Biomedical subjects

R M Epand

Publications and source records attributed to R M Epand.

At least 55 records · Page 3Linked to original sources

PubMed1999

Diversity of antimicrobial peptides and their mechanisms of action.

Antimicrobial peptides encompass a wide variety of structural motifs. Many peptides have alpha-helical structures. The majority of these peptides are cationic and amphipathic but there are also hydrophobic alpha-helical peptides which possess antimicrobial activity. In addition, some beta-sheet peptides have antimicrobial activity and even antimicrobial alpha-helical peptides which have been modified to possess a beta-structure retain part of their antimicrobial activity. There are also antimicrobial peptides which are rich in a certain specific amino acid such as Trp or His. In addition, antimicrobial peptides exist with thio-ether rings, which are lipopeptides or which have macrocyclic Cys knots. In spite of the structural diversity, a common feature of the cationic antimicrobial peptides is that they all have an amphipathic structure which allows them to bind to the membrane interface. Indeed, most antimicrobial peptides interact with membranes and may be cytotoxic as a result of disturbance of the bacterial inner or outer membranes. Alternatively, a necessary but not sufficient property of these peptides may be to be able to pass through the membrane to reach a target inside the cell. The interaction of these peptides with biological membranes is not just a function of the peptide but is also modulated by the lipid components of the membrane. It is not likely that this diverse group of peptides has a single mechanism of action, but interaction of the peptides with membranes is an important requirement for most, if not all, antimicrobial peptides.

Amino Acids↗

PubMed1999

The ectodomain of HA2 of influenza virus promotes rapid pH dependent membrane fusion.

To better understand the roles of different regions of influenza hemagglutinin in membrane fusion, we have studied the fusion properties of large unilamellar vesicles in the presence of constructs comprising the 127 amino acid ectodomain of the HA2 fragment (FHA2) as well as mutated forms of FHA2 containing single amino acid substitutions, the 95 amino acid truncated form of FHA2 lacking the N-terminal fusion peptide (SHA2), the 20 amino acid N-terminal fusion peptide and the ten amino acid peptide corresponding to the kinked loop region of FHA2. The 100 nm liposomes were made from dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine and cholesterol in equimolar ratio. At pH 5 a high rate of lipid mixing was observed with FHA2 present, even at very low molar concentrations, whereas much lower rates were observed using the shorter constructs: SHA2, the fusion peptide, and the loop peptide. Concentrations of FHA2 which promoted extensive lipid mixing also induced leakage of aqueous contents. Marked effects of FHA2 were also observed with liposomes of egg phosphatidylcholine. All of the changes observed with the liposomes were highly pH-dependent, with only negligible changes occurring at pH 7. The results demonstrate the potent action of FHA2 in promoting lipid mixing and demonstrate the contribution of other regions of the ectodomain of FHA2, in addition to the fusion peptide, to the mechanism of acceleration of membrane fusion. The results also indicate that the pH dependence of fusion is not due solely to changes in the interactions between the HA1 and HA2 subunits. Thus, the "spring loaded energy" is not required to bring about the apposition of the two membranes, considering that FHA2 is already in its thermostable conformation. The acidic amino acid residues in the kinked loop region appear to play a particularly important role in the pH-dependent fusion process as demonstrated by the marked loss of lipid mixing activity of mutant forms of FHA2.

Amino Acid Sequence↗

PubMed1999

Glucose-induced thermal stabilization of the native conformation of GLUT 1.

The glucose transporter, GLUT 1, was purified from erythrocyte membranes and incorporated into vesicles of erythrocyte lipids. These protein-containing vesicles were studied with differential scanning calorimetry. It was found that the protein underwent an irreversible denaturation at 68.5 +/- 0.2 degreesC (at a scan rate of 0.25 degreesC/min) which was shifted to 72.6 +/- 0.2 degreesC in the presence of 500 mM D-glucose, while 500 mM L-glucose or 10 microM cytochalasin B did not produce a significant shift. The calorimetric enthalpy was found to be 150 kcal/mol, independent of the presence of D-glucose. On a weight basis this value is lower than that for soluble proteins, but it is comparable to values obtained with other integral membrane proteins. The van't Hoff enthalpy is similar to the calorimetric enthalpy, within the experimental error, indicating that the transition is not likely to be cooperative. The activation energy is estimated from both the scan rate dependence of the transition temperature and from the shape of the DSC curve. The presence of 500 mM D-glucose slightly decreases the activation energy. It is concluded that the shift to a higher denaturation transition temperature in the presence of D-glucose is not a result of increased kinetic stability of GLUT 1.

