phospholipid membrane shows that most of them are concentrated in the inner side of phospholipid membrane, which also leads to the increase in tail order parameter and the thickness of phospholipid membrane. At the same time, we take into account the stress of cholesterol molecules to analyze the turnover of cholesterol molecules in different concentrations in the phospholipid membrane, and find that there is no obvious law of the stress on cholesterol molecules at different concentrations. Therefore, the turnover frequency of cholesterol molecules is mainly due to the stronger fluidity of the phospholipid membrane when the concentration of cholesterol increases. Our research can provide some help for a better understanding of the basic characteristics of the structure and kinetics of membranes containing cholesterol.

Structural Stringency and Optimal Nature of Cholesterol Requirement in the Function of the Serotonin1A Receptor.

The role of membrane cholesterol in modulating G protein-coupled receptor (GPCR) structure and function has emerged as a powerful theme in contemporary biology. In this paper, we report the subtlety and stringency involved in the interaction of sterols with the serotonin1A receptor. For this, we utilized two immediate biosynthetic precursors of cholesterol, 7-dehydrocholesterol (7-DHC) and desmosterol, which differ with cholesterol merely in a double bond in their chemical structures in a position-dependent manner. We show that whereas 7-DHC could not support the ligand binding function of the serotonin1A receptor in live cells, desmosterol could partially support it. Importantly, depletion and enrichment of membrane cholesterol over basal level resulted in an increase and reduction of the basal receptor activity, respectively. These results demonstrate the relevance of optimal membrane cholesterol in maintaining the activity of the serotonin1A receptor, thereby elucidating the relevance of cellular cholesterol homeostasis.

The elicitin β-cryptogein’s activity in tomato is mediated by jasmonic acid and ethylene signalling pathways independently of elicitin–sterol interactions

The level of resistance induced in different tomato genotypes after β-CRY treatment correlated with the upregulation of defence genes, but not sterol binding and involved ethylene and jasmonic acid signalling. Elicitins, a family of small proteins secreted by Phytophthora and Pythium spp., are the most well-known microbe-associated molecular patterns of oomycetes, a lineage of fungus-like organisms that include many economically significant crop pathogens. The responses of tomato plants to elicitin INF1 produced by Phytophthora infestans have been studied extensively. Here, we present studies on the responses of three tomato genotypes to β-cryptogein (β-CRY), a potent elicitin secreted by Phytophthora cryptogea that induces hypersensitive response (HR) cell death in tobacco plants and confers greater resistance to oomycete infection than acidic elicitins like INF1. We also studied β-CRY mutants impaired in sterol binding (Val84Phe) and interaction with the binding site on tobacco plasma membrane (Leu41Phe), because sterol binding was suggested to be important in INF1-induced resistance. Treatment with β-CRY or the Val84Phe mutant induced resistance to powdery mildew caused by the pathogen Pseudoidium neolycopersici, but not the HR cell death observed in tobacco and potato plants. The level of resistance induced in different tomato genotypes correlated with the upregulation of defence genes including defensins, β-1,3-glucanases, heveins, chitinases, osmotins, and PR1 proteins. Treatment with the Leu41Phe mutant did not induce this upregulation, suggesting similar elicitin recognition in tomato and tobacco. However, here β-CRY activated ethylene and jasmonic acid signalling, but not salicylic acid signalling, demonstrating that elicitins activate different downstream signalling processes in different plant species. This could potentially be exploited to enhance the resistance of Phytophthora-susceptible crops.

Solid-State NMR of highly 13C-enriched cholesterol in lipid bilayers.

