Aß Amyloid Fibers Drastically Alter the Topography and Mechanical Properties of Lipid Membranes.

Alzheimer's disease (AD) is related to the fibrillation of the Aß peptides at neuronal membranes, a process that depends on the lipid composition and may impart different physical states to the membrane. In the present work, we study the properties of the Aß peptide when mixed with a zwitterionic lipid (DMPC), using the Langmuir monolayer technique as an approach to control membrane physical conditions. First, we build on previous characterizations of pure Aß monolayers and observe that, in addition to high shear, these films present a pronounced compressional hysteresis. When Aß is assembled with DMPC in a binary film, the resulting membranes become heterogeneous, with a peptide-enriched phase distributed in a network-like pattern, and they exhibit a lateral transition that depends on the Aß content. At lower peptide proportions, the films segregate into two well-defined phases: one consisting of lipids and another enriched with peptides. The reflectivity of these phases differs from that obtained for pure Aß films. Thus, the formed fibers effectively cover most of the interface area and remain stable at higher pressures (from 20 to 30 mN m-1 depending on Aß content) compared to pure peptide films (17 mN m-1). Furthermore, such structures induce a compressional hysteresis in the film, similar to that of pure peptide films (which is nonexistent in the pure lipid monolayer), even at low peptide proportions. We claim that the mechanical properties at the interface are governed by the size of the fibril-like structures. Based on the low molar fractions and surface packing at which these phenomena were observed, we postulate that as a consequence of peptide intermolecular interactions, Aß may have drastic effects on the molecular arrangement and mechanical properties of a lipid membrane.

Aß-Amyloid Fibrils Are Self-Triggered by the Interfacial Lipid Environment and Low Peptide Content.

We studied the surface properties of Aß(1-40) amyloid peptides mixed with 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) (liquid state) or 1,2-disteraoyl-phosphatidylcholine (DSPC) (solid state) phospholipids by using nanostructured lipid/peptide films (Langmuir monolayers). Pure Aß(1-40) amyloid peptides form insoluble monolayers without forming fibril-like structures. In a lipid environment [phospholipid/Aß(1-40) peptide mixtures], we observed that both miscibility and stability of the films depend on the peptide content. At low Aß(1-40) amyloid peptide proportion (from 2.5 to 10% of peptide area proportion), we observed the formation of a fibril-like structure when mixed only with POPC lipids. The stability acquired by these mixed films is within 20-35 mN·m-1 compatible with the equivalent surface pressure postulated for natural biomembranes. Fibrils are clearly evidenced directly from the monolayers by using Brewster angle microscopy. The so-called nanostructured fibrils are thioflavin T positive when observed by fluorescence microscopy. The amyloid fibril network at the surface was also evidenced by atomic force microscopy when the films are transferred onto a mica support. Aß(1-40) amyloid mixed with the solid DSPC lipid showed an immiscible behavior in all peptide proportions without fibril formation. We postulated that the amyloid fibrillogenesis at the membrane can be dynamically nano-self-triggered at the surface by the quality of the interfacial environment, that is, the physical state of the water-lipid interface and the relative content of amyloid protein present at the interface.

V-Shaped Molecular Configuration of Wax Esters of Jojoba Oil in a Langmuir Film Model.

The aim of the present work was to understand the interfacial properties of a complex mixture of wax esters (WEs) obtained from Jojoba oil (JO). Previously, on the basis of molecular area measurements, a hairpin structure was proposed as the hypothetical configuration of WEs, allowing their organization as compressible monolayers at the air-water interface. In the present work, we contributed with further experimental evidence by combining surface pressure (π), surface potential (Δ V), and PM-IRRAS measurements of JO monolayers and molecular dynamic simulations (MD) on a modified JO model. WEs were self-assembled in Langmuir films. Compression isotherms exhibited πlift-off at 100 Å2/molecule mean molecular area ( Alift-off) and a collapse point at πc ≈ 2.2 mN/m and Ac ≈ 77 Å2/molecule. The Δ V profile reflected two dipolar reorganizations, with one of them at A > Alift-off due to the release of loosely bound water molecules and another one at Ac < A < Alift-off possibly due to reorientations of a more tightly bound water population. This was consistent with the maximal SP value that was calculated according to a model that considered two populations of oriented water and was very close to the experimental value. The orientation of the ester group that was assumed in that calculation was coherent with the PM-IRRAS behavior of the carbonyl group with the C═O oriented toward the water and the C-O oriented parallel to the surface and was in accordance with their orientational angles (∼45 and ∼90°, respectively) determined by MD simulations. Taken together, the present results confirm a V shape rather than a hairpin configuration of WEs at the air-water interface.

