1-2-oleoylphosphatidylcholine and 25-hydroxycholesterol

OSBP binds, extracts and transfers sterols and phosphatidylinositol-4-phosphate (PI(4)P between liposomes, but the sequence of steps at the membrane surface leading to ligand removal is poorly characterized. In this study, we used dual polarization interferometry (DPI), a label-free surface analytical technique, to characterize the interaction of recombinant, purified OSBP as it flows over immobilized dioleoyl-phosphatidylcholine (DOPC) bilayers containing PI(4)P, cholesterol or 25-hydroxycholesterol. Kinetics of membrane interaction were analyzed for PI(4)P-binding and phosphorylation mutants of OSBP. Wild-type OSBP demonstrated a distinctive association with immobilized DOPC bilayers containing 1-8 mol% PI(4)P that was characterized by initial saturable binding followed by desorption, indicative of PI(4)P extraction. In support of this conclusion, an OSBP mutant with impaired binding and extraction of PI(4)P was stably absorbed to PI(4)P-containing membranes, while a pleckstrin homology domain mutant did not associate with PI(4)P-containing membranes. The inclusion of >2 mol% cholesterol, but not 25-hydroxycholesterol, in membranes, enhanced the absorption of the wild-type OSBP. A phosphomimetic of OSBP with enhanced in vitro sterol binding activity displayed membrane interaction properties similar to wild-type. These real-time flow studies allow us to dissect the association of OSBP with PI(4)P into discrete components; initial recruitment to PI(4)P membranes by the PH domain, detection and extraction of PI(4)P, and desorption due to ligand depletion.

Topics: Animals; Cholesterol; Hydroxycholesterols; Interferometry; Lipid Bilayers; Phosphatidylcholines; Phosphatidylinositol Phosphates; Receptors, Steroid; Recombinant Proteins; Sf9 Cells; Spodoptera

Lipid bilayer membranes composed of DOPC, DPPC, and a series of sterols demix into coexisting liquid phases below a miscibility transition temperature. We use fluorescence microscopy to directly observe phase transitions in vesicles of 1:1:1 DOPC/DPPC/sterol within giant unilamellar vesicles. We show that vesicles containing the "promoter" sterols cholesterol, ergosterol, 25-hydroxycholesterol, epicholesterol, or dihydrocholesterol demix into coexisting liquid phases as temperature is lowered through the miscibility transition. In contrast, vesicles containing the "inhibitor" sterols androstenolone, coprostanol, cholestenone, or cholestane form coexisting gel (solid) and liquid phases. Vesicles containing lanosterol, a sterol found in the cholesterol and ergosterol synthesis pathways, do not exhibit coexisting phases over a wide range of temperatures and compositions. Although more detailed phase diagrams and precise distinctions between gel and liquid phases are required to fully define the phase behavior of these sterols in vesicles, we find that our classifications of promoter and inhibitor sterols are consistent with previous designations based on fluorescence quenching and detergent resistance. We find no trend in the liquid-liquid or gel-liquid transition temperatures of membranes with promoter or inhibitor sterols and measure the surface fraction of coexisting phases. We find that the vesicle phase behavior is related to the structure of the sterols. Promoter sterols have flat, fused rings, a hydroxyl headgroup, an alkyl tail, and a small molecular area, which are all attributes of "membrane active" sterols.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Biophysics; Cholestanes; Cholestanol; Cholestenones; Cholesterol; Dehydroepiandrosterone; Detergents; Ergosterol; Hydrogen; Hydroxycholesterols; Lipid Bilayers; Lipids; Macromolecular Substances; Microscopy, Fluorescence; Models, Chemical; Phosphatidylcholines; Sterols; Temperature