Calorimetry, Differential Scanning↗

PubMed1999

Re-analysis of magic angle spinning nuclear magnetic resonance determination of interlamellar waters in lipid bilayer dispersions.

A recent method to obtain the number of water molecules of hydration of multilamellar lipid vesicles using magic angle spinning nuclear magnetic resonance has been re-examined. The previous interpretation divided the water into bulk and interlamellar water and ignored water in defects (lakes) that are intrinsic to multilamellar lipid vesicles; the result was inconsistent with x-ray results for the lipid DOPC. The new interpretation takes advantage of the reduction of lake water with increased spinning and it uses osmotic pressure measurements to determine the loss of interlamellar water. The new result for DOPC from magic angle spinning is consistent with x-ray results.

Centrifugation↗

PubMed1999

Studies of phospholipid hydration by high-resolution magic-angle spinning nuclear magnetic resonance.

A sample preparation method using spherical glass ampoules has been used to achieve 1.5-Hz resolution in 1H magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of aqueous multilamellar dispersions of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), serving to differentiate between slowly exchanging interlamellar and bulk water and to reveal new molecular-level information about hydration phenomena in these model biological membranes. The average numbers of interlamellar water molecules in multilamellar vesicles (MLVs) of DOPC and POPC were found to be 37.5 +/- 1 and 37.2 +/- 1, respectively, at a spinning speed of 3 kHz. Even at speeds as high as 9 kHz, the number of interlamellar waters remained as high as 31, arguing against dehydration effects for DOPC and POPC. Both homonuclear and heteronuclear nuclear Overhauser enhancement spectroscopy (NOESY and HOESY) were used to establish the location of water near the headgroup of a PC bilayer. 1H NMR comparisons of DOPC with a lipid that can hydrogen bond (monomethyldioleoylphosphatidylethanolamine, MeDOPE) showed the following trends: 1) the interlamellar water resonance was shifted to lower frequency for DOPC but to higher frequency for MeDOPE, 2) the chemical shift variation with temperature for interlamellar water was less than that of bulk water for MeDOPE MLVs, 3) water exchange between the two lipids was rapid on the NMR time scale if they were mixed in the same bilayer, 4) water exchange was slow if they were present in separate MLVs, and 5) exchange between bulk and interlamellar water was found by two-dimensional exchange experiments to be slow, and the exchange rate should be less than 157 Hz. These results illustrate the utility of ultra-high-resolution 1H MAS NMR for determining the nature and extent of lipid hydration as well as the arrangement of nuclei at the membrane/water interface.

Biophysical Phenomena↗

PubMed1999

A "release" protocol for isothermal titration calorimetry.

Isothermal titration calorimetry (ITC) has become a standard method for investigating the binding of ligands to receptor molecules or the partitioning of solutes between water and lipid vesicles. Accordingly, solutes are mixed with membranes (or ligands with receptors), and the subsequent heats of incorporation (or binding) are measured. In this paper we derive a general formula for modeling ITC titration heats in both binding and partitioning systems that allows for the modeling of the classic incorporation or binding protocols, as well as of new protocols assessing the release of solute from previously solute-loaded vesicles (or the dissociation of ligand/receptor complexes) upon dilution. One major advantage of a simultaneous application of the incorporation/binding and release protocols is that it allows for the determination of whether a ligand is able to access the vesicle interior within the time scale of the ITC experiment. This information cannot be obtained from a classical partitioning experiment, but it must be known to determine the partition coefficient (or binding constant and stochiometry) and the transfer enthalpy. The approach is presented using the partitioning of the nonionic detergent C12EO7 to palmitoyloleoylphosphatidylcholine vesicles. The release protocol could also be advantageous in the case of receptors that are more stable in the ligand-saturated rather than the ligand-depleted state.

