Hexadecylphosphocholine interaction with lipid monolayers

The phospholipid analogue miltefosine or hexadecylphosphocholine (HePC) is a drug of high interest in the treatment for fatal visceral leishmaniasis (VL) due to Leishmania donovani particularly because of its activity by oral route. In this study, the interaction of HePC with a monolayer of beta-palmitoyl-gamma-oleyl-phosphatidylcholine (POPC) as membrane model or sterol (ergosterol or cholesterol) was investigated. At a constant pressure of 25 mN/m, the adsorption kinetics of HePC into the monolayers showed that HePC molecules are inserted into the monolayer of lipids as monomers until the critical micellar concentration (CMC). At HePC concentrations superior to the CMC, the micelles of HePC are deployed at the interface as groups of monomers into the POPC or sterol monolayer. The study of mixture of HePC/(POPC or sterol), spread at the air-water interface, shows that a simple miscibility between HePC and POPC is observed, whereas a high condensation appears between HePC and sterols showing a high affinity between HePC and sterols. In addition, HePC does not act as detergent disturbing membrane integrity.     

Cholesterol oxidase susceptibility of cholesterol and 5-androsten-3 beta-ol in pure sterol monolayers and in mixed monolayers containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

This study has examined the importance of the isocaproic side chain at C-17 of cholesterol to sterol/phospholipid interactions in monolayer membranes and to the cholesterol oxidase-susceptibility of cholesterol in pure and mixed monolayers at the air/water interface. The interactions between cholesterol or 5-androsten-3 beta-ol (which lacks the C-17 side chain) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in monolayers indicated that 5-androsten-3 beta-ol was not very efficient in causing condensation of the monolayer packing of POPC. Whereas cholesterol condensed the packing of POPC at all molar fractions examined (i.e., 0.25, 0.50 and 0.75 with regard to POPC), 5-androsten-3 beta-ol caused a slight condensing effect on POPC packing only in the equimolar mixture. The mean molecular area requirement of 5-androsten-3 beta-ol (in pure sterol monolayers at different lateral surface pressures) was 2.2-6.7% less than that observed for cholesterol. The pure 5-androsten-3 beta-ol monolayer also collapsed at lower lateral surface pressures compared with the pure cholesterol monolayer (34 mN/m and 45 mN/m, respectively). The cholesterol oxidase (Streptomyces sp.) catalyzed oxidation of cholesterol or 5-androsten-3 beta-ol in pure monolayers in the air/water interface (10 mN/m) proceeded with very similar rates, indicating that the enzyme did not recognize that the C-17 side chain of 5-androsten-3 beta-ol was missing. The oxidation of cholesterol or 5-androsten-3 beta-ol in mixed POPC-containing monolayers (equimolar mixture) also revealed similar reaction rates, although the reaction was slower in the mixed monolayer compared with the pure sterol monolayer. When the oxidation of cholesterol and 5-androsten-3 beta-ol was examined by monitoring the production of H2O2 (the sterol was solubilized in 2-propanol and the assay conducted in phosphate buffer), the maximal reaction rate observed with 5-androsten-3 beta-ol was only about 41% of that measured with cholesterol. From the cholesterol oxidase point-of-view, it can be concluded that the enzyme did not recognize the C-17 side chain of cholesterol (or lack of it in 5-androsten-3 beta-ol), when the sterol was properly oriented as a monolayer at the air/water interface. However, when the substrate was presented to the enzyme in a less controlled orientation (organic solvent in water), 5-androsten-3 beta-ol may have oriented itself unfavorably compared with the orientation of cholesterol, thereby leading to slower oxidation rates.     

Transmembrane Peptides Influence the Affinity of Sterols for Phospholipid Bilayers

