Biomedical subjects

Raquel F Epand

Publications and source records attributed to Raquel F Epand.

48 records · Page 3Linked to original sources

PubMed2003

Interactions of the antimicrobial beta-peptide beta-17 with phospholipid vesicles differ from membrane interactions of magainins.

We have studied the interaction of beta-17, a potent synthetic antimicrobial beta-peptide, with phospholipids. We find that unlike other antimicrobial peptides such as magainin II, beta-17 facilitates the formation of nonbilayer phases, indicating that the peptide promotes negative curvature. Studies of liposomal leakage also indicate a different mode of membrane interaction relative to magainin II, but both leakage and membrane binding show that beta-17, like magainin II, has strong affinity for membranes containing anionic lipids. This is likely to be an important factor contributing to the antimicrobial specificity of the beta-peptide.

Anti-Bacterial Agents↗

PubMed2002

Direct evidence for membrane pore formation by the apoptotic protein Bax.

Direct imaging of the interaction of the apoptotic protein, Bax, with membrane bilayers shows the presence of toroidal-shaped pores using atomic force microscopy. These pores are sufficiently large to allow passage of proteins from the intermitochondrial space. Both the perturbation of the membrane and the amount of protein bound to the bilayer are increased in the presence of calcium. The results from the imaging are consistent with leakage studies from liposomes of the same composition. The work shows that Bax by itself can form pores in membrane bilayers.

Apoptosis↗

PubMed2002

Membrane perturbations induced by the apoptotic Bax protein.

The apoptotic protein Bax, in oligomeric form, is effective in promoting both leakage and lipid mixing in liposomes composed of cardiolipin and phosphatidylethanolamine and/or phosphatidylcholine, upon the addition of calcium. In contrast, monomeric Bax is not active. At low concentrations at which caspase-8-cut Bid (tBid) alone has little effect on leakage, tBid augments the leakage caused by monomeric Bax. When solutions of oligomeric Bax are diluted to lower detergent concentrations than those required for Bax oligomerization, the protein is initially active in inducing liposomal leakage, indicating that the potency of the oligomeric form is not a consequence of being initially added to the liposomes in a high detergent concentration. However, in solutions of low detergent concentration, in the absence of liposomes, the oligomer gradually loses its lytic potency. This is accompanied by a loss of binding of bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulphonic acid), indicating the loss of exposed hydrophobic sites, as well as a loss of the ability of the protein to translocate to membranes. Membrane translocation was measured by an energy-transfer assay. It was demonstrated that membrane binding was greatly enhanced by oligomerization and by the presence of calcium. Thus the membrane-active form of Bax is unstable in the absence of detergent or lipid. In addition, we find that translocation to the membrane is enhanced by oligomerization as well as by the presence of high concentrations of calcium.

Apoptosis↗

PubMed2002

Position-dependent hydrophobicity of the antimicrobial magainin peptide affects the mode of peptide-lipid interactions and selective toxicity.

Cationic antimicrobial peptides are promising candidates as novel antibiotics of clinical usefulness. Magainin 2, a representative antimicrobial peptide isolated from the skin of the African clawed frog Xenopus leavis, electrostatically recognizes anionic lipids that are abundant in bacterial membranes, forming a peptide-lipid supramolecular complex pore, whereas the peptide does not effectively bind to zwitterionic phospholipids constituting the outer leaflets of mammalian cell membranes because of the low hydrophobicity of the peptide [Matsuzaki, K. (1999) Biochim. Biophys. Acta 1462, 1-10]. In this study, two magainin analogues with enhanced hydrophobicity, MG-H1 (GIKKFLHIIWKFIKAFVGEIMNS) and MG-H2 (IIKKFLHSIWKFGKAFVGEIMNI), with identical amino acid compositions were designed and interactions with lipid bilayers and biological activities were examined in comparison with those of MG (GIGKWLHSAKKFGKAFVGEIMNS = F5W-magainin 2). The apparent hydrophobicities and hydrophobic moments of MG-H1 and MG-H2, conventionally calculated assuming that all residues are involved in helix formation, were almost the same. MG-H2 behaved like MG except for greatly enhanced activity against zwitterionic membranes and erythrocytes. In contrast, despite a very similar calculated hydrophobicity, the observed hydrophobicity of MG-H1 was larger than that of MG-H2 because of a tendency toward helix fraying near the termini. Therefore, the physicochemical parameters of only the helical portion should be considered in characterizing peptide-lipid interactions, although this point was overlooked in most studies. Moreover, MG-H1 induced aggregation and/or fusion of negatively charged membranes. Furthermore, the peptide hydrophobicity was found to affect pore formation rate, pore size, and pore stability. These observations demonstrate that the hydrophobicity of the peptide also controls the mode of action and is dependent on the position of the hydrophobic amino acids in the peptide sequence.

