Cholesterol In Lipid Membranes Research Articles

A Measurement Method for Cytochrome P450 3A4 (CYP3A4)-mediated Oxidation of Cholesterol in Lipid Membranes

A Measurement Method for Cytochrome P450 3A4 (CYP3A4)-mediated Oxidation of Cholesterol in Lipid Membranes

Cholesterol provides nonsacrificial protection of membrane lipids from chemical damage at air–water interface

The role of cholesterol in bilayer and monolayer lipid membranes has been of great interest. On the biophysical front, cholesterol significantly increases the order of the lipid packing, lowers the membrane permeability, and maintains membrane fluidity by forming liquid-ordered-phase lipid rafts. However, direct observation of any influence on membrane chemistry related to these cholesterol-induced physical properties has been absent. Here we report that the addition of 30 mol % cholesterol to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) monolayers at the air-water interface greatly reduces the oxidation and ester linkage cleavage chemistries initiated by potent chemicals such as OH radicals and HCl vapor, respectively. These results shed light on the indispensable chemoprotective function of cholesterol in lipid membranes. Another significant finding is that OH oxidation of unsaturated lipids generates Criegee intermediate, which is an important radical involved in many atmospheric processes.

Cholesterol Modifies Huntingtin Binding to, Disruption of, and Aggregation on Lipid Membranes

Huntington's disease (HD) is an inherited neurodegenerative disease caused by abnormally long CAG-repeats in the huntingtin gene that encode an expanded polyglutamine (polyQ) domain near the N-terminus of the huntingtin (htt) protein. Expanded polyQ domains are directly correlated to disease-related htt aggregation. Htt is found highly associated with a variety of cellular and subcellular membranes that are predominantly comprised of lipids. Since cholesterol homeostasis is altered in HD, we investigated how varying cholesterol content modifies the interactions between htt and lipid membranes. A combination of Langmuir trough monolayer techniques, vesicle permeability and binding assays, and in situ atomic force microscopy were used to directly monitor the interaction of a model, synthetic htt peptide and a full-length htt-exon1 recombinant protein with model membranes comprised of total brain lipid extract (TBLE) and varying amounts of exogenously added cholesterol. As the cholesterol content of the membrane increased, the extent of htt insertion decreased. Vesicles containing extra cholesterol were resistant to htt-induced permeabilization. Morphological and mechanical changes in the bilayer associated with exposure to htt were also drastically altered by the presence of cholesterol. Disrupted regions of pure TBLE bilayers were grainy in appearance and associated with a large number of globular aggregates. In contrast, morphological changes induced by htt in bilayers enriched in cholesterol were plateau-like with a smooth appearance. Collectively, these observations suggest that the presence and amount of cholesterol in lipid membranes play a critical role in htt binding and aggregation on lipid membranes.

Cholesterol expels ibuprofen from the hydrophobic membrane core and stabilizes lamellar phases in lipid membranes containing ibuprofen.

There is increasing evidence that common drugs, such as aspirin and ibuprofen, interact with lipid membranes. Ibuprofen is one of the most common over the counter drugs in the world, and is used for relief of pain and fever. It interacts with the cyclooxygenase pathway leading to inhibition of prostaglandin synthesis. From X-ray diffraction of highly oriented model membranes containing between 0 and 20 mol% ibuprofen, 20 mol% cholesterol, and dimyristoylphosphatidylcholine (DMPC), we present evidence for a non-specific interaction between ibuprofen and cholesterol in lipid bilayers. At a low ibuprofen concentrations of 2 mol%, three different populations of ibuprofen molecules were found: two in the lipid head group region and one in the hydrophobic membrane core. At higher ibuprofen concentrations of 10 and 20 mol%, the lamellar bilayer structure is disrupted and a lamellar to cubic phase transition was observed. In the presence of 20 mol% cholesterol, ibuprofen (at 5 mol%) was found to be expelled from the membrane core and reside solely in the head group region of the bilayers. 20 mol% cholesterol was found to stabilize lamellar membrane structure and the formation of a cubic phase at 10 and 20 mol% ibuprofen was suppressed. The results demonstrate that ibuprofen interacts with lipid membranes and that the interaction is strongly dependent on the presence of cholesterol.

Electrochemical analysis of phase behavior of solid-supported lipid films. Influence of cholesterol

Diverse techniques of electroanalytical chemistry have already earned the reputation of dependable tools for research of biomembranes and lipid model systems. Among them, Electrical Impedance Spectroscopy (EIS) seems to be one of the most promising with respect to interface studies. Here we have made an attempt to use EIS for evaluation of the effect of cholesterol on the phase behavior of supported liquid films of lecithin. Our results showed that cholesterol promoted tight packing of lipid molecules in the different regions of the film. These findings are in consonance with the widely accepted condensing role of cholesterol in lipid membranes.