Biophysical Phenomena↗

PubMed1999

Fluorescent probes used to monitor membrane interfacial polarity.

The polarity of the interface between a lipid bilayer membrane and bulk water is an important physical parameter of the membrane. It is likely that several membrane-dependent biological functions are modulated by this property. However, interfacial polarity can be difficult to define because of an imprecise knowledge of the molecular nature of the interface. Nevertheless, attempts have been made to measure this quantity with the use of fluorescent probes which are sensitive to the solvent polarity. Often, however, other factors, such as the rate of solvent relaxation must be known in order to interpret the fluorescent properties in terms of the dielectric constant. In addition, the spatial orientation and location of the fluorophore are often not known precisely. Nevertheless, there have been successful efforts to gain a more accurate knowledge of this aspect of membrane physical properties and its relationship to biological phenomena is discussed.

Cell Membrane↗

PubMed1999

Relationship of membrane sidedness to the effects of the lipophosphoglycan of Leishmania donovani on the fusion of influenza virus.

Cells expressing the influenza hemagglutinin protein were fused to planar lipid bilayers containing the viral receptor GD1a at pH 5.0. An amphiphile known to alter membrane properties is lipophosphoglycan (LPG). This glycoconjugate was added from aqueous solution to either the cis or the trans monolayer to examine its effects on the fusion process. LPG markedly inhibited the formation of fusion pores when present in the cis monolayer but LPG in the trans monolayer had no effect on the parameters of pore formation or on the properties of the pores. The N-terminal segment of the HA2 subunit of the influenza hemagglutinin protein is important for membrane fusion. The effect of LPG on the conformation and membrane insertion of a synthetic 20-amino-acid peptide, corresponding to the influenza fusion peptide, was examined at pH 5.0 by attenuated total reflection Fourier transform infrared spectroscopy and by the fluorescence properties of the Trp residues of this peptide. It was found that cis LPG did not prevent insertion of the peptide into the membrane but it did alter the conformation of the membrane-inserted peptide from alpha-helix to beta-structure. The beta-structure was oriented along the bilayer normal. The effect of cis LPG on the conformation of the fusion peptide probably contributes to the observed inhibition of pore formation and lipid mixing. In contrast, trans LPG has no effect on the conformation or angle of membrane insertion of the peptide, nor does it affect pore formation by HA-expressing cells. The ineffectiveness of trans LPG, despite it having strong positive curvature-promoting properties, may be a consequence of the size of this amphiphile being too large to enter a fusion pore.

3T3 Cells↗

PubMed1999

The antimicrobial peptide trichogin and its interaction with phospholipid membranes.

The interaction of the antimicrobial peptide trichogin GA IV with phospholipid bilayers has been studied. A series of analogs of trichogin was synthesized in which the nitroxide spin label, 4-amino-4-carboxy-2,2,6,6-tetramethylpiperidino-1-oxyl (TOAC), replaced one of the three alpha-aminoisobutyric acid (Aib) residues in the sequence. These modified peptides were used to assess the location of different residues of the peptide in a phospholipid bilayer composed of egg phosphatidylcholine containing 0.4 mol% of a fluorescently labelled phospholipid. We demonstrate that the substitution of Aib residues with TOAC does not alter the manner in which the peptide affects membrane curvature or induces vesicle leakage. The proximity of the nitroxide group on the peptide to the 4,4-difluoro-4-bora-3a,4a-diaza-S-indacene (BODIPY) fluorophore attached to the phospholipid was estimated from the extent of quenching of the fluorescence. By this criterion it was concluded that the peptide penetrates into the bilayer and that Aib4 is the most deeply inserted of the Aib residues. The results suggest that the helix axis of the peptide is oriented along the plane of the membrane. All of the peptides were shown to raise the bilayer to the hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine, indicating that they promote positive membrane curvature. This is a property observed with peptides that do not penetrate deeply into the bilayer or are oriented along the bilayer normal. We also demonstrate trichogin-promoted leakage of the aqueous contents of liposomes. These results indicate that the peptides cause bilayer destabilization. The extent of leakage induced by trichogin is very sensitive to the peptide to lipid ratio over a narrow range.