Phase coexistence in films composed of DLPC and DPPC: a comparison between different model membrane systems.

For the biophysical study of membranes, a variety of model systems have been used to measure the different parameters and to extract general principles concerning processes that may occur in cellular membranes. However, there are very few reports in which the results obtained with the different models have been compared. In this investigation, we quantitatively compared the phase coexistence in Langmuir monolayers, freestanding bilayers and supported films composed of a lipid mixture of DLPC and DPPC. Two-phase segregation was observed in most of the systems for a wide range of lipid proportions using fluorescence microscopy. The lipid composition of the coexisting phases was determined and the distribution coefficient of the fluorescent probe in each phase was quantified, in order to explore their thermodynamic properties. The comparison between systems was carried out at 30mN/m, since it is accepted that at this or higher lateral pressures, the mean molecular area in bilayers is equivalent to that observed in monolayers. Our study showed that while Langmuir monolayers and giant unilamellar vesicles had a similar phase behavior, supported films showed a different composition of the phases with the distribution coefficient of the fluorescent probe being close to unity. Our results suggest that, in supported membranes, the presence of the rigid substrate may have led to a stiffening of the liquid-expanded phase due to a loss in the degrees of freedom of the lipids as a consequence of the proximity of the solid material.

Stiffness of lipid monolayers with phase coexistence.

The surface dilational modulus--or compressibility modulus--has been previously studied for monolayers composed of pure materials, where a jump in this modulus was related with the onset of percolation as a result of the establishment of a connected structure at the molecular level. In this work, we focused on monolayers composed of two components of low lateral miscibility. Our aim was to investigate the compressibility of mixed monolayers at pressures and compositions in the two-phase region of the phase diagram, in order to analyze the effect of the mechanical properties of each phase on the stiffness of the composite. In nine different systems with distinct molecular dipoles and charges, the stiffness of each phase and the texture at the plane of the monolayer were studied. In this way, we were able to analyze the general compressibility of two-phase lipid monolayers, regardless of the properties of their constituent parts. The results are discussed in the light of the following two hypotheses: first, the stiffness of the composite could be dominated by the stiffness of each phase as a weighted sum according to the percentage of each phase area, regardless of the distribution of the phases in the plane of the monolayer. Alternatively, the stiffness of the composite could be dominated by the mechanical properties of the continuous phase. Our results were better explained by this latter proposal, as in all the analyzed mixtures it was found that the mechanical properties of the percolating phase were the determining factors. The value of the compression modulus was closer to the value of the connected phase than to that of the dispersed phase, indicating that the bidimensional composites displayed mechanical properties that were related to the properties of each phases in a rather complex manner.

Melittin-solid phospholipid mixed films trigger amyloid-like nano-fibril arrangements at air-water interface.

We used the Langmuir monolayers technique to study the surface properties of melittin toxin mixed with either liquid-condensed DSPC or liquid-expanded POPC phospholipids. Pure melittin peptide forms stable insoluble monolayers at the air-water interface without interacting with Thioflavin T (Th-T), a sensitive probe to detect protein amyloid formation. When melittin peptide is mixed with DSPC lipid at 50 % of peptide area proportion at the surface, we observed the formation of fibril-like structures detected by Brewster angle microscopy (BAM), but they were not observable with POPC. The nano-structures in the melittin-DSPC mixtures became Th-T positive labeling when the arrangement was observed with fluorescence microscopy. In this condition, Th-T undergoes an unexpected shift in the typical emission wavelength of this amyloid marker when a 2D fluorescence analysis is conducted. Even when reflectivity analysis of BAM imaging evidenced that these structures would correspond to the DSPC lipid component of the mixture, the interpretation of ATR-FTIR and Th-T data suggested that both components were involved in a new lipid-peptide rearrangement. These nano-fibril arrangements were also evidenced by scanning electron and atomic force microscopy when the films were transferred to a mica support. The fibril formation was not detected when melittin was mixed with the liquid-expanded POPC lipid. We postulated that DSPC lipids can dynamically trigger the process of amyloid-like nano-arrangement formation at the interface. This process is favored by the relative peptide content, the quality of the interfacial environment, and the physical state of the lipid at the surface.

