Showing papers by "Richard M. Epand published in 2004"
TL;DR: The interactions of two amphiphilic and cationic, nine-residue β-peptides with liposomal membranes were studied and the biological potency and microbial specificity of the 14-helical β- peptides suggests that 14-helices can be particularly disruptive to microbial membranes.
Abstract: The interactions of two amphiphilic and cationic, nine-residue β-peptides with liposomal membranes were studied. These β-peptides are shown to form 14-helices in the presence of bilayers. Membrane binding and membrane permeabilization occur preferentially in the presence of anionic lipids. The β-peptides have the ability to cause tranbilayer diffusion of phospholipids, form pores, and promote lipid mixing between liposomes. These β-peptides have previously been shown to display antimicrobial activity comparable to that of a longer β-peptide, β-17, which adopts a different type of helical conformation (12-helix), and to the 23 amino acid (Ala8,13,18)-magainin-II-amide, which adopts an α-helical conformation. In addition, these 14-helical β-peptides show relatively low hemolytic activity. The biological potency and microbial specificity of the 14-helical β-peptides, despite their relatively short length, suggests that 14-helices can be particularly disruptive to microbial membranes.
98 citations
TL;DR: It is proposed that the arrangement of aromatic residues in class A amphipathic helical molecules regulates entry of reactive oxygen species into peptide-phospholipid complexes, thereby reducing the extent of monocyte chemotaxis, an important step in atherosclerosis.
91 citations
TL;DR: Lipid analysis of isolated nuclear membranes revealed the presence of a novel C26-substituted PI (phosphatidylinositol), which is the only C 26-containing glycerophospholipid that is detectable in wild-type yeast, and the C26 -substitution is highly specific for the sn-1 position of the glycerol backbone.
Abstract: Morphological analysis of a conditional yeast mutant in acetyl-CoA carboxylase acc1ts/mtr7, the rate-limiting enzyme of fatty acid synthesis, suggested that the synthesis of C26 VLCFAs (very-long-chain fatty acids) is important for maintaining the structure and function of the nuclear membrane. To characterize this C26-dependent pathway in more detail, we have now examined cells that are blocked in pathways that require C26. In yeast, ceramide synthesis and remodelling of GPI (glycosylphosphatidylinositol)-anchors are two pathways that incorporate C26 into lipids. Conditional mutants blocked in either ceramide synthesis or the synthesis of GPI anchors do not display the characteristic alterations of the nuclear envelope observed in acc1ts, indicating that the synthesis of another C26-containing lipid may be affected in acc1ts mutant cells. Lipid analysis of isolated nuclear membranes revealed the presence of a novel C26-substituted PI (phosphatidylinositol). This C26-PI accounts for approx. 1% of all the PI species, and is present in both the nuclear and the plasma membrane. Remarkably, this C26-PI is the only C26-containing glycerophospholipid that is detectable in wild-type yeast, and the C26-substitution is highly specific for the sn-1 position of the glycerol backbone. To characterize the biophysical properties of this lipid, it was chemically synthesized. In contrast to PIs with normal long-chain fatty acids (C16 or C18), the C26-PI greatly reduced the bilayer to hexagonal phase transition of liposomes composed of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE). The biophysical properties of this lipid are thus consistent with a possible role in stabilizing highly curved membrane domains.
83 citations
TL;DR: The preservation of a predicted loop structure in the fusion peptide of this unusual nonenveloped virus membrane fusion protein supports an imperative role for a kinked fused peptide motif in biological membrane fusion.
Abstract: The avian and Nelson Bay reoviruses are two of only a limited number of nonenveloped viruses capable of inducing cell-cell membrane fusion. These viruses encode the smallest known membrane fusion proteins (p10). We now show that a region of moderate hydrophobicity we call the hydrophobic patch (HP), present in the small N-terminal ectodomain of p10, shares the following characteristics with the fusion peptides of enveloped virus fusion proteins: (i) an abundance of glycine and alanine residues, (ii) a potential amphipathic secondary structure, (iii) membrane-seeking characteristics that correspond to the degree of hydrophobicity, and (iv) the ability to induce lipid mixing in a liposome fusion assay. The p10 HP is therefore predicted to provide a function in the mechanism of membrane fusion similar to those of the fusion peptides of enveloped virus fusion peptides, namely, association with and destabilization of opposing lipid bilayers. Mutational and biophysical analysis suggested that the internal fusion peptide of p10 lacks alpha-helical content and exists as a disulfide-stabilized loop structure. Similar kinked structures have been reported in the fusion peptides of several enveloped virus fusion proteins. The preservation of a predicted loop structure in the fusion peptide of this unusual nonenveloped virus membrane fusion protein supports an imperative role for a kinked fusion peptide motif in biological membrane fusion.
