Direct three-dimensional visualization of membrane disruption by amyloid fibrils.

Protein misfolding and aggregation cause serious degenerative conditions such as Alzheimer's, Parkinson, and prion diseases. Damage to membranes is thought to be one of the mechanisms underlying cellular toxicity of a range of amyloid assemblies. Previous studies have indicated that amyloid fibrils can cause membrane leakage and elicit cellular damage, and these effects are enhanced by fragmentation of the fibrils. Here we report direct 3D visualization of membrane damage by specific interactions of a lipid bilayer with amyloid-like fibrils formed in vitro from ß(2)-microglobulin (ß(2)m). Using cryoelectron tomography, we demonstrate that fragmented ß(2)m amyloid fibrils interact strongly with liposomes and cause distortions to the membranes. The normally spherical liposomes form pointed teardrop-like shapes with the fibril ends seen in proximity to the pointed regions on the membranes. Moreover, the tomograms indicated that the fibrils extract lipid from the membranes at these points of distortion by removal or blebbing of the outer membrane leaflet. Tiny (15-25 nm) vesicles, presumably formed from the extracted lipids, were observed to be decorating the fibrils. The findings highlight a potential role of fibrils, and particularly fibril ends, in amyloid pathology, and report a previously undescribed class of lipid-protein interactions in membrane remodelling.

Aggregation modulators interfere with membrane interactions of ß2-microglobulin fibrils.

Amyloid fibril accumulation is a pathological hallmark of several devastating disorders, including Alzheimer's disease, prion diseases, type II diabetes, and others. Although the molecular factors responsible for amyloid pathologies have not been deciphered, interactions of misfolded proteins with cell membranes appear to play important roles in these disorders. Despite increasing evidence for the involvement of membranes in amyloid-mediated cytotoxicity, the pursuit for therapeutic strategies has focused on preventing self-assembly of the proteins comprising the amyloid plaques. Here we present an investigation of the impact of fibrillation modulators upon membrane interactions of ß2-microglobulin (ß2m) fibrils. The experiments reveal that polyphenols (epigallocatechin gallate, bromophenol blue, and resveratrol) and glycosaminoglycans (heparin and heparin disaccharide) differentially affect membrane interactions of ß2m fibrils measured by dye-release experiments, fluorescence anisotropy of labeled lipid, and confocal and cryo-electron microscopies. Interestingly, whereas epigallocatechin gallate and heparin prevent membrane damage as judged by these assays, the other compounds tested had little, or no, effect. The results suggest a new dimension to the biological impact of fibrillation modulators that involves interference with membrane interactions of amyloid species, adding to contemporary strategies for combating amyloid diseases that focus on disruption or remodeling of amyloid aggregates.

Heparin inhibits membrane interactions and lipid-induced fibrillation of a prion amyloidogenic determinant.

Glycosaminoglycans (GAGs), particularly heparin, are known to reduce the toxicities of various amyloidogenic proteins. The molecular factors underlying the antitoxic effects of GAGs, however, are still not fully understood. Because interactions of amyloidogenic proteins and their aggregates with membranes are believed to play major roles in affecting amyloid pathogenesis, our objective in this study was to elucidate the effect of heparin on membrane interactions of the 21-residue amyloidogenic determinant of the prion protein [PrP(106-126)]. Indeed, the experimental results indicate that heparin significantly interferes in membrane interactions of the prion peptide. Specifically, we show that there is direct competition for binding of PrP(106-126) between heparin on the one hand and negatively charged phospholipids on the other hand. The data reveal that heparin, even in very low molar concentrations, exhibited high affinity towards PrP(106-126) and consequently suppressed interactions of the peptide with lipid vesicles. Interestingly, whereas heparin significantly inhibited lipid-induced PrP(106-126) fibrillation, it still promoted fibril formation in aqueous solutions independently of the lipid vesicles present. Our results strongly suggest that the primary effects of GAGs in attenuating amyloid toxicities are due to blocking of membrane interactions of the amyloidogenic proteins rather than modulation of their fibrillation properties.

Specific mutations alter fibrillation kinetics, fiber morphologies, and membrane interactions of pentapeptides derived from human calcitonin.