Biogenesis and Breakdown of Lipid Droplets in Pathological Conditions.

Lipid droplets (LD) have long been considered as mere fat drops; however, LD have lately been revealed to be ubiquitous, dynamic and to be present in diverse organelles in which they have a wide range of key functions. Although incompletely understood, the biogenesis of eukaryotic LD initiates with the synthesis of neutral lipids (NL) by enzymes located in the endoplasmic reticulum (ER). The accumulation of NL leads to their segregation into nanometric nuclei which then grow into lenses between the ER leaflets as they are further filled with NL. The lipid composition and interfacial tensions of both ER and the lenses modulate their shape which, together with specific ER proteins, determine the proneness of LD to bud from the ER toward the cytoplasm. The most important function of LD is the buffering of energy. But far beyond this, LD are actively integrated into physiological processes, such as lipid metabolism, control of protein homeostasis, sequestration of toxic lipid metabolic intermediates, protection from stress, and proliferation of tumours. Besides, LD may serve as platforms for pathogen replication and defense. To accomplish these functions, from biogenesis to breakdown, eukaryotic LD have developed mechanisms to travel within the cytoplasm and to establish contact with other organelles. When nutrient deprivation occurs, LD undergo breakdown (lipolysis), which begins with the LD-associated members of the perilipins family PLIN2 and PLIN3 chaperone-mediated autophagy degradation (CMA), a specific type of autophagy that selectively degrades a subset of cytosolic proteins in lysosomes. Indeed, PLINs CMA degradation is a prerequisite for further true lipolysis, which occurs via cytosolic lipases or by lysosome luminal lipases when autophagosomes engulf portions of LD and target them to lysosomes. LD play a crucial role in several pathophysiological processes. Increased accumulation of LD in non-adipose cells is commonly observed in numerous infectious diseases caused by intracellular pathogens including viral, bacterial, and parasite infections, and is gradually recognized as a prominent characteristic in a variety of cancers. This review discusses current evidence related to the modulation of LD biogenesis and breakdown caused by intracellular pathogens and cancer.

Langmuir films at the oil/water interface revisited.

We studied monomolecular layers at the oil/water interface (O/Wint) in a Langmuir interfacial trough using egg-yolk phosphatidylcholine (EPC) (the model phospholipid) and Vaseline (VAS) as oil phase. The temporal dynamics in the surface pressure (π) evolution depended on the method (spreading/adsorption) used for monolayers preparation and reflected the different distribution of EPC between all the system compartments (bulk phases and interfaces). We distinguished between EPC located either stable at the interface or hopping between the interface and bulk phases. The size order of the apparent mean molecular area, at constant π, of EPC at different interfaces (EPCO/W > EPC/VAS0.02;A/W > EPCA/W), suggested that VAS molecules intercalated between the hydrocarbon chains of EPCO/W, at a molar fraction xVAS > 0.02. However, EPC/VAS0.02;A/W showed the highest compressional free energy. This leaded us to study the EPC/VAS0.02 mixture at A/W by Brewster Angle Microscopy (BAM), finding that upon compression VAS segregated over the monolayer, forming non-coalescent lenses (as predicted by the spreading coefficient S = -13 mN/m) that remained after decompression and whose height changed (increase/decrease) accompanied the compression/decompression cycle. At the O/Wint, while some VAS molecules remained at the interface up to the collapse, others squeezed out towards the VAS bulk phase with an energy requirement lower than towards the air.

A surface active benzodiazepine receptor ligand for potential probing membrane order of GABAA-receptor surroundings.