Cholesterol (Chol) is vital for cell function as it is essential to a myriad of biochemical and biophysical processes. The atomistic details of Chol's interactions with phospholipids and proteins is therefore of fundamental interest, and NMR offers unique opportunities to interrogate these properties at high resolution. Towards this end, here we describe approaches for examining the structure and dynamics of Chol in lipid bilayers using high levels of 13C enrichment in combination with magic-angle spinning (MAS) methods. We quantify the incorporation levels and demonstrate high sensitivity and resolution in 2D 13C-13C and 1H-13C spectra, enabling de novo assignments and site-resolved order parameter measurements obtained in a fraction of the time required for experiments with natural abundance sterols. We envision many potential future applications of these methods to study sterol interactions with drugs, lipids and proteins.

Recombinant anthrax protective antigen: Observation of aggregation phenomena by TEM reveals specific effects of sterols

Negatively stained transmission electron microscope images are presented that depict the aggregation of recombinant anthrax protective antigen (rPA83 monomer and the PA63 prepore oligomer) under varying in vitro biochemical conditions. Heat treatment (50°C) of rPA83 produced clumped fibrils, but following heating the PA63 prepore formed disordered aggregates. Freeze-thaw treatment of the PA63 prepore generated linear flexuous aggregates of the heptameric oligomers. Aqueous suspensions of cholesterol microcrystals were shown to bind small rPA83 aggregates at the edges of the planar bilayers. With PA63 a more discrete binding of the prepores to the crystalline cholesterol bilayer edges occurs. Sodium deoxycholate (NaDOC) treatment of rPA83 produced quasi helical fibrillar aggregate, similar but not identical to that produced by heat treatment. Remarkably, NaDOC treatment of the PA63 prepores induced transformation into pores, with a characteristic extended ß-barrel. The PA63 pores aggregated as dimers, that aggregated further as angular chains and closed structures in higher NaDOC concentrations. The significance of the sterol interaction is discussed in relation to its likely importance for PA action in vivo.

Atomic Resolution Studies of Sterol Interactions by Solid-State NMR Spectroscopy

Sterols, vital and abundant components of plasma membranes, are involved in numerous cellular biochemical processes such as regulation of membrane properties and signaling events. However, these sterol interactions are not completely elucidated at the atomic level; this impedes further investigations into disorders related to these biological activities. Combining solid-state NMR (SSNMR), high yield biosynthesis of isotopically labeled sterols, and molecular dynamics, we aim to understand the key interactions that determine the sterol-specificity to amphotericin B (AmB), a powerful but toxic antifungal drug used to treat life-threatening fungal infections. AmB forms large aggregates that are neither crystalline nor soluble, making structural information difficult or impossible to obtain via x-ray crystallography or solution-state NMR. We have recently determined that AmB kills yeast cells primarily by binding to ergosterol1 (Erg) and forming a large extramembranous sterol sponge.2 These findings were enabled by the biosynthesis of fractionally 13C labeled Erg and state-of-the-art SSNMR techniques. The sterol sponge model hypothesizes that interactions of AmB with Erg determine its ability to kill yeast, whereas binding to cholesterol is responsible for determining its toxicity in human cells. SSNMR spectroscopy is uniquely able to detect and quantify the binding of sterols to AmB in the sterol sponge in atomistic detail. These studies provide a roadmap towards an improved therapeutic index3 for this drug while simultaneously elucidating additional details about the molecular mechanism of AmB-sterol interactions.(1)“Amphotericin primarily kills yeast by simply binding ergosterol”, Gray et al. Proc. Natl. Acad. Sci. 2011, 109, 2234-2239.(2)“Amphotericin forms an extramembranous sterol sponge”, Anderson et al. Nat. Chem. Bio. 2014, 10, 400-406.(3)“C2’-OH of amphotericin B plays an important role in binding the primary sterol of human cells but not yeast cells”, Wilcock et al. J. Am. Chem. Soc. 2013, 135, 8488-8491.

