Lipid membrane templates the ordering and induces the fibrillogenesis of Alzheimer's disease amyloid-beta peptide

The lipid membrane has been shown to mediate the fibrillogenesis and toxicity of Alzheimer's disease (AD) amyloid-beta (Abeta) peptide. Electrostatic interactions between Abeta40 and the phospholipid headgroup have been found to control the association and insertion of monomeric Abeta into lipid monolayers, where Abeta exhibited enhanced interactions with charged lipids compared with zwitterionic lipids. To elucidate the molecular-scale structural details of Abeta-membrane association, we have used complementary X-ray and neutron scattering techniques (grazing-incidence X-ray diffraction, X-ray reflectivity, and neutron reflectivity) in this study to investigate in situ the association of Abeta with lipid monolayers composed of either the anionic lipid 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), the zwitterionic lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or the cationic lipid 1,2-dipalmitoyl 3-trimethylammonium propane (DPTAP) at the air-buffer interface. We found that the anionic lipid DPPG uniquely induced crystalline ordering of Abeta at the membrane surface that closely mimicked the beta-sheet structure in fibrils, revealing an intriguing templated ordering effect of DPPG on Abeta. Furthermore, incubating Abeta with lipid vesicles containing the anionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) induced the formation of amyloid fibrils, confirming that the templated ordering of Abeta at the membrane surface seeded fibril formation. This study provides a detailed molecular-scale characterization of the early structural fluctuation and assembly events that may trigger the misfolding and aggregation of Abeta in vivo. Our results implicate that the adsorption of Abeta to anionic lipids, which could become exposed to the outer membrane leaflet by cell injury, may serve as an in vivo mechanism of templated-aggregation and drive the pathogenesis of AD.     

Lateral organization of GM1 in phase-separated monolayers visualized by scanning force microscopy

Phase separation of glycolipids in lipid mono- and bilayers is of great interest for the understanding of membrane function. The distribution of the ganglioside GM1 in sphingomyelin (SM)/1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), SM/1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DOPC) and SM/cholesterol/POPC Langmuir-Blodgett (LB) monolayers transferred at 36 mN/m has been studied by scanning force microscopy. Besides lateral organization of the glycolipid in LB monolayers as deduced from topography, material properties have been investigated by phase imaging, pulsed force mode and force modulation microscopy. It was shown that GM1 preferentially clusters in an ordered lipid matrix, i.e. the SM phase in the case of the SM/POPC and SM/DOPC mixture or in the ordered phase of POPC/SM/cholesterol monolayers. At higher local concentrations, three-dimensional protrusions enriched in GM1 occur, which may represent a precursor for the formation of micelles budding into the aqueous subphase. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00249-002-0232-4.     

The effect of cholesterol and monosialoganglioside (GM1) on the release and aggregation of amyloid beta-peptide from liposomes prepared from brain membrane-like lipids

In order to investigate the influence of cholesterol (Ch) and monosialoganglioside (GM1) on the release and subsequent deposition/aggregation of amyloid beta peptide (Abeta)-(1-40) and Abeta-(1-42), we have examined Abeta peptide model membrane interactions by circular dichroism, turbidity measurements, and transmission electron microscopy (TEM). Model liposomes containing Abeta peptide and a lipid mixture composition similar to that found in the cerebral cortex membranes (CCM-lipid) have been prepared. In all, four Abeta-containing liposomes were investigated: CCM-lipid; liposomes with no GM1 (GM1-free lipid); those with no cholesterol (Ch-free lipid); liposomes with neither cholesterol nor GM1 (Ch-GM1-free lipid). In CCM liposomes, Abeta was rapidly released from membranes to form a well defined fibril structure. However, for the GM1-free lipid, Abeta was first released to yield a fibril structure about the membrane surface, then the membrane became disrupted resulting in the formation of small vesicles. In Ch-free lipid, a fibril structure with a phospholipid membrane-like shadow formed, but this differed from the well defined fibril structure seen for CCM-lipid. In Ch-GM1-free lipid, no fibril structure formed, possibly because of membrane solubilization by Abeta. The absence of fibril structure was noted at physiological extracellular pH (7.4) and also at liposomal/endosomal pH (5.5). Our results suggest a possible role for both Ch and GM1 in the membrane release of Abeta from brain lipid bilayers.     