Cholesterol is distributed unevenly between different cellular membrane compartments, and the cholesterol content increases from the inner bilayers toward the plasma membrane. It has been suggested that this cholesterol gradient is important in the sorting of transmembrane proteins. Cholesterol has also been to shown play an important role in lateral organization of eukaryotic cell membranes. In this study the aim was to determine how transmembrane proteins influence the lateral distribution of cholesterol in phospholipid bilayers. Insight into this can be obtained by studying how cholesterol interacts with bilayer membranes of different composition in the presence of designed peptides that mimic the transmembrane helices of proteins. For this purpose we developed an assay in which the partitioning of the fluorescent cholesterol analog CTL between LUVs and mβCD can be measured. Comparison of how cholesterol and CTL partitioning between mβCD and phospholipid bilayers with different composition suggests that CTL sensed changes in bilayer composition similarly as cholesterol. Therefore, the results obtained with CTL can be used to understand cholesterol distribution in lipid bilayers. The effect of WALP23 on CTL partitioning between DMPC bilayers and mβCD was measured. From the results it was clear that WALP23 increased both the order in the bilayers (as seen from CTL and DPH anisotropy) and the affinity of the sterol for the bilayer in a concentration dependent way. Although WALP23 also increased the order in DLPC and POPC bilayers the effects on CTL partitioning was much smaller with these lipids. This indicates that proteins have the largest effect on sterol interactions with phospholipids that have longer and saturated acyl chains. KALP23 did not significantly affect the acyl chain order in the phospholipid bilayers, and inclusion of KALP23 into DMPC bilayers slightly decreased CTL partitioning into the bilayer. This shows that transmembrane proteins can both decrease and increase the affinity of sterols for the lipid bilayers surrounding proteins. This is likely to affect the sterol distribution within the bilayer and thereby the lateral organization in biomembranes.     

A fluorescence study of dehydroergosterol in phosphatidylcholine bilayer vesicles

The fluorescent sterol delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) was incorporated into 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) small unilamellar vesicles (SUV) with and without cholesterol in order to monitor sterol-sterol interactions in model membranes. In the range 0-5 mol % fluorescent sterol, dehydroergosterol underwent a concentration-dependent relaxation characterized by red-shifted wavelengths of maximum absorption as well as altered ratios of absorbance maxima and fluorescence excitation maxima at 338 nm/324 nm. Fluorescence intensity per mole of dehydroergosterol increased up to 5 mol % in POPC vesicles. In contrast, quantum yield, steady-state anisotropy, limiting anisotropy, lifetime, and rotational rate remained relatively constant in this concentration range. Similarly, addition of increasing cholesterol in the range 0-5 mol % in the presence of 3 mol % dehydroergosterol also increased the fluorescence intensity per mole of dehydroergosterol, red-shifted wavelengths of maximum absorption, and altered ratios of absorbance maxima. In POPC vesicles containing between 5 and 33 mol % dehydroergosterol, the fluorescent dehydroergosterol interacted to self-quench, thereby decreasing the fluorescence intensity, quantum yield, steady-state anisotropy, and limiting anisotropy and increasing the rotational rate (decreased rotational relaxation time) of the fluorescent sterol. The fluorescence lifetime of dehydroergosterol remained unchanged. The results were in accord with the interpretation that below 5 mol% sterol, the sterols behaved as monomers exposed to some degree to the aqueous solvent in POPC bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ergosterol in POPC membranes: physical properties and comparison with structurally similar sterols

The physical properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ergosterol bilayers in the liquid-crystalline phase were determined using deuterium nuclear magnetic resonance (²H NMR) and vesicle extrusion. For the ²H NMR experiments, the sn-1 chain of POPC was perdeuterated, and spectra were taken as a function of ergosterol concentration and temperature. Analysis of the liquid-crystalline spectra provides clear evidence that two types of liquid-crystalline domains, neither of which is a liquid-ordered phase, having distinct average chain conformations coexist in 80:20 and 75:25 POPC/ergosterol membranes over a wide temperature range (from −2 to at least 31°C). Adding ergosterol to a concentration of 25 mol % increases POPC-d₃₁ chain ordering as measured by the NMR spectral first moment M₁ and also increases the membrane lysis tension, obtained from vesicle extrusion. Further addition of ergosterol had no effect on either chain order or lysis tension. This behavior is in marked contrast to the effect of cholesterol on POPC membranes: POPC/cholesterol membranes have a linear dependence of chain order on sterol concentration to at least 40 mol %. To investigate further we compared the dependence on sterol structure and concentration of the NMR spectra and lysis tension for several POPC/sterol membranes at 25°C. For all POPC/sterol membranes investigated in this study, we observed a universal linear relation between lysis tension and M₁. This suggests that changes in acyl chain ordering directly affect the tensile properties of the membrane.     

Intracellular sterol transport in eukaryotes,a connection to mitochondrial function?