A conjugable analogue of the benzodiazepine 5-(2-hydroxiphenyl)-7-nitro-benzo[ e][1,4]diazepin-2(3 H)-one N 1-substituted with an aliphatic chain (CNZ acyl derivative, CAd) was synthesized. CAd inhibited FNZ binding to GABA A-R with an inhibition binding constant K i = 176 nM and expanded a model membrane packed up to 13 mN/m when penetrating from the aqueous phase. CAd exhibited surface activity with a collapse pressure pi = 18.8 mN/m and minimal molecular area A min = 49 A (2)/molecule at the closest molecular packing, resulting in full and nonideal mixing with a phospholipid in a monolayer up to a molar fraction x congruent with 0.1, decreasing its surface potential and contributing with a dipole that pointed its positive end toward the air and reoriented at the interface upon compression. These findings suggested that CAd could be stabilized at the membrane-water interface with its CNZ moiety stacked at the GABA A-R while its acyl chain can be inserted into the membrane depth.

The rheological properties of beta amyloid Langmuir monolayers: Comparative studies with melittin peptide.

We determined the rheological properties of ß-amyloid Langmuir films at the air/water interface, a peptide whose interfacial structure is extended ß-sheet, and compared them with those of films composed of Melittin (Mel), which adopts an α-helical conformation at neutral pH. To determine the dilatational and shear moduli we evaluated the response of pure peptide monolayers to an oscillatory anisotropic compressive work. Additionally, a micro-rheological characterization was performed by tracking the diffusion of micrometer sized latex beads onto the interface. This technique allowed us the detection of different rheological behaviour between monolayers presenting a low shear response. Monolayers of the ß-sheet structure-adopting peptides, such as ß-amyloid peptides, exhibited a marked shear (elastic) modulus even at low surface pressures. In contrast, Mel monolayers exhibited negligible shear modulus and the micro-rheological shear response was markedly lower than that observed for either Aß1-40 or Aß1-42 amyloid peptides. When Mel monolayers were formed at the interface of an aqueous solution at pH 11, we observed an increase in both the lateral stability and film viscosity as detected by a slower diffusion of the latex beads, in keeping with an increase in ß-sheet structure at this high pH (verified by ATR and FT-IR measurements). We suggest that the interactions responsible for the marked response upon shear observed for ß-amyloid peptide monolayers are the hydrogen bonds of the ß-sheet structure that can form an infinite planar network at the interface. Conversely, α-helical Mel peptide lack of these inter-molecular interactions and, therefore the shear contribution was negligible. We propose that the secondary structure is important for modulating the rheological behavior of short peptide monolayers regardless of the mass density or surface charge at the surface.

Reversing the peptide sequence impacts on molecular surface behaviour.

The protein's primary structure has all the information for specific protein/peptide folding and, in many cases, can define specific amphiphilic regions along molecules that are important for interaction with membranes. In order to shed light on how peptide sequence is important for the surface properties of amphiphilic peptides, we designed three pairs of peptides with the following characteristics: (1) all molecules have the same hydrophobic residues; (2) the couples differ from each other in their hydrophilic amino acids: positively, negatively and non-charged; (3) each pair has the same residues (same global molecular hydrophobicity) but the primary structure is reversed in comparison to its partner (retro-isomer), giving a molecule with a hydrophilic N or C-terminus and a hydrophobic C or N-terminus. Using the Langmuir monolayer approach, we observed that sequence reversal has a central role in the lateral stability of peptide monolayers, in the ability of the molecules to partition into the air-water interface and in the rheological properties of peptide films, whereas the peptide's secondary structure, determined by ATR-FTIR, was the same for all peptides. Reversing the sequence also gives a differential way of peptide/lipid interaction when peptides are in the presence of POPC lipid bilayers. Our results show how sequence inversion confers a distinctive peptide surface behaviour and lipid interaction for molecules with a similar structure.

Inter-domain interactions in charged lipid monolayers.

