Effective Parameters Controlling Sterol Transfer: A Time-Resolved Small-Angle Neutron Scattering Study.

Though cholesterol is the most prevalent and essential sterol in mammalian cellular membranes, its precursors, post-synthesis cholesterol products, as well as its oxidized derivatives play many other important physiological roles. Using a non-invasive in situ technique, time-resolved small angle neutron scattering, we report on the rate of membrane desorption and corresponding activation energy for this process for a series of sterol precursors and post-synthesis cholesterol products that vary from cholesterol by the number and position of double bonds in B ring of cholesterol's steroid core. In addition, we report on sterols that have oxidation modifications in ring A and ring B of the steroid core. We find that sterols that differ in position or the number of double bonds in ring B have similar time and energy characteristics, while oxysterols have faster transfer rates and lower activation energies than cholesterol in a manner generally consistent with known sterol characteristics, like Log P, the n-octanol/water partitioning coefficient. We find, however, that membrane/water partitioning which is dependent on lipid-sterol interactions is a better predictor, shown by the correlation of the sterols' tilt modulus with both the desorption rates and activation energy.

Lipoprotein-Induced Increases in Cholesterol and 7-Ketocholesterol Result in Opposite Molecular-Scale Biophysical Effects on Membrane Structure.

Under hypercholesterolemic conditions, exposure of cells to lipoproteins results in a subtle membrane increase in the levels of cholesterol and 7-ketocholesterol, as compared to normal conditions. The effect of these physiologically relevant concentration increases on multicomponent bilayer membranes was investigated using coarse-grained molecular dynamics simulations. Significant changes in the structural and dynamic properties of the bilayer membranes resulted from these subtle increases in sterol levels, with both sterol species inducing decreases in the lateral area and inhibiting lateral diffusion to varying extents. Cholesterol and 7-ketocholesterol, however, exhibited opposite effects on lipid packing and orientation. The results from this study indicate that the subtle increases in membrane sterol levels induced by exposure to lipoproteins result in molecular-scale biophysical perturbation of membrane structure.

Membrane modulatory effects of omega-3 fatty acids: Analysis of molecular level interactions.

Bioactive omega-3 polyunsaturated fatty acids have been shown to reduce the risk of death in patients with cardiovascular disease and alleviate the symptoms of other inflammatory diseases. However, the mechanisms of action of these effects remain unclear. It has been postulated that omega-3 polyunsaturated fatty acids modify cell membranes by incorporation into the membrane and altering the signaling properties of cellular receptors. In this chapter, we explore the effects of omega-3 polyunsaturated fatty acids on cell membrane structure and function. We present a review of the current evidence for the health benefits of these compounds and explore the molecular mechanisms through which omega-3 polyunsaturated fatty acids interact with membrane lipids and modulate bilayer structure. Using computational models of multicomponent phospholipid bilayers, we assess the consequences of incorporation of these fatty acids on membrane lipid packing, water permeation, and membrane structure.

Membrane Stiffening in Osmotic Swelling: Analysis of Membrane Tension and Elastic Modulus.

The effects of osmotic swelling on key cellular biomechanical properties are explored in this chapter. We present the governing equations and theoretical backgrounds of the models employed to estimate cell membrane tension and elastic moduli from experimental methods, and provide a summary of the prevailing experimental approaches used to obtain these biomechanical parameters. A detailed analysis of the current evidence of the effects of osmotic swelling on membrane tension and elastic moduli is provided. Briefly, due to the buffering effect of unfolding membrane reservoirs, mild hypotonic swelling does not change membrane tension or the adhesion of the membrane to the underlying cytoskeleton. Conversely, osmotic swelling causes the cell membrane envelope to stiffen, measured as an increase in the membrane elastic modulus.

Molecular Dynamics Simulations of Kir2.2 Interactions with an Ensemble of Cholesterol Molecules.

Cholesterol is a major regulator of multiple types of ion channels, but the specific mechanisms and the dynamics of its interactions with the channels are not well understood. Kir2 channels were shown to be sensitive to cholesterol through direct interactions with "cholesterol-sensitive" regions on the channel protein. In this work, we used Martini coarse-grained simulations to analyze the long (µs) timescale dynamics of cholesterol with Kir2.2 channels embedded into a model membrane containing POPC phospholipid with 30 mol% cholesterol. This approach allows us to simulate the dynamic, unbiased migration of cholesterol molecules from the lipid membrane environment to the protein surface of Kir2.2 and explore the favorability of cholesterol interactions at both surface sites and recessed pockets of the channel. We found that the cholesterol environment surrounding Kir channels forms a complex milieu of different short- and long-term interactions, with multiple cholesterol molecules concurrently interacting with the channel. Furthermore, utilizing principles from network theory, we identified four discrete cholesterol-binding sites within the previously identified cholesterol-sensitive region that exist depending on the conformational state of the channel-open or closed. We also discovered that a twofold decrease in the cholesterol level of the membrane, which we found earlier to increase Kir2 activity, results in a site-specific decrease of cholesterol occupancy at these sites in both the open and closed states: cholesterol molecules at the deepest of these discrete sites shows no change in occupancy at different cholesterol levels, whereas the remaining sites showed a marked decrease in occupancy.