Eukaryotic cells synthesize sterols in the endoplasmatic reticulum (ER) from where it needs to be efficiently transported to the plasma membrane, which harbors approximately 90% of the free sterol pool of the cell. Sterols that are being taken up from the environment, on the other hand, are transported back from the plasma membrane to the ER, where the free sterols are esterified to steryl esters. The molecular mechanisms that govern this bidirectional movement of sterols between the ER and the plasma membrane of eukaryotic cells are only poorly understood. Proper control of this transport is important for normal cell function and development as indicated by fatal human pathologies such as Niemann Pick type C disease and atherosclerosis, which are characterized by an over-accumulation of free sterols within endosomal membranes and the ER, respectively. Recently, a number of complementary approaches using Saccharomyces cerevisiae as a model organism lead to a more precise characterization of the pathways that control the subcellular transport of sterols and led to the identification of components that directly or indirectly affect sterol uptake at the plasma membrane and its transport back to the ER. A genetic approach that is based on the fact that yeast is a facultative anaerobic organism, which becomes auxotrophic for sterols in the absence of oxygen, resulted in the identification of 17 genes that are required for efficient uptake and/or transport of sterols. Unexpectedly, many of these genes are required for mitochondrial functions. A possible connection between mitochondrial biogenesis and sterol biosynthesis and uptake will be discussed in light of the fact that cholesterol transport into the inner membranes of mitochondria is a well established sterol transport route in vertebrates, where it is required to convert cholesterol into pregnenolone, the precursor of steroids.     

Organization and interaction of cholesterol and phosphatidylcholine in model bilayer membranes

The molecular organization of sterols in liposomes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at 37 degrees C is examined by utilizing the fluorescent analogue of cholesterol cholesta-5,7,9-trien-3 beta-ol (cholestatrienol). (1) Cholestatrienol is shown to be indistinguishable from native cholesterol in terms of its ability to condense POPC, as determined by (i) pressure/area studies of mixed-lipid monolayers and (ii) its ability to increase the order of POPC bilayers (determined by electron spin resonance studies) whether on its own or admixed with cholesterol at various ratios. (2) By analysis of the perturbation of the absorption spectra, cholestatrienol was found to be freely miscible in aggregates of cholesterol in buffer. In contrast, a lack of any detectable direct interaction of the sterol molecules in POPC bilayers was detected. (3) Fluorescence intensity and lifetime measurements of POPC/sterol (1:1 mol/mol) at various cholesterol/cholestratrienol molar ratios (0.5:1 up to 1:1 cholestatrienol/POPC) confirmed that sterol molecules in the membrane matrix were not associated to any great degree. (4) A quantitative estimate of how close sterol molecules approach each other in the membrane matrix was evaluated from the concentration dependence of the steady-state depolarization of fluorescence and was found to be 10.6 A. From geometrical considerations, the sterol/phospholipid phase at 1:1 mol/mol is depicted as each sterol having four POPC molecules as nearest neighbors. We term this arrangement of the lipid matrix an "ordered bimolecular mesomorphic lattice". (5) The concentration dependence of depolarization of fluorescence of cholestatrienol in POPC liposomes in the absence of cholesterol yielded results that were consistent with the cholestatrienol molecules being homogeneously dispersed throughout the phospholipid phase at sterol/POPC ratios of less than 1:1. (6) From qualitative calculations of the van der Walls' hydrophobic interactions of the lipid species, the phospholipid condensing effect of cholesterol is postulated to arise from increased interpenetration of the flexible methylene segments of the acyl chains, as a direct result of their greater mutual attraction compared to their attraction for neighboring sterol molecules. (7) The interdependence of the ordered bimolecular mesomorphic lattice and the acyl chain condensation is discussed in an effort to understand the ability of cholesterol to modulate the physical and mechanical properties of biological membranes.     

Displacement of sterols from sterol/sphingomyelin domains in fluid bilayer membranes by competing molecules

The formation of sterol and palmitoyl sphingomyelin enriched ordered domains in a fluid bilayer was examined using domain selective fluorescent reporter molecules (cholestatrienol and trans-parinaric acid containing lipids) together with a quencher molecule in the fluid phase. The aim of the study was to explore how stable the ordered domains were and how different, biologically interesting, membrane intercalators could affect domain stability and sterol distribution between domains. We show that sterols easily can be displaced from ordered domains by a variety of saturated, single- and double-chain membrane intercalators with a small polar group as a common denominator. Of the two-chain intercalators examined, both palmitoyl ceramide and palmitoyl dihydroceramide were effective in displacing sterols from ordered domains. Of the single-chain intercalators, hexadecanol and hexadecyl amide displaced the sterol from sterol/sphingomyelin domains, whereas palmitic acid, sphingosine and sphinganine failed to do so. All molecules examined stabilized the sphingomyelin-rich domains, as reported by trans-parinaric-sphingomyelin and by scanning calorimetry. Parallels between the displacement of sterol from ordered domains in our model membrane system and the ability of the above mentioned molecules to alter the chemical activity and distribution of sterols in biological membranes are discussed.     