Adult↗

PubMed2002

CTP:phosphocholine cytidylyltransferase and protein kinase C recognize different physical features of membranes: differential responses to an oxidized phosphatidylcholine.

Protein kinase C (PKC) and CTP:phosphocholine cytidylyltransferase (CT) are two examples of enzymes that are regulated by reversible binding to membranes, and this binding is influenced by membrane physical properties. CT activation by oxidized phosphatidylcholines was recently demonstrated and was linked to the acyl chain disordering effect of the oxidized species (Biochemistry 38, 15606). In this paper, we compare the responses of PKC and CT to an oxidized PC, and investigate the physical properties of lipid bilayers that modulate the activity of these enzymes. We show that 1-palmitoyl, 2-(11,15 dihydroxy) eicosatrienoyl PC (diOH-PAPC) caused less of an increase in the temperature of the lamellar to hexagonal II transition (T(H)) of an unsaturated PE, compared to its parent, PAPC. Using a polarity-sensitive interfacial probe, we also found evidence to suggest that this oxidized PC increases interfacial packing pressure. We found that whereas diOH-PAPC activates CT, it inhibits PKC relative to the parent PAPC. The activities of both CT and PKC are known to increase in the presence of non-lamellar forming lipids. The greater activating effect of diOH-PAPC compared with PAPC, is consistent with a stimulation of the activity of CT by negative curvature strain. However, this is not the case with PKC, for which we suggest that surface packing pressure is of prime importance.

Animals↗

PubMed2002

Thermal denaturation of influenza virus and its relationship to membrane fusion.

The X-31 strain of influenza virus was studied by differential scanning calorimetry (DSC), CD and SDS/PAGE analysis as a function of both temperature and pH. A bromelain-treated virus was also studied by these methods. The major transition observed in the intact virus was a result of the denaturation of the haemagglutinin (HA) protein. At pH 7.4, this transition was similar in the intact virus and the isolated HA, but was absent in the bromelain-treated virus. However, at pH 5 the denaturation temperature and enthalpy were both higher for HA in the virus than in the isolated protein, indicating that HA interacts with other molecular components in the intact virus. The transition observed by DSC occurs at a higher temperature than does the thermal transition observed by CD. The temperature of the CD transition coincides with the temperature at which the fusogenicity of the virus increases, and probably corresponds to the formation of an extended coiled-coil conformation. Analysis by SDS/PAGE at neutral pH under non-reducing conditions demonstrates a selective loss of the HA protein trimer, resulting in the formation of aggregates in the range of temperatures of 55 to 70 degrees C. In contrast, at acidic pH, the HA protein is largely in the monomeric form at 25 degrees C, and there is little change with temperature. There is thus a weakening of the quaternary structure of HA at acidic pH prior to heating. At the temperature at which the virus exhibits an increased fusogenicity at neutral pH, there is a loss of secondary structure and a beginning of a destabilization of the trimeric form of HA. This temperature is lower than that required for the major endothermic peak observed in DSC experiments. The results demonstrate that there is no kinetically trapped high-energy form of HA at neutral pH.

Bromelains↗

PubMed2002

The apoptotic protein tBid promotes leakage by altering membrane curvature.