Hopanoids as functional analogues of cholesterol in bacterial membranes

The functionality of cellular membranes relies on the molecular order imparted by lipids. In eukaryotes, sterols such as cholesterol modulate membrane order, yet they are not typically found in prokaryotes. The structurally similar bacterial hopanoids exhibit similar ordering properties as sterols in vitro, but their exact physiological role in living bacteria is relatively uncharted. We present evidence that hopanoids interact with glycolipids in bacterial outer membranes to form a highly ordered bilayer in a manner analogous to the interaction of sterols with sphingolipids in eukaryotic plasma membranes. Furthermore, multidrug transport is impaired in a hopanoid-deficient mutant of the gram-negative Methylobacterium extorquens, which introduces a link between membrane order and an energy-dependent, membrane-associated function in prokaryotes. Thus, we reveal a convergence in the architecture of bacterial and eukaryotic membranes and implicate the biosynthetic pathways of hopanoids and other order-modulating lipids as potential targets to fight pathogenic multidrug resistance.

Interactions of antiparasitic sterols with sterol 14α-demethylase (CYP51) of human pathogens.

Sterol 14α-demethylase is a validated and an attractive drug target in human protozoan parasites. Pharmacological inactivation of this important enzyme has proven very effective against fungal infections, and it is a target that is being exploited for new antitrypanosomal and antileishmanial chemotherapy. We have used in silico calculations to identify previously reported antiparasitic sterol-like compounds and their structural congeners that have preferential and high docking affinity for CYP51. The sterol 14α-demethylase from Trypanosoma cruzi and Leishmania infantum, in particular, preferentially dock to taraxerol, epi-oleanolic acid, and α/β-amyrim structural scaffolds. These structural information and predicted interactions can be exploited for fragment/structure-based antiprotozoal drug design.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-679) contains supplementary material, which is available to authorized users.

Neural HO-1/sterol interactions in vivo: Implications for Alzheimer’s disease

Background: Up-regulation of heme oxygenase-1 (HO-1) and altered cholesterol (CH) metabolism are characteristic of Alzheimer-diseased (AD) neural tissues. We previously provided evidence of significant HO-1/sterol interactions in vitro (cultured rat astroglia) and in post-mortem human AD brain (Religious Orders Study). Methods: The current experiments were designed to further delineate these interactions in vivo by comparing the behavior of HO-1/sterol interactions in two mouse models; (1) a novel HO-1 transgenic mouse (GFAP.HMOX1) engineered to selectively express human HO-1 in the astrocytic compartment and (2) the previously described triple transgenic AD mouse (3xTg-AD). In samples of frontal cortex, total CH, CH precursors and relevant oxysterols were quantified by gas chromatography–mass spectrometry (GC–MS) and HO-1 protein expression was assessed by ELISA. The relationships of HO-1 expression to total CH, CH precursors and total oxysterols were determined for both mouse models using linear regression analysis. Results: HO-1 expression is increased in GFAP.HMOX1 mice relative to wild type and in 11–12-month-old 3xTg-AD mice (with AD-like phenotype) relative to control mice and 5–6-month-old 3xTg-AD mice (no AD-like phenotype). Total oxysterols significantly decreased as HO-1 expression increased in GFAP.HMOX1 mice expressing high levels of HO-1, whereas total oxysterols increased as HO-1 expression increased in aged 3xTg-AD mice. Total CH and total CH precursors increased as HO-1 protein expression increased in 11–12-month-old 3xTg-AD mice relative to 5–6-month old 3xTg-AD mice. Conclusions: Our findings indicate a differential impact of HO-1 on patterns of brain sterol and redox homeostasis that is contingent on the presence or absence of AD-like neuropathology. These data provide fresh insight concerning the regulation of sterol homeostasis within the aging and degenerating CNS which may inform the development of novel therapeutic and preventive strategies for the management of AD and related conditions.