51 citations
TL;DR: It is proposed that the majority of the p14 ectodomain is composed of a fusion peptide motif, the first such motif dependent on myristoylation for membrane fusion activity.
51 citations
TL;DR: A brief review of some of the diverse methodologies used to identify the presence of domains in membranes is given and some of these domains are enriched in cholesterol.
50 citations
TL;DR: The results with these model systems demonstrate changes in peptide-lipid interactions that may be related to the observed biological properties of the 18-amino acid amphipathic helical peptide.
Abstract: The 18-amino acid amphipathic helical peptide Ac-DWFKAFYDKVAEKFKEAF-NH(2) promotes the separation of cholesterol from the phospholipid, resulting in the formation of cholesterol crystallites, even at mole fractions of cholesterol as low as 0.3. The peptide exerts a greater degree of penetration into membranes of pure phosphatidylcholine in the absence of cholesterol than into bilayers of phosphatidylcholine and cholesterol. The circular dichroism spectrum of the peptide in buffer indicates that it self-associates, leading to the formation of structures with higher helical content. However, in the presence of lipid, the peptide remains helical over a larger concentration range. The peptide undergoes a thermal transition on heating. Cholesterol has little effect on the secondary structure of the peptide; however, increased Trp emission intensity in the absence of cholesterol indicates a deeper penetration of the helix upon removal of cholesterol from the membrane. The results with these model systems demonstrate changes in peptide-lipid interactions that may be related to the observed biological properties of this peptide.
48 citations
TL;DR: It is indicated that oleoyl-sphingomyelin, unlike saturated forms of sphingomylin, does not form segregated domains with cholesterol because of its greater miscibility with phosphatidylcholine.
45 citations
TL;DR: Both fluorescence spectroscopy and total internal reflectance fluorescence microscopy revealed the cholesterol-dependent nature of PtdIns(4,5) P2-enriched membrane-domain formation.
Abstract: A myristoylated peptide corresponding to the N-terminus of NAP-22 (neuronal axonal myristoylated membrane protein of 22 kDa) causes the quenching of the fluorescence of BODIPY®-TMR-labelled PtdIns(4,5) P 2 in bilayers of 1-palmitoyl-2-oleoyl phosphatidylcholine containing 40 mol% cholesterol and 0.1 mol% BODIPY®–PtdIns(4,5) 2 . Both fluorescence spectroscopy and total internal reflectance fluorescence microscopy revealed the cholesterol-dependent nature of PtdIns(4,5) P 2 -enriched membrane-domain formation.
43 citations
TL;DR: 3F-2 had a greater effect in altering the properties of the hydrocarbon region of the membrane, consistent with the proposed model of the effect of peptide shape on the nature of the difference in peptide insertion into the bilayer.
26 citations
TL;DR: It is demonstrated that Bax can induce leakage from liposomes at ion concentrations resembling those found physiologically, and the action of Bax is strongly modulated by the presence of bivalent cations that can act synergistically, as well as by micelle-forming lipid components that can be either stimulatory or inhibitory.
Abstract: Fatty acids are known promoters of apoptosis. In the present study, the direct role of fatty acids with regard to their ability to cause membrane permeabilization by Bax was explored. Addition of fatty acids to liposomes in the presence of cations greatly enhanced the permeabilizing activity of Bax, a pro-apoptotic Bcl-2 protein. This provides a putative mechanism for the role of fatty acids in apoptosis. It is not a result of detergent-like properties of fatty acids, since a different micelle-forming amphiphile, dilysocardiolipin, was strongly inhibitory. We also demonstrate that there is a synergistic effect on Bax-induced permeabilization between Ca(2+) and Mg(2+), both on the binding of Bax to liposomes as well as on the induction of the leakage of liposomal contents. Micromolar concentrations of Ca(2+) added externally or submicromolar concentrations of free Ca(2+) present in the medium were sufficient to promote Bax-induced permeabilization synergistically with externally added Mg(2+). These results indicate that Bax can induce leakage from liposomes at ion concentrations resembling those found physiologically. The synergistic effects of Ca(2+) and Mg(2+) were observed with liposomes with different lipid compositions. Thus the action of Bax is strongly modulated by the presence of bivalent cations that can act synergistically, as well as by micelle-forming lipid components that can be either stimulatory or inhibitory.