Protein misfolding and fibrillation are fundamental facets underlying a diverse group of amyloid disorders and diseases. The molecular factors responsible for amyloid protein toxicity and pathological consequences, however, are still not fully understood. The involvement of specific residues or sequence elements in fibril formation and the interactions of amyloid protein aggregates with membranes are believed to constitute two critical parameters contributing to amyloidogenesis and amyloid pathologies. This work aims to elucidate sequence determinants and membrane-protein interactions of five-residue peptide fragments derived from a core amyloidogenic sequence of human calcitonin. We show that single-residue mutations within the native pentapeptide sequence significantly modulate the kinetics of peptide self-assembly, alter beta-sheet organization between parallel and antiparallel arrangements, and modify fibrillar morphologies. We further demonstrate that hydrophobic or aromatic interactions are not prerequisites for peptide fiber formation. The experiments also disclose pronounced effects of lipid vesicles comprising cholesterol and negatively charged phospholipids on the rate of fibrillation and fiber structures formed by the short peptides. This work indicates that the structural and kinetic properties of peptide fibrils as well as lipid interactions of fibrillar species are interrelated and are significantly affected by specific residues within amyloid peptide sequences.

Membrane interactions and lipid binding of casein oligomers and early aggregates.

Caseins constitute the main protein components in mammalian milk and have critical functions in calcium transport and prevention of protein aggregation. Fibrillation and aggregation of kappa-casein, a phenomenon which has only recently been detected, might be associated with malfunctions of milk secretion and amyloidosis phenomena in the mammary glands. This study employs a newly-designed chromatic biomimetic vesicle assay to investigate the occurrence and the parameters affecting membrane interactions of casein aggregates and the contribution of individual casein members to membrane binding. We show that physiological casein colloids exhibit membrane activity, as well as early globular aggregates of kappa-casein, a prominent casein isoform. Furthermore, inhibition of kappa-casein fibrillation through complexation with alphaS-casein and beta-casein, respectively, was found to go hand in hand with induction of enhanced membrane binding; these data are important in the context of casein biology since in secreted milk kappa-casein is found only in assemblies containing also alphaS-casein and beta-casein. The chromatic experiments, complemented by transmission electron microscopy analysis and fluorescence quenching assays, also revealed significantly higher affinity early spherical aggregates of k-casein to anionic phosphatidylglycerol-lipids, as compared to zwitterionic phospholipids. Overall, this study suggests that lipid interactions play important roles in maintaining the essential physiological functions of caseins in mammalian milk.

Mechanisms of alpha-defensin bactericidal action: comparative membrane disruption by Cryptdin-4 and its disulfide-null analogue.

Mammalian alpha-defensins all have a conserved triple-stranded beta-sheet structure that is constrained by an invariant tridisulfide array, and the peptides exert bactericidal effects by permeabilizing the target cell envelope. Curiously, the disordered, disulfide-null variant of mouse alpha-defensin cryptdin-4 (Crp4), termed (6C/A)-Crp4, has bactericidal activity equal to or greater than that of the native peptide, providing a rationale for comparing the mechanisms by which the peptides interact with and disrupt phospholipid vesicles of defined composition. For both live Escherichia coli ML35 cells and model membranes, disordered (6C/A)-Crp4 induced leakage in a manner similar to that of Crp4 but had less overall membrane permeabilizing activity. Crp4 induction of the leakage of the fluorophore from electronegative liposomes was strongly dependent on vesicle lipid charge and composition, and the incorporation of cardiolipin into liposomes of low electronegative charge to mimic bacterial membrane composition conferred sensitivity to Crp4- and (6C/A)-Crp4-mediated vesicle lysis. Membrane perturbation studies using biomimetic lipid/polydiacetylene vesicles showed that Crp4 inserts more pronouncedly into membranes containing a high fraction of electronegative lipids or cardiolipin than (6C/A)-Crp4 does, correlating directly with measurements of induced leakage. Fluorescence resonance energy transfer experiments provided evidence that Crp4 translocates across highly charged or cardiolipin-containing membranes, in a process coupled with membrane permeabilization, but (6C/A)-Crp4 did not translocate across lipid bilayers and consistently displayed membrane surface association. Thus, despite the greater in vitro bactericidal activity of (6C/A)-Crp4, native, beta-sheet-containing Crp4 induces membrane permeabilization more effectively than disulfide-null Crp4 by translocating and forming transient membrane defects. (6C/A)-Crp4, on the other hand, appears to induce greater membrane disintegration.

Effect of structural and conformation modifications, including backbone cyclization, of hydrophilic hexapeptides on their intestinal permeability and enzymatic stability.

A library of 18 hexapeptide analogs was synthesized, including sub-libraries of N- or C-methylation of the parent hexapeptide Phe-Gly-Gly-Gly-Gly-Phe, as well as backbone cyclized analogs of each linear analog with various ring sizes. N- or C-methylation as well as cyclization (but not backbone cyclization) have been suggested to improve intestinal permeability and metabolic stability of peptides in general. Here we aimed to assess their applicability to hydrophilic peptides. The intestinal permeability (Papp) of the 18-peptide library was in the range of 0.2-6.8 x 10-6 cm/sec. Based on several tests, we concluded that the absorption mechanism of all tested analogs is paracellular, regardless of the structural or conformational modifications. In all cases, backbone cyclization increased Papp (5-fold) in comparison to the linear analogs due to the smaller 3D size and also dramatically decreased peptide proteolysis by brush border enzymes. N- or C-methylation did not enhance the permeability of the linear analogs in this series.