Octyl-beta-D-glucopyranoside partitioning into lipid bilayers: thermodynamics of binding and structural changes of the bilayer.

The interaction of the nonionic detergent octyl-beta-D-glucopyranoside (OG) with lipid bilayers was studied with high-sensitivity isothermal titration calorimetry (ITC) and solid-state 2H-NMR spectroscopy. The transfer of OG from the aqueous phase to lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) can be investigated by employing detergent at concentrations below the critical micellar concentration; it can be defined by a surface partition equilibrium with a partition coefficient of K = 120 +/- 10 M-1, a molar binding enthalpy of delta H degrees D = 1.3 +/- 0.15 kcal/mol, and a free energy of binding of delta G degrees D = -5.2 kcal/mol. The heat of transfer is temperature dependent, with a molar heat capacity of delta CP = -75 cal K-1 mol-1. The large heat capacity and the near-zero delta H are typical for a hydrophobic binding equilibrium. The partition constant K decreased to approximately 100 M-1 for POPC membranes mixed with either negatively charged lipids or cholesterol, but was independent of membrane curvature. In contrast, a much larger variation was observed in the partition enthalpy. delta H degrees D increased by about 50% for large vesicles and by 75% for membranes containing 50 mol% cholesterol. Structural changes in the lipid bilayer were investigated with solid-state 2H-NMR. POPC was selectively deuterated at the headgroup segments and at different positions of the fatty acyl chains, and the measurement of the quadrupolar splittings provided information on the conformation and the order of the bilayer membrane. Addition of OG had almost no influence on the lipid headgroup region, even at concentrations close to bilayer disruption. In contrast, the fluctuations of fatty acyl chain segments located in the inner part of the bilayer increased strongly with increasing OG concentration. The 2H-NMR results demonstrate that the headgroup region is the most stable structural element of the lipid membrane, remaining intact until the disordering of the chains reaches a critical limit. The perturbing effect of OG is thus different from that of another nonionic detergent, octaethyleneglycol mono-n-dodecylether (C12E8), which produces a general disordering at all levels of the lipid bilayer. The OG-POPC interaction was also investigated with POPC monolayers, using a Langmuir trough. In the absence of lipid, the measurement of the Gibbs adsorption isotherm for pure OG solutions yielded an OG surface area of AS = 51 +/- 3 A2. On the other hand, the insertion area AI of OG in a POPC monolayer was determined by a monolayer expansion technique as AI = 58 +/- 10 A2. The similar area requirements with AS approximately AI indicate an almost complete insertion of OG into the lipid monolayer. The OG partition constant for a POPC monolayer at 32 mN/m was Kp approximately 320 M-1 and thus was larger than that for a POPC bilayer.     

Influence of lipid saturation grade and headgroup charge: a refined lung surfactant adsorption model

Rapid adsorption of surfactant material to the air/liquid interface of the lung is essential for maintaining normal lung function. The detailed mechanism of this process, however, remains unclear. In this study, we elucidate the influence of lipid saturation grade and headgroup charge of surface layer lipids on surfactant protein (SP)-induced vesicle insertion into monolayers spread at the air/water interface of a film balance. We used dipalmitoylphosphatidlycholine (DPPC),1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) as monolayer lipids doped with either hydrophobic surfactant-specific protein SP-B or SP-C (0.2 and 0.4 mol %, respectively). Vesicles consisting of DPPC/DPPG (4:1, mol ratio) were injected into a stirred subphase to quantify adsorption kinetics. Based on kinetic film balance and fluorescence measurements, a refined model describing distinct steps of vesicle adsorption to surfactant monolayers is presented. First, in a protein-independent step, lipids from vesicles bridged to the interfacial film by Ca²⁺ ions are inserted into defects of a disordered monolayer at low surface pressures. Second, in a SP-facilitated step, active material insertion involving an SP-B- or SP-C-induced flip-flop of lipids occurs at higher surface pressures. Negatively charged lipids obviously influence the threshold pressures at which this second protein-mediated adsorption mechanism takes place.     