The apoptotic protein tBid is effective in promoting both leakage and lipid mixing in liposomes composed of cardiolipin and phosphatidylcholine at a molar ratio of 1:2 in the presence of calcium. When half of the phosphatidylcholine component of these liposomes is replaced with phosphatidylethanolamine, a lipid that promotes negative membrane curvature, the rates of both leakage and lipid mixing caused by tBid are substantially increased. Replacement of cardiolipin with phosphatidylglycerol, a lipid that is structurally similar to cardiolipin but does not promote negative membrane curvature in the presence of calcium, prevents the tBid from promoting leakage. The promotion of leakage by tBid is also inhibited by several substances that promote positive membrane curvature, including lysophosphatidylcholine, tritrpticin, a potent antimicrobial peptide, and cyclosporin A, a known inhibitor of cytochrome c release from mitochondria. We directly measured the effect of tBid on membrane curvature by (31)P NMR. We found that tBid promotes the formation of highly curved non-lamellar phases. All of these data are consistent with the hypothesis that tBid promotes negative curvature, and as a result it destabilizes bilayer membranes. Bcl-X(L) inhibits leakage and lipid mixing induced by tBid. Bcl-X(L) is anti-apoptotic. It reduces the promotion of non-bilayer phases by tBid, although by itself Bcl-X(L) is capable of promoting their formation. Bcl-X(L) has little effect on liposomal integrity. Our results suggest that the anti-apoptotic activity of Bcl-X(L) is not a consequence of its interaction with membranes, but rather with other proteins, such as tBid.

Apoptosis↗

PubMed2002

Sendai virus N-terminal fusion peptide consists of two similar repeats, both of which contribute to membrane fusion.

The N-terminal fusion peptide of Sendai virus F1 envelope glycoprotein is a stretch of 14 amino acids, most of which are hydrophobic. Following this region, we detected a segment of 11 residues that are strikingly similar to the N-terminal fusion peptide. We found that, when anchored to the membrane by palmitoylation of its N-terminus, this segment (WT-palm-19-33) induces membrane fusion of large unilamellar liposomes to almost the same extent as a segment that includes the N-terminal fusion peptide. The activity of WT-palm-19-33 was dependent on its specific sequence, as a palmitoylated peptide with the same amino-acid composition but a scrambled sequence was inactive. Interestingly, two mutations (G7A and G12A) known to increase F1- induced cell-cell fusion, also increased the homology between the N-terminal fusion peptide and WT-palm-19-33. The role of the amino-acid sequence on the fusogenicity, secondary structure, and mechanism of membrane fusion was analyzed by comparing a peptide comprising both homologous segments (WT 1-33), a G12A mutant (G12A 1-33), a G7A-G12A double mutant (G7A-G12A 1-33), and a peptide with a scrambled sequence (SC 1-33). Based on these experiments, we postulate that replacement of Gly 7 and Gly12 by Ala increases the alpha helical content of the N-terminal region, with a concomitant increase in its fusogenic activity. Furthermore, the dissimilar abilities of the different peptides to induce membrane negative curvature as well as to promote isotropic 31P NMR signals, suggest that these mutations might also alter the extent of membrane penetration of the 33-residue peptide. Interestingly, our results serve to explain the effect of the G7A and G12A mutations on the fusogenic activity of the parent F1 protein in vivo.

Calorimetry, Differential Scanning↗

PubMed2002

Towards a structure-function analysis of bovine lactoferricin and related tryptophan- and arginine-containing peptides.

The iron-binding protein lactoferrin is a multifunctional protein that has antibacterial, antifungal, antiviral, antitumour, anti-inflammatory, and immunoregulatory properties. All of these additional properties appear to be related to its highly basic N-terminal region. This part of the protein can be released in the stomach by pepsin cleavage at acid pH. The 25-residue antimicrobial peptide that is released is called lactoferricin. In this work, we review our knowledge about the structure of the peptide and attempt to relate this to its many functions. Microcalorimetry and fluorescence spectroscopy data regarding the interaction of the peptide with model membranes show that binding to net negatively charged bacterial and cancer cell membranes is preferred over neutral eukaryotic membranes. Binding of the peptide destabilizes the regular membrane bilayer structure. Residues that are of particular importance for the activity of lactoferricin are tryptophan and arginine. These two amino acids are also prevalent in "penetratins", which are regions of proteins or synthetic peptides that can spontaneously cross membranes and in short hexapeptide antimicrobial peptides derived through combinatorial chemistry. While the antimicrobial, antifungal, antitumour, and antiviral properties of lactoferricin can be related to the Trp/Arg-rich portion of the peptide, we suggest that the anti-inflammatory and immunomodulating properties are more related to a positively charged region of the molecule, which, like the alpha- and beta-defensins, may act as a chemokine. Few small peptides are involved in as wide a range of host defense functions as bovine and human lactoferricin.