TL;DR: It is suggested that sequential cycles lead to a gradual loss in magnitude of the heat effect upon pressure perturbation and can be explained by the formation of a metastable glass-like state that converts to a stable phase at temperatures removed from the region of the phase transition.
TL;DR: Mixtures of cholesterol with phosphatidylcholine species containing the polyunsaturated acyl chains arachidonoyl or docosahexaenoyl were studied using both cross‐polarization and direct polarization, by 31P NMR and by differential scanning calorimetry.
Abstract: Mixtures of cholesterol with phosphatidylcholine species containing the polyunsaturated acyl chains arachidonoyl or docosahexaenoyl were studied by 13C magic angle spinning (MAS) NMR using both cross-polarization and direct polarization, by 31P NMR and by differential scanning calorimetry. Several unique features of these systems were observed. The separation of cholesterol in crystalline form occurred at much lower molar fractions than with other forms of phosphatidylcholine. The crystals that were formed were sensitive to the history of the sample. At cholesterol molar fractions below 0.5, they dissolved into the membrane by sequential heating and cooling scans. With higher molar fractions of cholesterol, larger amounts of anhydrous crystals were formed after the first heating. This was accompanied by the formation of non-lamellar phases. The cholesterol crystals that were formed generally were not observed by direct polarization 13C MAS NMR, even with delay times of 100 s. This suggests that the cholesterol crystals are in a more rigid state in mixtures with these lipids. This is in contrast with the terminal methyl group of the acyl chains that is too mobile to allow cross-polarization using 1 ms contact times. Copyright © 2004 John Wiley & Sons, Ltd.
TL;DR: Liposomal assays show that the DGKalpha and, to a lesser extent, DGKzeta preferentially act on substrates containing an arachidonoyl group when this group is incorporated into alkylacylglycerols, demonstrating that the acyl chain specificity is affected by the structure of the lipid headgroup.
Abstract: We compared the diacylglycerol kinase (DGK) catalyzed phosphorylation of 1-O-hexanoyl-2-oleoylglycerol (HOG) with 1-O-hexanoyl-2-arachidonoylglycerol (HAG) We assayed the activity of DGKalpha and DGKzeta using a liposomal-based assay system Liposomal assays show that the DGKalpha and, to a lesser extent, DGKzeta preferentially act on substrates containing an arachidonoyl group when this group is incorporated into alkylacylglycerols The activity of DGKalpha was 82 times greater with HAG compared to HOG DGKzeta is 10 times more active in catalyzing the phosphorylation of HAG compared to HOG Although diacylglycerols were better substrates for both DGKalpha and DGKzeta than the alkylacylglycerols, no specificity was exhibited for arachidonoyl-containing diacylglycerols However, this specificity for HAG over HOG is modulated by the phospholipid composition of the liposome Addition of cholesterol and/or phosphatidylethanolamine partially reduces the substrate selectivity We also analyzed the kinetic constants for the phosphorylation of both diacylglycerol and 1-alkyl-2-acylglycerol catalyzed by the alpha, epsilon, or zeta isoforms using a soluble Triton mixed micelle system We found that all three isoforms of DGK can phosphorylate 1-alkyl-2-acylglycerols but generally at a lower rate than for the corresponding diacylglycerol The specificity of DGKepsilon for diacylglycerols containing an arachidonoyl group was retained when the ester group in the C-1 position is replaced with an ether linkage In contrast, DGKalpha and, to a lesser extent, DGKzeta had greater specificity for arachidonoyl-containing 1-alkyl-2-acylglycerols than for arachidonoyl-containing diacylglycerols This demonstrates that the acyl chain specificity is affected by the structure of the lipid headgroup
TL;DR: The ability of the analogs to form helical structures in the presence of dimyristoylphosphatidylglycerol as well as their ability to lower the enthalpy of the calorimetric phase transition of this phospholipid correlates well with the hypocalcemic potency of the peptide.
Abstract: The conformational and lipid binding properties of several calcitonin analogs were compared. These analogs were designed to have the central amphipathic helical region of salmon calcitonin and N- and C-terminal segments similar to human calcitonin. The various analogs differed from one another either by removal of Leu19 from this hybrid analog, replacement of Leu19 with Gly19 or having a carboxyl terminus more closely related to salmon calcitonin. It had been found that replacement of Leu19 with Gly19 caused a marked reduction in the hypocalemic activity of the analog. The ability of the analogs to form helical structures in the presence of dimyristoylphosphatidylglycerol as well as their ability to lower the enthalpy of the calorimetric phase transition of this phospholipid correlates well with the hypocalcemic potency of the peptide. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004