Phase coexistence is common in model biomembranes with the presence of domains formed by lipids in a dense phase state modulating lateral diffusion of species through hydrodynamic and electrostatic interactions. In this study, interdomain interactions in monolayers of charged surfactants were analyzed and compared with neutral systems. Interactions were investigated at different interdomain distances and by varying the ionic strength (I) of the subphase. At low percentages of condensed area (%Ac), i.e., high interdomain distances, domains were approximated as point charges or dipoles, and a comparison between the simulated and experimental results was made. At high %Ac, domains were arranged in a distorted hexagonal lattice, and the energy of a domain around its equilibrium position in the lattice was modeled using a harmonic potential and the spring constant determined. On subphases of high I, charged domains interacted in a manner similar to neutral domains with domain motion being precluded at high percentages of condensed area. At low I, a higher interdomain repulsion was observed along with a lower domain motion and, therefore, a higher apparent viscosity at comparable %Ac. Interestingly, this effect was observed at conditions where the Debye-Hückel length was still 2 orders lower than the interdomain distances.

Probing the combined effect of flunitrazepam and lidocaine on the stability and organization of bilayer lipid membranes. A differential scanning calorimetry and dynamic light scattering study.

Combined effects of flunitrazepam (FNZ) and lidocaine (LDC) were studied on the thermotropic equilibrium of dipalmitoyl phosphatidylcholine (dpPC) bilayers. This adds a thermodynamic dimension to previously reported geometric analysis in the erythrocyte model. LDC decreased the enthalpy and temperature for dpPC pre- and main-transitions (ΔHp, ΔHm, Tp, Tm) and decreased the cooperativity of the main-transition (ΔT(1/2,m)). FNZ decreased ΔHm and, at least up to 59 µM, also decreased ΔHp. In conjunction with LDC, FNZ induced a recovery of ∆T(1/2,m) control values and increased ΔHm even above the control level. The deconvolution of the main-transition peak at high LDC concentrations revealed three components possibly represented by: a self-segregated fraction of pure dpPC, a dpPC-LDC mixture and a phase with a lipid structure of intermediate stability associated with LDC self-aggregation within the lipid phase. Some LDC effects on thermodynamic parameters were reverted at proper LDC/FNZ molar ratios, suggesting that FNZ restricts the maximal availability of the LDC partitioned into the lipid phase. Thus, beyond its complexity, the lipid-LDC mixture can be rationalized as an equilibrium of coexisting phases which gains homogeneity in the presence of FNZ. This work stresses the relevance of nonspecific drug-membrane binding on LDC-FNZ pharmacological interactions and would have pharmaceutical applications in liposomal multidrug-delivery.

Phosphatidylcholine/vegetable oil pseudo-binary mixtures at the air-water interface: predictive formulation of oil blends with selected surface behavior.

The present work is an attempt to define how to formulate oil blends with an expected surface behavior using easily accessible data such as chemical compositions. Hence, we determined average surface properties of triglycerides (TG) from olive (O), soybean (S), and walnut (W) oils self-organized in Langmuir films alone or in pseudo-binary mixtures with phosphatidylcholines (PC). Collapse pressure (pi(c)), compressibility modulus (K) and molecular area at the closest packing (A(min)) were determined from pi-mean molecular area (Mma) isotherms. The pi(c)-composition phase diagrams of TG-PC mixtures provided information about oils solubility limit with PCs in the monolayer phase. A thermodynamic equilibrium model was fitted to the line joining points of monolayer-TG(liquid phase) coexistence and allowed to obtain interaction parameters, omega, which consistently with those of excess surface energy (Delta G(ex)) and Mma deviations from ideality, contributed to describe interfacial intermolecular interactions. Oil molar fractions (x(TG)) for TGs-PCs self-assembling into vesicles were estimated from x(TG) values at pi(c) congruent with 30 mN/m (equilibrium pi of bilayers), which resulted higher in egg PC (0.15, 0.2, 0.15 for O, S and W, respectively) than in dipalmitoyl-PC (0.125, 0.075, 0.1). Principal component analysis performed on surface parameters, grouped S and W separated from O. This result was mainly influenced by variables estimating the effect of unsaturation degrees of fatty acids sterified at TGs, A(min) and pi(c). Peanut oils surface data interpolated in pi(c)-C16/C18 and A(min)-DBI correlation lines obtained with O-S mixtures (TG(mix)) and with TG(mix)-PC supported C16/C18 ratio and DBI as predictors to formulate oil blends with selected surface behavior.