Glass-supported lipid/polydiacetylene films for colour sensing of membrane-active compounds.

Glass-supported biomimetic lipid/polydiacetylene films were employed for colourimetric detection and analysis of amphiphilic and membrane-active molecules. The sensor films comprise lipid monolayers that constitute a biomimetic membrane platform, interspersed within polydiacetylene domains that function as the colour reporter. The optical detection scheme is based on visible blue-red transitions of polydiacetylene, induced by amphiphilic analytes interacting with the film. The colour transitions of the lipid/polydiacetylene films can be either detected by the naked eye, recorded spectroscopically, or registered through digital image analysis using conventional scanning devices. Digital image analysis, in particular, allows quantification of the colourimetric transformations. Detection threshold of micromolar concentration of a membrane-active cytolytic peptide is demonstrated.

ß2-Microglobulin amyloid fibril-induced membrane disruption is enhanced by endosomal lipids and acidic pH.

Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of ß2-microglobulin (ß2m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which ß2m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of ß2m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that ß2m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between ß2m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of ß2m amyloid-associated osteoarticular tissue destruction in DRA.

N-terminal aromatic residues closely impact the cytolytic activity of cupiennin 1a, a major spider venom peptide.

Cupiennins are small cationic α-helical peptides from the venom of the ctenid spider Cupiennius salei which are characterized by high bactericidal as well as hemolytic activities. To gain insight into the determinants responsible for the broad cytolytic activities, two analogues of cupiennin 1a with different N-terminal hydrophobicities were designed. The insecticidal, bactericidal and hemolytic activities of these analogues were assayed and compared to the native peptide. Specifically, substitution of two N-terminal Phe residues by Ala results in less pronounced insecticidal and cytolytic activity, whereas a substitution by Lys reduces strongly its bactericidal activity and completely diminishes its hemolytic activity up to very high tested concentrations. Biophysical analyses of peptide/bilayer membrane interactions point to distinct interactions of the analogues with lipid bilayers, and dependence upon membrane surface charge. Indeed, we find that lower hemolytic activity was correlated with less surface association of the analogues. In contrast, our data indicate that the reduced bactericidal activity of the two cupiennin 1a analogues likely correspond to greater bilayer-surface localization of the peptides. Overall, ultimate insertion and destruction of the host cell membrane is highly dependent on the presence of Phe-2 and Phe-6 (Cu 1a) or Leu-6 (Cu 2a) in the N-terminal sequences of native cupiennins.

Amyloid - membrane interactions: experimental approaches and techniques.

The growing interest in membrane interactions of amyloidogenic peptides and proteins emanates from the realization that lipids and membranes play important, potentially central, roles in the toxicity and pathological pathways of amyloid diseases. Expanding body of evidence indicates that lipid binding of amyloidogenic peptides and amyloid peptide association with cellular membranes is critical in the onset and progression of amyloid diseases. Advancing the understanding in this field goes hand in hand with application of varied biophysical and biological techniques designed to probe the characteristics and underlying mechanisms of membrane-peptide interactions. This review summarizes experimental approaches and technical aspects employed in recent years for investigating the interaction of amyloid peptides and fibrillar species with lipid bilayers, and the reciprocal contribution of membranes to fibrillation processes and amyloidogenesis.

Lipid-induced calcitonin fibrillation blocks membrane interactions of a peptide antibiotic.

Interactions between membranes and amyloid proteins are believed to be a major factor contributing to pathogenesis in amyloid diseases. Furthermore, membranes have been shown to closely affect fibrillation processes of varied amyloidogenic peptides. Here we describe an intriguing phenomenon in which bilayer-induced fibrillation of human calcitonin (hCT) gave rise to significant inhibition of membrane interactions of alamethicin, an antibiotic, membrane-permeating peptide. This "membrane shielding" effect was apparent only when fibrillation of hCT occurred in the presence of cholesterol-containing vesicles; no interference with membrane binding was detected when hCT fibrillar species were formed in noncholesterol lipid environments, or when hCT amyloid aggregates were separately added to lipid bilayers. The experimental data indicate that cholesterol-promoted formation of amyloid fibril network at the bilayer interface is most likely responsible for the shielding effect. This phenomenon might point to a role of amyloid fibers in preventing membrane disruption by antibiotic peptides and other toxic species.