The Interaction between Amyloid-β Peptides and Anionic Lipid Membranes Containing Cholesterol and Melatonin

One of the hallmarks of Alzheimer's disease is the formation of senile plaques, primarily consisting of amyloid- (A) peptides. Peptide-membrane and peptide-lipid interactions are thought to be crucial in this process. We studied the interaction of A and A peptides with anionic lipid membranes made of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphoserine (DMPS) using X-ray diffraction. We compare the experimentally determined electron densities in the gel state of the membranes with density calculations from peptide structures reported in the Protein Data Bank in order to determine the position of the peptide in the bilayers. The full length peptide A was found to embed in the hydrocarbon core of the anionic lipid bilayers. Two populations were found for the A peptide: (1) membrane-bound states in the hydrophilic head group region of the bilayers, where the peptides align parallel to the membranes, and (2) an embedded state in the bilayer center. Aging plays an important role in the development of Alzheimer's, in particular with respect to changes in cholesterol and melatonin levels in the brain tissue. Immiscible cholesterol plaques were created by addition of 30 mol% cholesterol to the anionic membranes. The A peptides were found to strongly interact with the lipid bilayers, displacing further cholesterol molecules into the plaques, effectively lowering the cholesterol concentration in the membranes and increasing the total fraction of cholesterol plaques. Addition of 30 mol% melatonin molecules to the anionic membranes drastically reduced the population of the membrane-embedded A state. These results present experimental evidence for an interaction between A peptides, melatonin and cholesterol in lipid membranes.

Solubility of cholesterol in lipid membranes and the formation of immiscible cholesterol plaques at high cholesterol concentrations

The molecular in-plane and out-of-plane structure of dimyristoylphosphatidylcholine (DMPC) membranes containing up to 60 mol% of cholesterol was studied using X-ray diffraction. Up to 37.5 mol% cholesterol could be dissolved in the membranes, resulting in a disordered lateral membrane structure. Highly ordered cholesterol structures were observed at cholesterol concentrations of more than 40 mol% cholesterol. These structures were characterized as immiscible cholesterol plaques, i.e., bilayers of cholesterol molecules coexisting with the lipid bilayer. The cholesterol molecules were found to form a monoclinic structure at 40 mol% cholesterol, which transformed into a triclinic arrangement at the highest concentration of 60 mol%. Monoclinic and triclinic structures were found to coexist at cholesterol concentrations between 50 and 55 mol%.

Hydrogen-bonding patterns of cholesterol in lipid membranes

Correlation between the rotation of the cholesterol hydroxyl group and the formation of hydrogen bonds with its lipid environment is examined through molecular dynamics (MD) simulations and compared with recently reported NMR experiments. All atom MD simulations of a fully hydrated 1:2 cholesterol–dimyristoylphosphatidylcholine bilayer have been performed. Precise reproduction of the cholesterol cell parameters via simulation of its P1-group crystal validates the force field utilized. The lipid–cholesterol hydrogen-bonding pattern reflects the coexistence of alternative dimer motifs with comparable conformer populations, in line with the estimated free energy differences for the rotamers of the cholesterol C O bond.

Desmosterol May Replace Cholesterol in Lipid Membranes

Recently, knockout mice entirely lacking cholesterol have been described as showing only a mild phenotype. For these animals, synthesis of cholesterol was interrupted at the level of its immediate precursor, desmosterol. Since cholesterol is a major and essential constituent of mammalian cellular membranes, we asked whether cholesterol with its specific impact on membrane properties might be replaced by desmosterol. By employing various approaches of NMR, fluorescence, and EPR spectroscopy, we found that the properties of phospholipid membranes like lipid packing in the presence of cholesterol or desmosterol are very similar. However, for lanosterol, a more distant precursor of cholesterol synthesis, we found significant differences in comparison with cholesterol and desmosterol. Our results show that, from the point of view of membrane biophysics, cholesterol and desmosterol behave identically and, therefore, replacement of cholesterol by desmosterol may not impact organism homeostasis.