Characterization of the binary mixed monolayers of α-tocopherol with phospholipids at the air-water interface

The mixed Langmuir monolayers composed of model constituents of biological membranes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), were investigated to provide information on the intermolecular interactions between these membrane components and the physiologically active vitamin E–α-tocopherol (TF), as well as on the phase behavior of these mixed systems. Additionally, topography of these monolayers transferred onto the mica support was investigated by the inverted metallurgical microscope. Morphological characteristics were directly observed by Brewster angle microscopy (BAM). From the surface pressure–area isotherms and the analysis of physicochemical parameters (compressibility and mean molecular area at the maximum compressibility) it was found that depending on the acyl chains saturation degree, TF has different effect on the phospholipids. In the case of DPPC, the addition of TF to the phospholipid film causes destabilization of the ordered hydrocarbon chains, while in the POPC/DOPC–TF systems, the attractive interactions are responsible for the monolayer increased stability. Thus, the results of these studies confirm the hypothesis that α-tocopherol may play a role in the stabilization of biological membranes.     

A long-lived amphiphilic fluorescent probe studied in POPC air-water monolayer and solution bilayer systems.

A novel amphiphilic fluorescent probe (Fluorazophore-L) with a strongly dipolar, nonionic azoalkane as headgroup and a palmitoyl tail has been synthesized and characterized. Pure Fluorazophore-L was found to be sufficiently amphiphilic to form stable air-water monolayers. An analysis of the surface pressure versus area suggests an area per molecule of about 34+/-2 A(2) at 29 mN m(-1). The partitioning into a lipid membrane model was quantified at the air-water interface by spreading 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) monolayers. Measurements with different molar fractions of Fluorazophore-L revealed a small but significant reduction of the mean area in the mixed monolayer. The excess free energy of mixing (-0.5+/-0.1 kT) indicated a weakly attractive interaction slightly above thermal energy, suggesting a good miscibility of the fluorescent probe within the lipid monolayer without major structural modifications. Spectroscopic measurements confirmed the incorporation of Fluorazophore-L into POPC vesicles. The fluorescence lifetime was very long (125+/-5 ns under air) with monoexponential fluorescence decays.     

Cholesterol oxidase susceptibility of cholesterol and 5-androsten-3 beta-ol in pure sterol monolayers and in mixed monolayers containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