Miscibility and interactions of animal and plant sterols with choline plasmalogen in binary and multicomponent model systems

In this work miscibility and interactions of sterols with choline plasmalogen (PC-plasm) in Langmuir monolayers were studied. Moreover, the properties of cholesterol/phosphatidylcholine/plasmalogen mixtures of different PC-plasm concentration were investigated. The foregoing systems were treated as a model of cancer cell membranes, which are of higher plasmalogen level than normal cells. Finally, the influence of β-sitosterol and stigmasterol (phytosterols differing in anticancer potency) on these mixtures was verified. The properties of monolayers were analyzed based on the parameters derived from the surface pressure-area isotherms and images taken with Brewster Angle Microscope. It was found that at 30% of sterol in sterol/plasmalogen monolayer the lipids are immiscible and 3D crystallites are formed within the film. Cholesterol molecules mix favorably with PC-plasm at Xchol≥0.5, while the investigated phytosterols only at their prevailing proportion in binary system. The increase of choline plasmalogen in cholesterol/phosphatidylcholine monolayer causes destabilization of the system. Moreover, the incorporation of phytosterols into cholesterol/phosphatidylcholine+PC-plasm mixtures disturbed membrane morphology and this effect was stronger for β-sitosterol as compared to stigmasterol. It was concluded that the presence of vinyl ether bond at sn-1 position in PC-plasm molecule strongly affects miscibility of choline plasmalogen with sterols. The comparison of the collected data with those reported in literature allowed one to conclude that miscibility and interactions of sterols with PC-plasm are less favorable than those with phosphatidylcholine. It was also suggested that overexpression of plasmalogens in cancer cell membranes may be a factor differentiating sensitivity of cells to anticancer effect of phytosterols.

Synthesis of cholesterol analogs having varying length alkyl side chains including cholesterol-23, 23, 24, 24, 25, 26, 26, 26, 27, 27, 27-d11 as probes of cholesterol's functions and properties

Cholesterol-23, 23, 24, 24, 25, 26, 26, 26, 27, 27, 27-d11 and nondeuterated long-chain analogs of cholesterol were prepared by alkylation of cyano-containing sterols with isopentyl-d11 4-methylbenzenesulfonate (1.0equiv.) or with isoalkyl bromides, followed by reductive decyanation with excess potassium metal and a crown ether in toluene. The products are potentially useful probes of the role of the side-chain of cholesterol in the sterol's interactions with membrane lipids and proteins.

Differential Interaction of Bovine Alpha-Lactalbumin with Membranes: Interplay of Negatively Charged Lipids and Cholesterol

Many soluble proteins are known to interact with membranes and the mechanism of such interactions in cellular processes is beginning to be understood. Bovine alpha-lactalbumin (BLA) is a soluble protein and yet it interacts with membranes. We recently reported the specific binding of apo-BLA with negatively charged membranes using a variety of fluorescence approaches [1]. A novel finding is that BLA exhibits an enhanced binding to negatively charged membranes in the presence of cholesterol and it possesses cholesterol recognition/interaction amino acid consensus (CRAC), a motif recognized for preferential association with cholesterol in many proteins. We monitored the specificity of sterol interaction by replacing cholesterol with 7-dehydrocholesterol (an immediate biosynthetic precursor of cholesterol), and observed that BLA-sterol interaction is specific to membrane cholesterol. Significant Fluorescence Resonance Energy Transfer (FRET) was observed between the tryptophan residues of BLA and dehydroergosterol (a naturally occurring fluorescent analog of cholesterol) in presence of negatively charged membranes, indicating close proximity between them. Dipole potential measurements upon BLA binding to membranes and docking studies of cholesterol with BLA provide useful insight into the lipid selectivity of BLA binding to membranes. Depth analysis by the parallax approach provides evidence for interfacial localization of tryptophans of BLA when bound to membranes. Taken together, our results assume significance in the light of tumoricidal and antimicrobial functions of α-lactalbumin [2,3].

