1-palmitoyl-2-oleoylphosphatidylcholine and 25-hydroxycholesterol

Oxysterols are oxidized derivatives of cholesterol with many important biological functions. Trafficking of oxysterols in and between cells is not well studied, largely due to the lack of appropriate oxysterol analogs. Intrinsically fluorescent oxysterols present a new route towards direct observation of intracellular oxysterol trafficking by fluorescence microscopy. We characterize the fluorescence properties of the existing fluorescent 25-hydroxycholesterol analog 25-hydroxycholestatrienol, and propose a new probe with an extended conjugated system. The location of both probes inside a membrane is analyzed and compared with that of 25-hydroxycholesterol using molecular dynamics simulations. The analogs' one- and two-photon absorption properties inside the membrane are evaluated using electronic structure calculations with polarizable embedding. Due to predicted keto-enol tautomerisation of the new oxysterol analog, we also evaluate the keto form. Both analogs are found to be good probe candidates for 25-hydroxycholesterol, provided that the new analog remains in the enol-form. Only the new analog with extended conjugated system shows significant two-photon absorption, which is strongly enhanced by the presence of the membrane.

Topics: Hydroxycholesterols; Liposomes; Microscopy, Fluorescence; Molecular Dynamics Simulation; Oxysterols; Phosphatidylcholines; Spectrophotometry, Ultraviolet

Side-chain oxysterols are enzymatically generated oxidation products of cholesterol that serve a central role in mediating cholesterol homeostasis. Recent work has shown that side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), alter membrane structure in very different ways from cholesterol, suggesting a possible mechanism for how these oxysterols regulate cholesterol homeostasis. Here we extend our previous work by using molecular-dynamics simulations of 25-HC and cholesterol mixtures in 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayers to examine the combined effects of 25-HC and cholesterol in the same bilayer. 25-HC causes larger changes in membrane structure when added to cholesterol-containing membranes than when added to cholesterol-free membranes. We also find that the presence of 25-HC changes the position, orientation, and solvent accessibility of cholesterol, shifting it into the water interface and thus increasing its availability to external acceptors. This is consistent with experimental results showing that oxysterols can trigger cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. These effects provide a potential mechanism for 25-HC-mediated regulation of cholesterol trafficking and homeostasis through modulation of cholesterol availability.

Topics: Biological Availability; Cholesterol; Hydrogen Bonding; Hydroxycholesterols; Lipid Bilayers; Membranes, Artificial; Molecular Dynamics Simulation; Phosphatidylcholines; Phospholipids; Solvents

Cholesterol is essential for proper function and regulation of eukaryotic membranes, and significant amounts of metabolic energy are dedicated to controlling cellular cholesterol levels. Oxidation products of cholesterol, the oxysterols, are enzymatically produced molecules that play a major role in mediating cholesterol homeostasis through mechanisms which have not yet been fully elucidated. Certain oxysterols are known to have direct effects on membrane permeability and structure, effects that are strikingly different from that of cholesterol. We use molecular dynamics simulations of these oxysterols in 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC) bilayers to explain the structural origins for the differing effects of cholesterol and 25-hydroxycholesterol on bilayer properties. In particular, we demonstrate that the source for these differing perturbations is the much wider range of molecular orientations accessible to 25-hydroxycholesterol when compared to cholesterol. This study shows that direct membrane perturbation by side-chain oxysterols is significant and suggests that these membrane perturbations may play a role in the oxysterol regulation of cholesterol homeostasis.

Topics: Cholesterol; Compressive Strength; Computer Simulation; Elasticity; Hydrogen Bonding; Hydroxycholesterols; Lipid Bilayers; Models, Biological; Models, Molecular; Molecular Structure; Oxidation-Reduction; Phosphatidylcholines

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