Externalization of phosphatidylserine from inner to outer layer may alter the effect of plant sterols on human erythrocyte membrane - The Langmuir monolayer studies

One of the factors, which can strongly modify the cell membrane composition, is disordering in membrane asymmetry, resulting from redistribution of lipids from inner to outer layer. Such a disturbance may affect the behavior of various biologically active compounds incorporating into membranes. In this contribution, the relationship between the amounts of phosphatidylserine (PS) in the model outer layer of human erythrocyte (RBC) membrane and the effect induced by a plant sterol (β-sitosterol) was verified. The experiments were performed on multicomponent Langmuir films imitating red blood cell (RBC) membrane, differing in the contents of PS (0%; 5% and 10%) into which the plant sterol was incorporated in various concentrations. The analysis of experimental results (surface pressure-area isotherms complemented with Brewster Angle Microscopy (BAM) proved that the presence of phosphatidylserine molecules, depending on their contents in the mixed monolayer mimicking RBC membrane, changes its properties and exerts influence on the effect of plant sterol on the model system. The addition of phytosterol into the monolayer that lacks or contains only 5% of PS was found to be of rather weak effect on the properties of the system. However, in the case of the model membrane containing the increased amount (10%) of PS, the incorporation of plant sterol strongly affects the interactions between molecules and caused thermodynamic destabilization of the monolayer imitating RBC membrane. These results allow one to suggest that externalization of phosphatidyserine to the outer membrane leaflet may differentiate the effect of plant sterols on cell membranes of various origins.     

Externalization of phosphatidylserine from inner to outer layer may alter the effect of plant sterols on human erythrocyte membrane — The Langmuir monolayer studies

One of the factors, which can strongly modify the cell membrane composition, is disordering in membrane asymmetry, resulting from redistribution of lipids from inner to outer layer. Such a disturbance may affect the behavior of various biologically active compounds incorporating into membranes. In this contribution, the relationship between the amounts of phosphatidylserine (PS) in the model outer layer of human erythrocyte (RBC) membrane and the effect induced by a plant sterol (β-sitosterol) was verified. The experiments were performed on multicomponent Langmuir films imitating red blood cell (RBC) membrane, differing in the contents of PS (0%; 5% and 10%) into which the plant sterol was incorporated in various concentrations. The analysis of experimental results (surface pressure–area isotherms complemented with Brewster Angle Microscopy (BAM) proved that the presence of phosphatidylserine molecules, depending on their contents in the mixed monolayer mimicking RBC membrane, changes its properties and exerts influence on the effect of plant sterol on the model system. The addition of phytosterol into the monolayer that lacks or contains only 5% of PS was found to be of rather weak effect on the properties of the system. However, in the case of the model membrane containing the increased amount (10%) of PS, the incorporation of plant sterol strongly affects the interactions between molecules and caused thermodynamic destabilization of the monolayer imitating RBC membrane. These results allow one to suggest that externalization of phosphatidyserine to the outer membrane leaflet may differentiate the effect of plant sterols on cell membranes of various origins.     

Sterol Composition and Ecdysteroid Content of Eggs of the Root-knot Nematodes Meloidogyne incognita and M. arenaria

Free and esterified sterols of eggs of the root-knot nematodes Meloidogyne incognita races 2 and 3 and M. arenaria race 1 were isolated and identified by gas-liquid chromatography-mass spectrometry. The major sterols of eggs of each race were 24-ethylcholesterol (33.4-38.8% of total sterol), 24-ethylcholestanol (18.3-25.3%), 24-methylcholesterol (8.6-11.7%), 24-methylcholestanol (7.7-12.5%), and cholesterol (4.6-11.6%). Consequently, the major metabolic transformation performed by Meloidogyne females or eggs upon host sterols appeared to be saturation of the sterol nucleus. The free and esterified sterols of the same race did not differ appreciably, except for a slight enrichment of the steryl esters in cholesterol. Although the sterol composition of Meloidogyne eggs differed from that of other life stages of other genera of plant-parasitic nematodes, the three Meloidogyne races could not be distinguished from each other by their egg sterols. Ecdysteroids, compounds with hormonal function in insects, were not detected by radioimmunoassay in the Meloidogyne eggs either as free ecdysteroids or as polar conjugates.     