Differential Effects of Sterols on Ion Channels: Stereospecific Binding vs Stereospecific Response.

Numerous ion channels have been shown to be regulated by the level of membrane cholesterol, but the mechanisms responsible for these effects are still not well understood. The key question in the field is how to discriminate between the contributions of the two central mechanisms that might be responsible for the sensitivity of ion channels to cholesterol: specific sterol-protein interactions or regulation of channels by the bilayer physical properties. Comparative analysis of cholesterol and its isomers on the function of an ion channel is a powerful tool to achieve this goal. An increasing number of studies show that cholesterol regulates several types of ion channels in a stereospecific manner, suggesting an involvement of specific sterol-protein interactions. However in this chapter, we present evidence that the stereospecificity of cholesterol-ion channel interactions may be mediated, not by a lack of binding, as has been generally assumed, but by the specificity of the interaction, which results in a functional effect, in the case of native cholesterol, and a lack of functional effect, in the case of a cholesterol isomer. In other words, accumulating evidence suggests that the structural requirements of ion channel cholesterol-binding sites are lax, allowing chiral isomers of cholesterol to bind to the same site in a nonstereospecific way, but the ability of a sterol to confer a functional effect on the channel activity can still be stereospecific. This is an important distinction both conceptually and methodologically. Indeed, our analysis shows that the orientations of cholesterol and its chiral isomer ent-cholesterol within a hydrophobic binding pocket of Kir2.2 are significantly different, and we propose that this difference may underlie distinct functional outcomes.

oxLDL induces endothelial cell proliferation via Rho/ROCK/Akt/p27kip1 signaling: opposite effects of oxLDL and cholesterol loading.

Oxidized modifications of LDL (oxLDL) play a key role in the development of endothelial dysfunction and atherosclerosis. However, the underlying mechanisms of oxLDL-mediated cellular behavior are not completely understood. Here, we compared the effects of two major types of oxLDL, copper-oxidized LDL (Cu2+-oxLDL) and lipoxygenase-oxidized LDL (LPO-oxLDL), on proliferation of human aortic endothelial cells (HAECs). Cu2+-oxLDL enhanced HAECs' proliferation in a dose- and degree of oxidation-dependent manner. Similarly, LPO-oxLDL also enhanced HAEC proliferation. Mechanistically, both Cu2+-oxLDL and LPO-oxLDL enhance HAEC proliferation via activation of Rho, Akt phosphorylation, and a decrease in the expression of cyclin-dependent kinase inhibitor 1B (p27kip1). Both Cu2+-oxLDL or LPO-oxLDL significantly increased Akt phosphorylation, whereas an Akt inhibitor, MK2206, blocked oxLDL-induced increase in HAEC proliferation. Blocking Rho with C3 or its downstream target ROCK with Y27632 significantly inhibited oxLDL-induced Akt phosphorylation and proliferation mediated by both Cu2+- and LPO-oxLDL. Activation of RhoA was blocked by Rho-GDI-1, which also abrogated oxLDL-induced Akt phosphorylation and HAEC proliferation. In contrast, blocking Rac1 in these cells had no effect on oxLDL-induced Akt phosphorylation or cell proliferation. Moreover, oxLDL-induced Rho/Akt signaling downregulated cell cycle inhibitor p27kip1 Preloading these cells with cholesterol, however, prevented oxLDL-induced Akt phosphorylation and HAEC proliferation. These findings provide a new understanding of the effects of oxLDL on endothelial proliferation, which is essential for developing new treatments against neovascularization and progression of atherosclerosis.

Molecular-Scale Biophysical Modulation of an Endothelial Membrane by Oxidized Phospholipids.

The influence of two bioactive oxidized phospholipids on model bilayer properties, membrane packing, and endothelial cell biomechanics was investigated computationally and experimentally. The truncated tail phospholipids, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), are two major oxidation products of the unsaturated phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphocholine. A combination of coarse-grained molecular dynamics simulations, Laurdan multiphoton imaging, and atomic force microscopy microindentation experiments was used to determine the impact of POVPC and PGPC on the structure of a multicomponent phospholipid bilayer and to assess the consequences of their incorporation on membrane packing and endothelial cell stiffness. Molecular simulations predicted differential bilayer perturbation effects of the two oxidized phospholipids based on the chemical identities of their truncated tails, including decreased bilayer packing, decreased bilayer bending modulus, and increased water penetration. Disruption of lipid order was consistent with Laurdan imaging results indicating that POVPC and PGPC decrease the lipid packing of both ordered and disordered membrane domains. Computational predictions of a larger membrane perturbation effect by PGPC correspond to greater stiffness of PGPC-treated endothelial cells observed by measuring cellular elastic moduli using atomic force microscopy. Our results suggest that disruptions in membrane structure by oxidized phospholipids play a role in the regulation of overall endothelial cell stiffness.