This study has examined the importance of the isocaproic side chain at C-17 of cholesterol to sterol/phospholipid interactions in monolayer membranes and to the cholesterol oxidase-susceptibility of cholesterol in pure and mixed monolayers at the air/water interface. The interactions between cholesterol or 5-androsten-3 beta-ol (which lacks the C-17 side chain) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in monolayers indicated that 5-androsten-3 beta-ol was not very efficient in causing condensation of the monolayer packing of POPC. Whereas cholesterol condensed the packing of POPC at all molar fractions examined (i.e., 0.25, 0.50 and 0.75 with regard to POPC), 5-androsten-3 beta-ol caused a slight condensing effect on POPC packing only in the equimolar mixture. The mean molecular area requirement of 5-androsten-3 beta-ol (in pure sterol monolayers at different lateral surface pressures) was 2.2-6.7% less than that observed for cholesterol. The pure 5-androsten-3 beta-ol monolayer also collapsed at lower lateral surface pressures compared with the pure cholesterol monolayer (34 mN/m and 45 mN/m, respectively). The cholesterol oxidase (Streptomyces sp.) catalyzed oxidation of cholesterol or 5-androsten-3 beta-ol in pure monolayers in the air/water interface (10 mN/m) proceeded with very similar rates, indicating that the enzyme did not recognize that the C-17 side chain of 5-androsten-3 beta-ol was missing. The oxidation of cholesterol or 5-androsten-3 beta-ol in mixed POPC-containing monolayers (equimolar mixture) also revealed similar reaction rates, although the reaction was slower in the mixed monolayer compared with the pure sterol monolayer. When the oxidation of cholesterol and 5-androsten-3 beta-ol was examined by monitoring the production of H2O2 (the sterol was solubilized in 2-propanol and the assay conducted in phosphate buffer), the maximal reaction rate observed with 5-androsten-3 beta-ol was only about 41% of that measured with cholesterol. From the cholesterol oxidase point-of-view, it can be concluded that the enzyme did not recognize the C-17 side chain of cholesterol (or lack of it in 5-androsten-3 beta-ol), when the sterol was properly oriented as a monolayer at the air/water interface. However, when the substrate was presented to the enzyme in a less controlled orientation (organic solvent in water), 5-androsten-3 beta-ol may have oriented itself unfavorably compared with the orientation of cholesterol, thereby leading to slower oxidation rates.     

Early synergy between Abeta42 and oxidatively damaged membranes in promoting amyloid fibril formation by Abeta40

Oxidative lipid membrane damage is known to promote the misfolding of Abeta42 into pathological beta structure. In fully developed senile plaques of Alzheimer's disease, however, it is the shorter and more soluble amyloid beta protein, Abeta40, that predominates. To investigate the role of oxidative membrane damage in the misfolding of Abeta40, we have examined its interaction with supported lipid monolayer membranes using internal reflection infrared spectroscopy. Oxidatively damaged lipids modestly increased Abeta40 accumulation, with adsorption kinetics and a conformation that are distinct from that of Abeta42. In stark contrast, pretreatment of oxidatively damaged monolayer membranes with Abeta42 vigorously promoted Abeta40 accumulation and misfolding. Pretreatment of saturated or undamaged membranes with Abeta42 had no such effect. Parallel studies of lipid bilayer vesicles using a dye binding assay to detect fibril formation and electron microscopy to examine morphology demonstrated that Abeta42 pretreatment of oxidatively damaged membranes promoted the formation of mature Abeta40 amyloid fibrils. We conclude that oxidative membrane damage and Abeta42 act synergistically at an early stage to promote fibril formation by Abeta40. This synergy could be detected within minutes using internal reflection spectroscopy, whereas a dye-binding assay required several days and much higher protein concentrations to demonstrate this synergy.     

Atomic force microscopy and force spectroscopy study of Langmuir-Blodgett films formed by heteroacid phospholipids of biological interest

Langmuir-Blodgett (LB) films of two heteroacid phospholipids of biological interest 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), as well as a mixed monolayer with chi(POPC)=0.4, were transferred onto mica in order to investigate by a combination of atomic force microscopy (AFM) and force spectroscopy (FS) their height, and particularly, their nanomechanical properties. AFM images of such monolayers extracted at 30 mN m(-1) revealed a smooth and defect-free topography except for the POPE monolayer. Since scratching such soft monolayers in contact mode was proved unsuccessful, their molecular height was measured by means of the width of the jump present in the respective force-extension curves. While for pure POPC a small jump occurs near zero force, for the mixed monolayer with chi(POPC)=0.4 the jump occurs at approximately 800 pN. Widths of approximately 2 nm could be established for POPC and chi(POPC)=0.4, but not for POPE monolayer at this extracting pressure. Such different mechanical stability allowed us to directly measure the threshold area/lipid range value needed to induce mechanical stability to the monolayers. AFM imaging and FS were next applied to get further structural and mechanical insight into the POPE phase transition (LC-LC') occurring at pressures >36.5 mN m(-1). This phase transition was intimately related to a sudden decrease in the area/molecule value, resulting in a jump in the force curve occurring at high force ( approximately 1.72 nN). FS reveals to be the unique experimental technique able to unveil structural and nanomechanical properties for such soft phospholipid monolayers. The biological implications of the nanomechanical properties of the systems under investigation are discussed considering that the annular phospholipids region of some transmembrane proteins is enriched in POPE.     