Role of sterol superlattice in free radical-induced sterol oxidation in lipid membranes

We developed a new fluorescence assay for sterol oxidation and used it to study the relationship between free radical-induced sterol oxidation and membrane sterol lateral organization. This assay used dehydroergosterol (DHE) as both a membrane probe and a membrane component. Sterol oxidation was induced by a free radical generator, AAPH (2,2'-azobis(2-amidinopropane)dihydrochloride). Using this new assay, we found that, in unilamellar vesicles composed of DHE and 1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine (POPC), the initial rate of DHE oxidation induced by AAPH changed with membrane sterol content in an alternating manner, exhibiting a local maximum at 20.3, 22.2, 25.0, 32.3, and 40.0 mol % DHE. These mole fractions correspond to the critical sterol mole fractions C(r) predicted for maximal sterol superlattice formation. In three-component bilayers composed of POPC, cholesterol, and DHE (fixed at 1 and 5 mol %), the initial rate of AAPH-induced DHE oxidation exhibited a biphasic change whenever the total sterol mole fraction, irrespective of the DHE content, was near C(r), indicating that the correlation between sterol oxidation and sterol superlattice formation revealed in this study is not an artifact due to the use of the fluorescent cholesterol analogue DHE. The alternating variation of AAPH-induced sterol oxidation with sterol content also appeared in multicomponent unilamellar vesicles containing bovine brain sphingomyelins (bbSPM), POPC, and DHE. The present work and our previous study on cholesterol oxidase-induced sterol oxidation [Wang et al. (2004) Biochemistry 43, 2159-2166] suggest that sterol oxidation in general, either by reactive oxygen species or by enzymes, may be regulated by the extent of sterol superlattice in the membrane and thus regulated by the membrane sterol content in a fine-tuning manner.     

Sphingomyelin analogs with branched N-acyl chains: The position of branching dramatically affects acyl chain order and sterol interactions in bilayer membranes

Sphingolipids have been found to have single methyl branchings both in their long-chain base and in their N-linked acyl chains. In this study we determined how methyl-branching in the N-linked acyl chain of sphingomyelin (SM) affected their membrane properties. SM analogs with a single methyl-branching at carbon 15 (of a 17:0 acyl chain; anteiso) had a lower gel-liquid transition temperature as compared to an iso-branched SM analog. Phytanoyl SM (methyls at carbons 3, 7, 11 and 15) as well as a SM analog with a methyl on carbon 10 in a hexadecanoyl chain failed to show a gel-liquid transition above 10 °C. Only the two distally branched SM analogs (iso and anteiso) formed ordered domains with cholesterol in a 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer. However, domains formed by the branched SM analogs appeared to contain less sterol when compared to palmitoyl SM (PSM) as the saturated phospholipid. Sterol-enriched domains formed by the anteiso SM analog were also less stable against temperature than domains formed by PSM. Both the 10-methyl and phytanoyl SM analogs failed to form sterol-enriched domains in the POPC bilayer. Acyl chain branching weakened SM/sterol interactions markedly when compared to PSM, as also evidenced from the decreased affinity of cholestatrienol to bilayers containing branched SM analogs. Our results show that methyl-branching weakened intermolecular interactions in a position-dependent manner.     

The replacement of cholesterol by phytosterols and the increase of total sterol content in model erythrocyte membranes

The activity of phytosterols on human organism includes the ability of these compounds to incorporate into membranes. In the consequence the plant sterols are able to increase total sterol concentration in membrane or/and to replace cholesterol molecules. The aim of this work was to compare the influence of both these effects on the properties of model erythrocyte membranes. Moreover, the interactions between the plant sterols (β-sitosterol and stigmasterol) and saturated–monounsaturated phosphatidylcholine were investigated and the condensing and ordering potency of these phytocompounds on membrane phospholipids were thoroughly analyzed. It was found that the addition of the plant sterols into model membrane modifies the condensation, ordering and interactions in the system. Moreover, the replacement of mammalian sterol by phytosterol more strongly influences the model system than even a 10% increase of total sterol concentration induced by the incorporation of the plant sterol, at constant content of cholesterol. The investigated plant sterols at their lower concentration in the mixed system are of similar effect on its properties. At higher content stigmasterol was found to modify the properties of model membrane more strongly than β-sitosterol.     