In artificial membrane bilayers, saturated long acyl chain-containing phospholipids and cholesterol (Chol) interact to form more ordered domains than those in phospholipids with unsaturated or short fatty acyl chains. We have extended the fluorescence techniques of London et al. [Xu, X., and London, E. (2000) Biochemistry 39, 843-849; Xu, X., Bittman, R., Duportail, G., Heissler, D., Vilchezes, C., and London, E. (2001) J. Biol. Chem. 276, 33540-33546] to study the propensity of several steroids to form or disrupt such ordered lipid domains. Temperature-dependent fluorescence quenching and steady-state polarization of the extrinsic fluorescent probe diphenylhexatriene (DPH) in model membranes composed of dipalmitoylphosphatidylcholine (or sphingomyelin), a nitroxide spin-labeled phosphatidylcholine (12-SLPC), and a given steroid were combined to study the influence of the latter on (a) ordered lipid domain formation, (b) stabilization, and (c) the extension of the ordered lipid assemblies. The results of the two totally independent methods, fluorescence quenching by 12-SLPC and fluorescence polarization of DPH, show that all steroids examined, except for Chol and 25-hydroycholesterol, behave as lipid domain-disrupting compounds. Additionally, we found a positive correlation between the hydrophobicity of steroids and their ordered lipid domain-promoting activity. Comparison of the chemical structures disclosed some distinctive traits of ordered lipid domain-promoting steroids: (i) the presence of an isooctyl side chain bond at C17; (ii) the absence of carbons attached to C23 (i.e., C24-C27) in any of the other (domain-disrupting) steroids; (iii) the presence of a small polar group at position C3; and (iv) the absence of polar groups in the fused rings, with the exception of substitutions at position C3 in the A ring.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; Diphenylhexatriene; Fluorescence Polarization; Fluorescent Dyes; Hydrophobic and Hydrophilic Interactions; Hydroxycholesterols; Liposomes; Membrane Microdomains; Models, Chemical; Nitrogen Oxides; Phosphatidylcholines; Phospholipids; Pregnenolone; Spectrometry, Fluorescence; Spin Labels; Steroids; Structure-Activity Relationship

Planar oriented membranes of 1-palmitoyl, 2-oleoyl-phosphatidylcholine (POPC) containing cholesterol, 19-hydroxycholesterol, 22S-hydroxycholesterol, or 25-hydroxycholesterol in concentrations up to 5 mol % were investigated with angle-resolved fluorescence depolarization and electron spin resonance measurements. Analyses of the data with the Brownian diffusion model show that the oxysterols have structural effects similar to those of cholesterol: an increase in molecular order and no change in the rotational diffusion coefficients of the probe molecules. Time-resolved fluorescence anisotropy measurements on diphenylhexatriene (DPH) in small unilamellar vesicles of POPC and DOPC were performed using oxysterols commonly found in oxidized low density lipoproteins (LDL) in comparison to membranes containing cholesterol or no sterols. Analyses using the Brownian rotational diffusion model show that most LDL-oxysterols affect the vesicle physical structure in a manner similar to cholesterol, viz. an increase in molecular order and a decrease in the dynamics. Cholesterol-alpha-epoxide has a much smaller ordering effect than cholesterol in POPC-vesicles. A similar effect was found for 7 beta-hydroxycholesterol in DOPC-vesicles. The tendency of the oxysterols to influence the molecular order as compared to pure cholesterol may contribute to cell membrane permeability changes affecting crucial cell functions and events leading to vascular cell injury. Increased LDL oxysterol levels may account for some of the structural changes noted for oxidatively modified LDL as well as its toxicity to vascular cells.

Topics: Cholesterol; Cholesterol, LDL; Electron Spin Resonance Spectroscopy; Fluorescence Polarization; Hydroxycholesterols; Lipid Bilayers; Oxidation-Reduction; Phosphatidylcholines; Spectrometry, Fluorescence

The homogenizing effect of cholesterol and its oxidative derivatives, 7-ketocholesterol, cholesterol 5 alpha, 6 alpha-epoxide and 25-hydroxycholesterol, in liquid-crystalline 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC) bilayer vesicles was studied using the fluorescence lifetimes of 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-p hosphocholine (DPH-PC). The phase and modulation data were fitted either to discrete exponential models or to models characterized by continuous distributional lifetimes. Among all the models tested, it was found that the best one to account for the experimental data was the unimodal Lorentzian distribution. Thus, the DPH-PC lifetime was adequately described by a distributional center and a full width at half-maximum, for DOPC vesicles these values being 6.23 and 0.48 ns, respectively. Increasing the concentration of cholesterol, 7-ketocholesterol, or cholesterol 5 alpha, 6 alpha-epoxide from 0 to 30 mol% resulted in an increase of the lifetime center (e.g., 7.16 ns at 30 mol% cholesterol) and a decrease of the distributional width (e.g., 0.05 ns at 30 mol% cholesterol). On the other hand, up to 30 mol% of 25-hydroxycholesterol incorporated into the bilayer vesicles showed little influence on both lifetime parameters. Our results support the use of lifetime distributional width to evaluate membrane heterogeneity and suggest that oxysterols, depending on their molecular structural particulars, may exert cholesterol-like homogenizing effect in membranes.

Topics: Cholesterol; Diphenylhexatriene; Hydroxycholesterols; Ketocholesterols; Lipid Bilayers; Membrane Fluidity; Membrane Lipids; Phosphatidylcholines; Spectrometry, Fluorescence