Interfacial properties of most monofluorinated bile acids deviate markedly from the natural congeners: studies with the Langmuir-Pockels surface balance

We characterized the air-water interfacial properties of four monofluorinated bile acids alone and in binary mixtures with a common lecithin, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), using an automated Langmuir-Pockels surface balance. We compared 7alpha-fluoromurocholic acid (FMCA), 7alpha-fluorohyodeoxycholic acid (FHDCA), 6alpha-fluoroursodeoxycholic acid (FUDCA), and 6alpha-fluorochenodeoxycholic acid (FCDCA) with their natural dihydroxy homologs, murocholic acid (MCA), hyodeoxycholic acid (HDCA), ursodeoxycholic acid (UDCA), and chenodeoxycholic acid (CDCA). For further comparison, two trihydroxy bile acids, 3alpha,6beta,7alpha-trihydroxycholanoic acid [alpha-muricholic acid (alpha-MCA)] and 3alpha,6alpha,7beta-trihydroxycholanoic acid [omega-muricholic acid (omega-MCA)], with isologous OH polar functions to FMCA and FUDCA were also studied. Pressure-area isotherms of MCA, HDCA, UDCA, CDCA, and FMCA displayed sharp collapse points. In contrast, FHDCA, FUDCA, and FCDCA formed monolayers that were less stable than the trihydroxy bile acids, displaying second-order phase transitions in their isotherms. All natural and fluorinated bile acids condensed mixed monolayers with POPC, with maximal effects at molar bile acid concentrations between 30 and 50 mol%. Examination of molecular models revealed that the 7alpha-F atom of the interfacially stable FMCA projects away from the 6beta-OH function, resulting in minimal steric interactions, whereas in FHDCA, FUDCA, and FCDCA, close vicinal interactions between OH and F polar functions result in progressive bulk solubility upon monolayer compression. These results provide a framework for designing F-modified bile acids to mimic or diverge from the natural compounds in vivo.     

Distribution of ganglioside GM1 in L-alpha-dipalmitoylphosphatidylcholine/cholesterol monolayers: a model for lipid rafts

The distribution of low concentrations of ganglioside GM1 in L-alpha-dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol monolayers supported on mica has been studied using atomic force microscopy (AFM). The monolayers studied correspond to a pure gel phase and a mixture of liquid-expanded (LE) and liquid-condensed (LC) phases for DPPC and to a single homogeneous liquid-ordered phase for 2:1 DPPC/cholesterol. The addition of 2.5-5% GM1 to phase-separated DPPC monolayers resulted in small round ganglioside-rich microdomains in the center and at the edges of the LC domains. Higher amounts of GM1 (10%) give numerous filaments in the center of the LC domains and larger patches at the edges. A gel phase DPPC monolayer containing GM1 showed large domains containing a network of GM1-rich filaments. The addition of GM1 to a liquid-ordered 2:1 DPPC/cholesterol monolayer gives small, round domains that vary in size from 50 to 150 nm for a range of surface pressures. Larger amounts of GM1 lead to coalescence of the small, round domains to give longer filaments that cover 30-40% of the monolayer surface for 10 mol % GM1. The results indicate that biologically relevant GM1 concentrations lead to submicron-sized domains in a cholesterol-rich liquid-ordered phase that is analogous to that found in detergent-insoluble membrane fractions, and are thought to be important in membrane microdomains or rafts. This demonstrates that AFM studies of model monolayers and bilayers provide a powerful method for the direct detection of microdomains that are too small for study with most other techniques.     