Influence of stigmastanol and stigmastanyl-phosphorylcholine, two plasma cholesterol lowering substances, on synthetic phospholipid membranes. A 2H- and 31P-NMR study.

Cholesterol, stigmastanol, and stigmastanyl-phosphorylcholine (ST-PC) were incorporated into model membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). POPC and ST-PC were deuterated at the lipid headgroup, DOPC at the cis-double bonds. The influence of the three sterols on the motion and conformation of the lipid headgroups and the hydrocarbon chains was monitored with 2H- and 31P-NMR. All three sterols were freely miscible with the lipid matrix in concentrations of up to 50 mol% without inducing phase separations or nonbilayer structures. However, the molecules exert quite different effects on the phospholipid bilayer. Cholesterol and stigmastanol are largely buried in the hydrocarbon part of the membrane, distinctly restricting the flexing motions of the fatty acyl chains whereas the conformation of the phospholipid headgroups is little affected. In contrast, ST-PC is anchored with its headgroup in the layer of phospholipid dipoles, preventing an extensive penetration of the sterol ring into the hydrocarbon layer. Hence ST-PC has almost no effect on the hydrocarbon chains but induces a characteristic conformational change of the phospholipid headgroups. The 2H- and 31P-NMR spectra of mixed phospholipid/ST-PC membranes further demonstrate that the PC headgroup of ST-PC has a similar orientation as the surrounding phosphatidylcholine headgroups. For both types of molecules the -P-N+ dipole is essentially parallel to the membrane surface. Addition of ST-PC induces a small rotation of the POPC headgroup towards the water phase.     

Survival strategies of a sterol auxotroph

The high sterol concentration in eukaryotic cell membranes is thought to influence membrane properties such as permeability, fluidity and microdomain formation. Drosophila cannot synthesize sterols, but do require them for development. Does this simply reflect a requirement for sterols in steroid hormone biosynthesis, or is bulk membrane sterol also essential in Drosophila? If the latter is true, how do they survive fluctuations in sterol availability and maintain membrane homeostasis? Here, we show that Drosophila require both bulk membrane sterol and steroid hormones in order to complete adult development. When sterol availability is restricted, Drosophila larvae modulate their growth to maintain membrane sterol levels within tight limits. When dietary sterol drops below a minimal threshold, larvae arrest growth and development in a reversible manner. Strikingly, membrane sterol levels in arrested larvae are dramatically reduced (dropping sixfold on average) in most tissues except the nervous system. Thus, sterols are dispensable for maintaining the basic membrane biophysical properties required for cell viability; these functions can be performed by non-sterol lipids when sterols are unavailable. However, bulk membrane sterol is likely to have essential functions in specific tissues during development. In tissues in which sterol levels drop, the overall level of sphingolipids increases and the proportion of different sphingolipid variants is altered. These changes allow survival, but not growth, when membrane sterol levels are low. This relationship between sterols and sphingolipids could be an ancient and conserved principle of membrane homeostasis.     

The influence of terpinen-4-ol and eucalyptol – The essential oil components - on fungi and plant sterol monolayers

Antifungal and herbicidal activity of terpenes, being the components of the essential oils, is directly related to the incorporation of these compounds into cellular membranes. Thus, the differences in the lipid composition of various pathogenic membranes may be the factor determining the activity of these molecules. One of the class of lipids, which form the membrane environment are sterols. The aim of this work was to compare the effect of two terpenes: terpinen-4-ol and eucalyptol on the monolayers formed by ergosterol and β – sitosterol, which are the components of fungi and plant membranes, respectively. The modifications in the sterol monolayer properties were investigated in the surface pressure-area measurements and penetration studies as well as in a micrometer scale (Brewster angle microscopy experiments) and in nanoscale (GIXD technique). It was evidenced that although at higher surface pressure the terpene molecules are in part removed from the interface, they are able to substantially modify the condensation, morphology and molecular organization of the sterol film. It was also found that the incorporation of terpenes into sterol films is comparable for both sterols, however, β – sitosterol monolayers properties are affected more strongly than ergosterol films. Finally, the analysis of the results of the studies performed on model membrane systems and the results of antimicrobial studies reported in literature, enabled us to suggest that the activity of terpenes depends on the membrane composition and that the sterol concentration may be important from the point of view of antifungal effect of terpinen-4-ol and eucalyptol.     