Amino Acid Sequence↗

PubMed2002

Tryptophan-rich antimicrobial peptides: comparative properties and membrane interactions.

The interaction of several tryptophan (Trp)-rich cationic antimicrobial peptides with membranes was investigated. These peptides included tritrpticin, indolicidin, lactoferricin B (Lfcin B), and a shorter fragment of lactoferricin (LfcinB4-9). The average environment of the Trp residues of these peptides was assessed from their fluorescence properties, both the wavelength of maximal emission as well as the red edge effect. The insertion of the peptides into vesicles of differing composition was examined using quenching of the Trp fluorescence, with both soluble acrylamide and nitroxide-labelled phospholipids as well as by chemical modification of the Trp residues with N-bromosuccinimide. The results were consistent with the Trp side chains positioned mostly near the membrane-water interface. The extent of burial of the Trp side chains appears to be greater in vesicles containing phospholipids with the anionic phosphatidylglycerol headgroup. Leakage of the aqueous contents of liposomes was also measured using the 8-aminonaphthalene-1,3,6-trisulfonic acid--p-xylene-bis-pyridinium bromide assay. Tritrpticin, which demonstrated the greatest red edge shift, also displayed the largest amount of leakage from liposomes. Taken together, the results illustrate that cationic Trp-rich antimicrobial peptides preferentially disrupt large unilamellar vesicles with a net negative charge following their insertion into the interfacial region of the phospholipid bilayer.

Anti-Infective Agents↗

PubMedDate not supplied

Trichogin: a paradigm for lipopeptaibols.

Lipopeptaibols are members of a novel family of naturally occurring, short peptides with antimicrobial activity, characterized by a lipophilic acyl chain at the N-terminus, a high content of turn/helix inducing alpha-aminoisobutyric acid and a 1,2-amino alcohol at the C-terminus. Using solution methods, the prototypical lipopeptaibol trichogin GA IV and a large series of appropriately designed analogues were synthesized, which allow: (i) determination of the minimal lipid chain and peptide main-chain lengths for the onset of membrane activity, and (ii) exploitation of a number of physico-chemical techniques aimed at assessing the trichogin preferred conformation under a variety of conditions and at investigating its mechanism of interaction with the phospholipid membranes.

Amino Acid Sequence↗

PubMedDate not supplied

Bacterial species selective toxicity of two isomeric alpha/beta-peptides: role of membrane lipids.

We have studied how membrane interactions of two synthetic cationic antimicrobial peptides with alternating alpha- and beta-amino acid residues ("alpha/beta-peptides") impact toxicity to different prokaryotes. Electron microscopic examination of thin sections of Escherichia coli and of Bacillus subtilis exposed to these two alpha/beta-peptides reveals different structural changes in the membranes of these bacteria. These two peptides also have very different effects on the morphology of liposomes composed of phosphatidylethanolamine and phosphatidylglycerol in a 2:1 molar ratio. Freeze fracture electron microscopy indicates that with this lipid mixture, alpha/beta-peptide I induces the formation of a sponge phase. 31P NMR and X-ray diffraction are consistent with this conclusion. In contrast, with alpha/beta-peptide II and this same lipid mixture, a lamellar phase is maintained, but with a drastically reduced d-spacing. alpha/beta-Peptide II is more lytic to liposomes composed of these lipids than is I. These findings are consistent with the greater toxicity of alpha/beta-peptide II, relative to alpha/beta-peptide I, to E. coli, a bacterium having a high content of phosphatidylethanolamine. In contrast, both alpha/beta-peptides display similar toxicity toward B. subtilis, in accord with the greater anionic lipid composition in its membrane. This work shows that variations in the selectivity of these peptidic antimicrobial peptides toward different strains of bacteria can be partly determined by the lipid composition of the bacterial cell membrane.

Anti-Infective Agents↗