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Here, we examined the effects of phosphatidylserine (PS) and cholesterol on the fibril-forming properties of the N-terminal 1‒83 fragment of an amyloidogenic G26R variant of apoA-I bound to small unilamellar vesicles. A thioflavin T fluorescence assay together with microscopic observations showed that PS significantly retards the nucleation step in fibril formation by apoA-I 1‒83/G26R, whereas cholesterol slightly enhances fibril formation. Circular dichroism analyses demonstrated that PS facilitates a structural transition from random coil to α-helix in apoA-I 1‒83/G26R with great stabilization of the α-helical structure upon lipid binding. Isothermal titration calorimetry measurements revealed that PS induces a marked increase in capacity for binding of apoA-I 1‒83/G26R to the membrane surface, perhaps due to electrostatic interactions of positively charged amino acids in apoA-I with PS. Such effects of PS to enhance lipid interactions and inhibit fibril formation of apoA-I were also observed for the amyloidogenic region-containing apoA-I 8‒33/G26R peptide. Fluorescence measurements using environment-sensitive probes indicated that PS induces a more solvent-exposed, membrane-bound conformation in the amyloidogenic region of apoA-I without affecting membrane fluidity. Since cell membranes have highly heterogeneous lipid compositions, our findings may provide a molecular basis for the preferential deposition of apoA-I amyloid fibrils in tissues and organs.

The transfer of 22-NBD-cholesterol (22-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol) between two liposome membranes was quantitatively analyzed by using the fluorescence resonance energy transfer (FRET) method. Liposomes labeled with both 22-NBD-cholesterol and a rhodamine-labeled phosphatidylethanolamine (Rh-DHPE) were used as donor liposomes, and the 22-NBD-cholesterol transfer from these donor liposomes to acceptor liposomes prepared from same type of phosphatidylcholine was monitored. The transfer kinetics was found to be composed of a fast and a slow phase, and all kinetic measurements could be fitted with a bi-exponential model. The results obtained indicate that the 22-NBD-cholesterol transfer kinetics between liposome membranes depends on the fluidity of the liposome used and that the curvature may affect the kinetics. Furthermore, the behavior of 22-NBD-cholesterol in lipid membrane is similar to that of the oxysterol 25-hydroxycholesterol rather than cholesterol. It is proposed that 22-NBD-cholesterol can be a useful fluorescent probe to mimic the intermembrane transfer of oxidized cholesterols like 25-hydroxycholesterol, rather than that of cholesterol itself., (2010 Elsevier B.V. All rights reserved.)

The amyloid beta protein with 42 amino acid residues (Abeta), which is a causative protein of Alzheimer's disease (AD), forms the complex with copper (II) to induce the cholesterol oxidase-like activity by the proton transfer from the cholesterol. In this study, the oxidation of cholesterol by Abeta/Cu complex was investigated on the surface of the zwitterionic phospholipid liposome including the bound water advantageous for the enhancement of the proton transfer. The bound water was pooled by the formation of cholesterol-rich domain within liposomes. The resulting reactivity was enhanced by the proton transfer mediated by the bound water., (Copyright (c) 2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

A catalytic reaction of H2O2 production by an amyloid beta-peptide (Abeta)-Cu complex with cholesterol incorporated in a liposome was kinetically analyzed. The Michaelis-Menten model was applied to the H2O2 production reaction using cholesterol as the substrate catalyzed by the Abeta-Cu complex. The Km value for the Abeta-Cu complex catalytic reaction with cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes (Km=0.436 microM for Abeta(1-40); Km=0.641 microM for Abeta(1-42)) was found to be smaller than that with cholesterol-containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes (Km=0.585 microM for Abeta(1-40), Km=0.890 microM for Abeta(1-42)). The results imply that membrane properties could play an important role in the interactions of the Abeta-Cu complex with cholesterol in these liposomes. Considering the physical states of the cholesterol/POPC (liquid disordered phase) and cholesterol/DPPC (liquid ordered phase) liposomes in the present reaction conditions, the data obtained suggests that the H2O2-generating activity of the Abeta-Cu complex, accompanied by oxidation of membrane-incorporated cholesterol, could be effected by the phase of the liposome membranes.

A new fluorescent arrayed biosensor has been developed to discriminate species and concentrations of target proteins by using plural different phospholipid liposome species encapsulating fluorescent molecules, utilizing differences in permeation of the fluorescent molecules through the membrane to modulate liposome-target protein interactions. This approach proposes a basically new label-free fluorescent sensor, compared with the common technique of developed fluorescent array sensors with labeling. We have confirmed a high output intensity of fluorescence emission related to characteristics of the fluorescent molecules dependent on their concentrations when they leak from inside the liposomes through the perturbed lipid membrane. After taking an array image of the fluorescence emission from the sensor using a CMOS imager, the output intensities of the fluorescence were analyzed by a principal component analysis (PCA) statistical method. It is found from PCA plots that different protein species with several concentrations were successfully discriminated by using the different lipid membranes with high cumulative contribution ratio. We also confirmed that the accuracy of the discrimination by the array sensor with a single shot is higher than that of a single sensor with multiple shots. [ABSTRACT FROM AUTHOR]

Abstract: The surface properties of Span 80 vesicles at various cholesterol contents, together with those of various liposomes, were characterized by using fluorescence probes. The membrane fluidity of the Span 80 vesicles was measured by 1,6-diphenyl-1.3.5-hexatriene (DPH) and trimethlyammonium-DPH (TMA-DPH), and the results suggested that the surface of the Span 80 vesicles was fluid due to the lateral diffusion of Span 80 molecules. The depolarization measured by TMA-DPH and the headgroup mobility measured by dielectric dispersion analysis indicated the high mobility of the head group of Span 80 vesicles. This suggested that the surface of Span 80 vesicles was flexible due to the head group structure of Span 80, sorbitol. In addition, spectrophotometric analysis with 6-dodecanoyl-N, N-dimethyl-2-naphthylamine and 8-anilino-1-naphthalenesulfonic acid indicated that the water molecules could easily invade into the interior of the Span 80 vesicle membrane, suggesting that the membrane surface was more wet than the liposome surface. These surface properties indicated that the protein could interact with the interior of vesicle membranes, which was similar to the case of cholesterol. Thus the present results confirmed that the Span 80 vesicle surfaces showed the unique characteristics of fluidity, flexibility, and “wetness”, whereas the liposome surfaces did not. [Copyright &y& Elsevier]