Sterol affinity for bilayer membranes is affected by their ceramide content and the ceramide chain length

It is known that ceramides can influence the lateral organization in biological membranes. In particular ceramides have been shown to alter the composition of cholesterol and sphingolipid enriched nanoscopic domains, by displacing cholesterol, and forming gel phase domains with sphingomyelin. Here we have investigated how the bilayer content of ceramides and their chain length influence sterol partitioning into the membranes. The effect of ceramides with saturated chains ranging from 4 to 24 carbons in length was investigated. In addition, unsaturated 18:1- and 24:1-ceramides were also examined. The sterol partitioning into bilayer membranes was studied by measuring the distribution of cholestatrienol, a fluorescent cholesterol analogue, between methyl-β-cyclodextrin and large unilamellar vesicle with defined lipid composition. Up to 15 mol% ceramide was added to bilayers composed of DOPC:PSM:cholesterol (3:1:1), and the effect on sterol partitioning was measured. Both at 23 and 37 °C addition of ceramide affected the sterol partitioning in a chain length dependent manner, so that the ceramides with intermediate chain lengths were the most effective in reducing sterol partitioning into the membranes. At 23 °C the 18:1-ceramide was not as effective at inhibiting sterol partitioning into the vesicles as its saturated equivalent, but at 37 °C the additional double bond had no effect. The longer 24:1-ceramide behaved as 24:0-ceramide at both temperatures. In conclusion, this work shows how the distribution of sterols within sphingomyelin-containing membranes is affected by the acyl chain composition in ceramides. The overall membrane partitioning measured in this study reflects the differential partitioning of sterol into ordered domains where ceramides compete with the sterol for association with sphingomyelin.     

Phospholipase A2 hydrolysis of supported phospholipid bilayers: a neutron reflectivity and ellipsometry study

We have investigated the phospholipase A(2) catalyzed hydrolysis of supported phospholipid bilayers using neutron reflection and ellipsometry. At the hydrophilic silica-water interface, hydrolysis of phosphatidylcholine bilayers by phospholipase A(2) from Naja mossambica mossambica venom is accompanied by destruction of the bilayer at an initial rate, which is comparable for DOPC and DPPC but is doubled for POPC. The extent of bilayer destruction at 25 degrees C decreases from DOPC to POPC and is dramatically reduced for DPPC. Neutron reflectivity measurements indicate that the enzyme penetrates into the bilayers in increasing order for DOPC, POPC, and DPPC, while the amount of enzyme adsorbed at the interface is smallest for DPPC and exhibits a maximum for POPC. Penetration into the hydrophobic chain region in the bilayer is further supported by the fact that the enzyme adsorbs strongly and irreversibly to a hydrophobic monolayer of octadecyltrichlorosilane. These results are rationalized in terms of the properties of the reaction products and the effect of their accumulation in the membrane on the kinetics of enzyme catalysis.     

Sterol biosynthesis and prokaryotes-to-eukaryotes evolution

It has been widely accepted that there exists a correlation between prokaryotes-to-eukaryotes evolution and atmospheric oxygen rise. However, it is a great challenge to elucidate the mechanisms underlying the correlation. Considering the facts that cellular communication of eukaryotes depends largely on membrane functions (e.g., endo- and exocytosis) and sterols play a key role in fulfilling these functions, we propose that the biosynthesis of sterols represents a critical step in the prokaryotes-to-eukaryotes evolution. Indeed, sterol biosynthesis is nearly ubiquitous among eukaryotes, but is generally excluded by prokaryotes. More importantly, during the biosynthesis of sterols, oxygen is absolutely required. Therefore, the missing link between prokaryotes-to-eukaryotes evolution and atmospheric oxygen rise is likely to reside in, at least in part, sterol biosynthesis, i.e., high atmospheric oxygen concentration facilitates the generation of sterols and thus benefits the birth of complex organisms.