Hexadecylphosphocholine interaction with lipid monolayers

The phospholipid analogue miltefosine or hexadecylphosphocholine (HePC) is a drug of high interest in the treatment for fatal visceral leishmaniasis (VL) due to Leishmania donovani particularly because of its activity by oral route. In this study, the interaction of HePC with a monolayer of beta-palmitoyl-gamma-oleyl-phosphatidylcholine (POPC) as membrane model or sterol (ergosterol or cholesterol) was investigated. At a constant pressure of 25 mN/m, the adsorption kinetics of HePC into the monolayers showed that HePC molecules are inserted into the monolayer of lipids as monomers until the critical micellar concentration (CMC). At HePC concentrations superior to the CMC, the micelles of HePC are deployed at the interface as groups of monomers into the POPC or sterol monolayer. The study of mixture of HePC/(POPC or sterol), spread at the air-water interface, shows that a simple miscibility between HePC and POPC is observed, whereas a high condensation appears between HePC and sterols showing a high affinity between HePC and sterols. In addition, HePC does not act as detergent disturbing membrane integrity.     

The effect of ceramide on phosphatidylcholine membranes: a deuterium NMR study

Biological membranes contain domains having distinct physical properties. We study defined mixtures of phosphoglycerolipids and sphingolipids to ascertain the fundamental interactions governing these lipids in the absence of other cell membrane components. By using (2)H-NMR we have determined the temperature and composition dependencies of membrane structure and phase behavior for aqueous dispersions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the ceramide (Cer) N-palmitoyl-sphingosine. It is found that gel and liquid-crystalline phases coexist over a wide range of temperature and composition. Domains of different composition and phase state are present in POPC/Cer membranes at physiological temperature for Cer concentrations exceeding 15 mol %. The acyl chains of liquid crystalline phase POPC are ordered by the presence of Cer. Moreover, Cer's chain ordering is greater than that of POPC in the liquid crystalline phase. However, there is no evidence of liquid-liquid phase separation in the liquid crystalline region of the POPC/Cer phase diagram.     

Merocyanine 540 as a probe to monitor the molecular packing of phosphatidylcholine: a monolayer epifluorescence microscopy and spectroscopy study.

The characteristics of the fluorescent dye, merocyanine 540 (MC-540), incorporated in monolayers of 1,2-dipalmitoyl-phosphatidylcholine (DPPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were studied in different states of molecular packing. Conditions for phase separation in these monolayers were defined by their pressure/area (pi-A) isotherms. Within the liquid expanded (LE) and the liquid condensed (LC) coexisting phases of DPPC monolayers, low light level epifluorescence microscopy revealed 'dark' discoid domains embedded in a 'bright' matrix. Under the same conditions, and at temperatures as low as 12 degrees C, the pi-A isotherms of POPC demonstrate the existence of a single phase, and no fluorescent domains were observed. Fluorescence spectra of MC-540 labelled monolayers, recorded in different structural states, reveal three distinct emission peaks: a 572 nm peak, present for monolayer packing conditions at low surface pressures; a 585 nm peak, similar to that obtained from dye molecules in fluid phase lipid bilayers, and observed here within the respective area/molecule ranges of 54-62 A2 and 62-69 A2 for monolayers of DPPC and POPC with diminishing intensity at increasing surface pressure; and finally, a peak at 560 nm, which predominates in densely packed POPC monolayers. Our results are interpreted on the basis of dye partitioning between monolayer and subphase, and different orientations of the dye with respect to the monolayer in various structural states. The usefulness of MC-540 to differentiate lipid packing in cell membranes is discussed.     