The Potential of Fluorescent and Spin-labeled Steroid Analogs to Mimic Natural Cholesterol

Cholesterol analogs are often used to investigate lipid trafficking and membrane organization of native cholesterol. Here, the potential of various spin (doxyl moiety) and fluorescent (7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group) labeled cholesterol analogs as well as of fluorescent cholestatrienol and the naturally occurring dehydroergosterol to mimic the unique properties of native cholesterol in lipid membranes was studied in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes by electron paramagnetic resonance, nuclear magnetic resonance, and fluorescence spectroscopy. As cholesterol, all analogs undergo fluctuating motions of large amplitude parallel to the bilayer normal. Native cholesterol keeps a strict orientation in the membrane with the long axis parallel to the bilayer normal. Depending on the chemical modification or the position of the label, cholesterol analogs may adopt an "up-side-down" orientation in the membrane or may even fluctuate between "upright" and up-side-down orientation by rotational motions about the short axis not typical for native cholesterol. Those analogs are not able to induce a comparable condensation of phospholipid membranes as known for native cholesterol revealed by 2H nuclear magnetic resonance. However, cholesterol-induced lipid condensation is one of the key properties of native cholesterol, and, therefore, a well suited parameter to assess the potential of steroid analogs to mimic cholesterol. The study points to extreme caution when studying cholesterol behavior by the respective analogs. Among seven analogs investigated, only a spin-labeled cholesterol with the doxyl group at the end of the acyl chain and the fluorophore cholestatrienol mimic cholesterol satisfactorily. Dehydroergosterol has a similar upright orientation as cholesterol and could be used at low concentration (about 1 mol %), at which its lower potential to enhance lipid packing density does not perturb membrane organization.

Influence of Cholesterol on the Interaction of α-Crystallin with Phospholipids

The influence of cholesterol on the binding of α-crystallin to pure phospholipid membranes was studied. The rationale of this investigation stems from two unique aspects of human lens cells: an unusually high level of cholesterol in the membranes and the specific binding of α-crystallin to membranes. In the absence of cholesterol, binding of α-crystallin liposomes composed of either sphingomyelin, disteroylphosphatidylcholine or egg-phosphatidylcholine caused a decrease in the fluorescence intensity and anisotropy of the fluorophore NBD-PE. Since this fluorescence probe resides in the polar headgroup region of the membrane, the observed changes indicated that the binding of α-crystallin affected the structure of these membrane regions. The ability of α-crystallin to modulate membrane structure suggests yet another potential role for this lens protein. Addition of cholesterol markedly decreased the binding of α-crystallin to liposomes composed of either sphingomyelin or disteroylphosphatidylcholine and antagonized the capacity of bound α-crystallin to decrease membrane surface order. This antagonism could be explained by the ability of cholesterol to directly decrease the anisotropy of the fluorophore in sphingomyelin membranes unexposed to α-crystallin. Thus, with cholesterol present, a further decrease in membrane order upon subsequent binding of α-crystallin was less likely. The results obtained with the sphingomyelin liposomes are considered most meaningful, since sphingomyelins are the principal phospholipids in the human lens nuclear membrane and cholesterol preferentially interacts with sphingomyelin. We conclude that cholesterol in lipid membranes can antagonize the binding of α-crystallin and thus interfere with the capacity of bound α-crystallin to alter membrane order. We suggest that such actions of cholesterol might serve to preserve lens membrane structure in the physiological state where the concentration of soluble α-crystallin is great.

Structural and dynamical aspects of membrane immunochemistry using model membranes

Three different phospholipid haptens have been synthesized, in which the haptenic group is the paramagnetic nitroxide (spin-label) group. These lipid haptens differ from one another in the length and composition of the molecular chain linking the 2,2,6,6-tetramethylpiperidinyl-N-oxy moiety to the phosphodiester group of the lipid. These lipid haptens have been incorporated at low molar concentrations (0.01 to 0.5 mol %) in liposomes containing various proportions of cholesterol and dipalmitoylphosphatidylcholine (DPPC). A study has been made of specific antinitroxide IgG (and Fab) binding to these liposomes, and the fixation of complement. From these studies we conclude: (a) For lipid haptens whose possible extension above the bilayer plane is limited (e.g., approximately 10-20 A), antibody binding and complement fixation depend strongly on the hapten structure and host lipid composition, because of steric limitations on the accessibility of lipid haptens to the binding sites in the protein. (b) Complement fixation by specific IgG antibodies directed against the nitroxide group as part of a lipid hapten depends strongly on the lateral mobility of the lipid hapten when its molar concentration in the plane of the membrane is of the order of 0.1 mol % or less. It is likely that this conclusion applies to many lipid haptens, and possibly other membrane components. (c) The inclusion of cholesterol in lipid membranes has at least two distinct effects on complement fixation involving lipid haptens. Through a steric effect on bilayer structure (probably involving lateral molecular ordering) cholesterol in phosphatidylcholine bilayers can enhance hapten exposure to antibody binding sites, enhance antibody binding, and thereby enhance complement fixation. It is likely that cholesterol also affects complement fixation at low hapten concentrations through a modification of membrane fluidity.

The role of cholesterol in lipid membranes

The role of cholesterol in lipid membranes