Tania Sheynis

Mechanisms of α-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.

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.

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.

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.

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.

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.

A paradigm for solvent and temperature induced conformational changes.

Knowing the chemical formula of small molecules, which are the basic building blocks of organic and inorganic materials, is not always enough to predict their behavior because also the environment can have a significant influence. Distinct molecular conformations can arise from free rotations around the bonds resulting in a number of potential threedimensional conformations (stereoisomers) and differences in activities can be induced, despite the fact that the atomic composition of the original molecule is unchanged and the number of single bonds conserved. Examples include the surprising results that biological enzymes in neat organic solvents can show increased stability and profound changes in selectivity compared to aqueous environments. The stabilization or recognition of a single molecular conformation over others is necessary in divers phenomena, such as: bioseparations, the ability of peptides to pass membranes, and many protein behaviors such as: translocation, folding, and substrate or inhibitor binding. Herein we examine a small organic molecule, 4-[(tert-butoxycarbonyl)amino]-2-hydroxybutanoic acid (Boc-amine; Figure 1) that exhibits multiple conformations. The conformations observed depend on solvent and temperature. Bocamine is explored via X-ray, NMR, membrane–activity assays, and DFT calculations. In addition, its behaviors are contrasted with those of other small isomeric organic molecules, including three Boc-amine analogues (Figure 2) with the ultimate goal of understanding the physical and chemical details of how the environment induces the observed conformational isomerism. The crystal structure can be described by two different chains (Figure 1). Each individual chain contains conformationally identical molecules (“open” and “closed”) hydrogen bonded via their hydroxyl and carboxylate groups and extending by the screw axis along b (Figure 1 a). No solvent molecules are present in the crystal structure.