Binding of a neuropeptide, substance P, to neutral and negatively charged lipids

The binding of substance P (SP), a positively charged neurotransmitter peptide, to neutral and to negatively charged phospholipids has been investigated by means of a monolayer technique. Monolayers formed at room temperature from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or mixtures of the two, were maintained throughout the course of a binding experiment at a constant surface pressure while the monolayer surface area was monitored. Injection of SP into the aqueous subphase (154 mM NaCl, 10 mM Tris adjusted to pH 7.4) led to an expansion of the monolayer surface area that was attributed to a spontaneous insertion of SP between the lipid molecules. A quantitative evaluation of the area increase at constant pressure yielded SP insertion isotherms that showed that levels of SP insertion increased directly with the monolayer POPG content and decreased to negligible levels at surface pressures above 35 +/- 1 mN/m. If electrostatic effects were ignored, these data showed biphasic behavior in Scatchard plots. The apparent binding constants ranged, at 20 mN/m, from (3.2 +/- 0.3) X 10(4) M-1 for 100% POPG monolayers to (2.0 +/- 0.05) X 10(3) M-1 for 25% POPG/75% POPC monolayers. At 32 mN/m, a monolayer surface pressure that mimics bilayer conditions, the apparent binding constant for a 100% POPG monolayer was measured to be (1.1 +/- 0.05) X 10(3) M-1. However, for a monolayer containing only 25% charged lipids, corresponding to a natural membrane composition, K app at 32 mN/m was estimated to be at most 41 M-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of Bax and tBid with Lipid Monolayers

The release of cytochrome c from mitochondria to the cytosol is a crucial step of apoptosis that involves interactions of Bax and tBid proteins with the mitochondrial membrane. We investigated Bax and tBid interactions with (i) phosphatidylcholine (PC) monolayer as the main component of the outer leaflet of the outer membrane, (ii) with phosphatidylethanolamine (PE) and phosphatidylserine (PS) that are present in the inner leaflet and (iii) with a mixed PC/PE/Cardiolipin (CL) monolayer of the contact sites between the outer and inner membranes. These interactions were studied by measuring the increase of the lipidic monolayer surface pressure induced by the proteins. Our measurements suggest that tBid interacts strongly with the POPC/DOPE/CL, whereas Bax interaction with this monolayer is about 12 times weaker. Both tBid and Bax interact moderately half as strongly with negatively charged DOPS and non-lamellar DOPE monolayers. TBid also slightly interacts with DOPC. Our results suggest that tBid but not Bax interacts with the PC-containing outer membrane. Subsequent insertion of these proteins may occur at the PC/PE/CL sites of contact between the outer and inner membranes. It was also shown that Bax and tBid being mixed in solution inhibit their insertion into POPC/DOPE/CL monolayer. The known 3-D structures of Bax and Bid allowed us to propose a structural interpretation of these experimental results.     

Effect of N-terminal acetylation on lytic activity and lipid-packing perturbation induced in model membranes by a mastoparan-like peptide

L1A (IDGLKAIWKKVADLLKNT-NH2) is a peptide that displays a selective antibacterial activity to Gram-negative bacteria without being hemolytic. Its lytic activity in anionic lipid vesicles was strongly enhanced when its N-terminus was acetylated (ac-L1A). This modification seems to favor the perturbation of the lipid core of the bilayer by the peptide, resulting in higher membrane lysis. In the present study, we used lipid monolayers and bilayers as membrane model systems to explore the impact of acetylation on the L1A lytic activity and its correlation with lipid-packing perturbation. The lytic activity investigated in giant unilamellar vesicles (GUVs) revealed that the acetylated peptide permeated the membrane at higher rates compared with L1A, and modified the membrane's mechanical properties, promoting shape changes. The peptide secondary structure and the changes in the environment of the tryptophan upon adsorption to large unilamellar vesicles (LUVs) were monitored by circular dichroism (CD) and red-edge excitation shift experiments (REES), respectively. These experiments showed that the N-terminus acetylation has an important effect on both, peptide secondary structure and peptide insertion into the bilayer. This was also confirmed by experiments of insertion into lipid monolayers. Compression isotherms for peptide/lipid mixed films revealed that ac-L1A dragged lipid molecules to the more disordered phase, generating a more favorable environment and preventing the lipid molecules from forming stiff films. Enthalpy changes in the main phase transition of the lipid membrane upon peptide insertion suggested that the acetylated peptide induced higher impact than the non-acetylated one on the thermotropic behavior of anionic vesicles.     