22-NBD-cholesterol, a new fluorescent substrate of bacterial cholesterol-oxidases

Fluorescent analogs of sterols are convenient agents for studying the transport, distribution, and protein—ligand interaction of natural sterols in cells [1]. However, the effect of the fluorescent moiety on the interaction parameters with cholesterol-transforming enzymes such as bacterial cholesterol-oxidases or mammalian steroid-hydroxylating cytochrome P450 has not been addressed in the literature. Herein we report data for the interaction of the fluorescent cholesterol analog 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-23,24-bisnor-5-cholen-3 -ol [22-NBD-cholesterol (1)] with cholesteroloxidases (EC 1.1.3.6). Cholesterol-oxidases are FAD-dependent enzymes that catalyze the transformation of 3 -hydroxy5steroids and are widely used for laboratory and clinical cholesterol determinations [2]. We calculated the interaction of 1 with the enzyme based on the three-dimensional structure of Brevibacterium sterolicum cholesterol-oxidase (code PDB 1COY) using the Autodock 4.0 programs and MGLTools. It was found with the help of molecular docking that 1 can bind to the active center of cholesterol-oxidase such that its 3 -hydroxy group is situated in close proximity to the functionally important His447 and Glu361 residues [3] and the redox-active groups of the FAD molecule. The calculated binding energies for cholesterol and 1 were practically the same (–11.43 and –11.94 kcal/mol, respectively). Next selective transformation of 1 was demonstrated using cholesterol-oxidase in vitro. After biotransformation of fluorescent substrate 1 with purified cholesterol-oxidase, the formation of 2, which retained the fluorescent properties of 1 (Scheme 1) was detected by HPLC (Shimadzu LC10-AD chromatograph with an RF-10A 1 fluorimetric detector and SPD-10A spectrophotometric detector; CH3CN:H2O:i-PrOH, 84:16:5; LiChroCART C18 column, Merck, 250 4 mm).

Sterol Properties Required for Microdomain Formation: From Model Systems to Living Yeast and Mammalian Cells

This work presents new insights into the evolution of sterol-enriched domains in the plasma membrane (PM) of eukaryotes. The recent finding of sphingolipid-enriched gel domains in the PM of Saccharomyces cerevisiae(1) demands novel strategies to study sterol-enriched liquid ordered domains in this organism. Therefore, we employed di-4-ANEPPS fluorescence, here shown to be sensitive to S. cerevisiae sterols and Fluorescence Lifetime Imaging Microscopy (FLIM) for the first time in S. cerevisiae PM. In vivo studies were performed with S. cerevisiae (wild type and erg6Δ), and animal (CHO) cells, modeled by liposomes containing ergosterol, zymosterol, or cholesterol, respectively. Different fluorescent probes were used, showing that these sterols had comparable effects regarding lipid bilayer order and complete solubilization of gel domains. However, confocal fluorescence microscopy of Giant Unilamellar Vesicles showed that zymosterol, a biosynthetic precursor of ergosterol and cholesterol, has no ability to form lipid rafts.In living cells, sterol-induced emission blue-shift and longer fluorescence lifetime of di-4-ANEPPS increased as: wild type<erg6Δ<CHO. Concomitantly, in model systems, cholesterol and zymosterol had much larger effects than ergosterol in di-4-ANEPPS fluorescence. However, zymosterol was unique because it yielded overlapping results for both saturated and unsaturated lipids. The two other sterols produced much stronger alterations with a saturated lipid, even though the absolute effect in di-4-ANEPPS fluorescence was often smaller than that of zymosterol. Hence, the differential regarding the interaction of sterols with saturated versus unsaturated lipids emerges as a requisite for lipid raft formation and as an important factor in sterol evolution.(1) F. Aresta-Branco, A.M. Cordeiro, H.S. Marinho, L. Cyrne, F. Antunes, R.F.M. de Almeida (2011) “Gel domains in the plasma membrane of Saccharomyces cerevisiae: highly ordered, ergosterol-free, sphingolipid-enriched lipid rafts.” J. Biol. Chem. 286:5043-5054.Acknowledgements: PTDC/QUI-BIQ/104311/2008 and Ciencia2007 (FCT, Portugal).