Interactions of amyloid beta-protein with various gangliosides in raft-like membranes: importance of GM1 ganglioside-bound form as an endogenous seed for Alzheimer amyloid

GM1 ganglioside-bound amyloid beta-protein (GM1-Abeta), found in brains exhibiting early pathological changes of Alzheimer's disease (AD) plaques, has been suggested to accelerate amyloid fibril formation by acting as a seed. We have previously found using dye-labeled Abeta that Abeta recognizes a GM1 cluster, the formation of which is facilitated by cholesterol [Kakio, A., Nishimoto, S., Yanagisawa, K., Kozutsumi, Y., and Matsuzaki, K. (2001) J. Biol. Chem. 276, 24985-24990]. In this study, we investigated the ganglioside species-specificity in its potency to induce a conformational change of Abeta, by which ganglioside-bound Abeta acts as a seed for Abeta fibrillogenesis, using a major ganglioside occurring in brains (GM1, GD1a, GD1b, and GT1b) in raft-like membranes composed of cholesterol and sphingomyelin. Abeta recognized ganglioside clusters, the density of which increased with the number of sialic acid residues. Interestingly, however, mixing of gangliosides inhibited cluster formation. In contrast, the affinities of the protein for the clusters were similar irrespective of lipid composition and of the order of 10(6) M(-)(1) at 37 degrees C. Abeta underwent a conformational transition from an alpha-helix-rich structure to a beta-sheet-rich structure with the increase in protein density on the membrane. Ganglioside-bound Abeta proteins exhibited seeding abilities for amyloid formation. GM1-Abeta exhibited the strongest seeding potential, especially under beta-sheet-forming conditions. This study suggested that lipid composition including gangliosides and cholesterol strictly controls amyloid formation.     

Interactions of Alkylphosphocholines with Model Membranes—The Langmuir Monolayer Study

Alkylphosphocholines (APCs) belong to a class of synthetic antitumor lipids, which are new-generation anticancer agents. In contrast to traditional antitumor drugs, they do not attack the cell nucleus but, rather, the cellular membrane; however, their mechanism of action is not fully understood. This work compared the interactions of selected APCs [namely, hexadecylphosphocholine (miltefosine), octadecylphosphocholine and erucylphosphocholine] with the most important membrane lipids [cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)] and examined their influence on a model membrane of tumor and normal cells. As a simple model of membranes, Langmuir monolayers prepared by mixing cholesterol either with a saturated phosphatidylcholine (DPPC), for a normal cell membrane, or with an unsaturated one (POPC), for a tumor cell membrane, have been applied. The APC–lipid interactions, based on experimental surface pressure (π) versus mean molecular area (A) isotherms, were analyzed qualitatively (with mean molecular area values) as well as quantitatively (with the ΔG ^exc function). Strong attractive interactions were observed for mixtures of APCs with cholesterol, contrary to the investigated phosphatidylcholines, for which the interactions were found to be weak with a tendency to separation of film components. In ternary monolayers it has been found that the investigated model systems (cholesterol/DPPC/APC vs cholesterol/POPC/APC) differ significantly as regards the interactions between film-forming molecules. The results demonstrate stronger interactions between the components of cholesterol/POPC/APC monolayers compared to cholesterol/POPC film, mimicking tumor cell membranes. In contrast, the interactions in cholesterol/DPPC/APC films were found to be weaker than those in the cholesterol/DPPC system, serving as a model of healthy cell membranes, thus proving that the incorporation of APCs is, from a thermodynamic point of view, unfavorable for binary cholesterol/DPPC monolayers. It can be concluded that the composition of healthy cell membranes is a natural barrier preventing the incorporation of APCs into normal cells.