Amino Acid Sequence↗

PubMed1998

Structural properties of the putative fusion peptide of fertilin, a protein active in sperm-egg fusion, upon interaction with the lipid bilayer.

We recently demonstrated that a peptide representing the putative fusion domain of fertilin, a surface membrane protein of sperm involved in sperm-egg fusion, induces fusion of large unilamellar vesicles containing negatively charged lipids [Martin, I., and Ruysschaert, J. M. (1997) FEBS Lett. 405, 351-355]. In the present work, we demonstrate that increasing the concentration in negatively charged lipids strongly enhances the binding of the fertilin fusion peptide to the membrane, suggesting that electrostatic attractions play a crucial role in the binding process. While no significant change of the secondary structure content is observed by increasing the amounts of negatively charged lipids in the bilayer, the orientation of the alpha-helix changes from a parallel to an oblique orientation in the membrane. This topological change is confirmed by amide II hydrogen/deuterium exchange measurements that monitor the accessibility of the peptide to the water medium. Differential scanning calorimetry data also suggest that the fertilin fusion peptide lowers the bilayer to hexagonal phase transition temperature of model membranes composed of mixtures of dipalmitoleoylphosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylserine and therefore promotes negative curvature in lipid vesicles. A comparison of the biophysical properties and the membrane-perturbing activities of fertilin and of viral fusion peptides is discussed in terms of sperm-egg fusion and virus cell fusion.

ADAM Proteins↗

PubMed1998

Lipophosphoglycan of Leishmania donovani inhibits lipid vesicle fusion induced by the N-terminal extremity of viral fusogenic simian immunodeficiency virus protein.

Lipophosphoglycan (LPG), the major glycoconjugate of Leishmania parasites, was recently shown to be a potent inhibitor of viral infection. The mechanism by which this natural membrane amphiphile compound inhibits membrane fusion was investigated in this study using a simple model membrane system and a synthetic peptide corresponding to the fusion peptide of simian immunodeficiency virus (SIV). At low concentration (< 10 microM), LPG inhibits SIV-induced lipid mixing of large unilamellar vesicles composed of an equimolar mixture of egg phosphatidylcholine and egg phosphatidylethanolamine. Importantly, this inhibition was observed regardless of which LPG was inserted in the inner monolayer, the outer monolayer or both sides of the membrane, suggesting that the inner monolayer plays a determining role in membrane fusion. Fourier transform infrared spectroscopy revealed that LPG induced a conformational change of SIV fusion peptide without affecting its capacity to interact with the lipid membrane. This structural change was shown not to depend on the LPG localization and was observed even when LPG was exclusively associated to the inner lipid membrane.

Animals↗

PubMed1998

Lipid polymorphism and protein-lipid interactions.

Non-lamellar-forming lipids play an important role in determining the physical properties of membranes. They affect the activity of membrane proteins and peptides. In addition, peptides which lyse membranes as well as those which promote membrane fusion facilitate the formation of non-lamellar phases, either micelles, cubic or hexagonal phases. The relationship of these diverse effects on membrane curvature is discussed in relation to the function of certain peptides and proteins. Specific examples of ionophoric peptides, cytotoxic peptides and viral fusion peptides are given. In addition, we compare the modulation of the rate of photoisomerisation of an integral membrane protein, rhodopsin, by non-lamellar-forming lipids with the effects of these lipids on an amphitropic protein, protein kinase C. Among these diverse systems it is frequently observed that the modulation of biological activity can be described in terms of the effect of the peptide or protein on the relative stability of lamellar and non-lamellar structures.

Animals↗

PubMed1998

Fusion of Sendai virus and individual host cells and inhibition of fusion by lipophosphoglycan measured with image correlation spectroscopy.

Fusion between Sendai virus (SV) and individual host cells was investigated with confocal laser scanning microscopy (CLSM) and image correlation spectroscopy (ICS). SV was labeled with the fluorescent probe 7-octadecylamino-4-nitrobenz-2-oxa-1,3-diazole (NBD-NH-C18) and was allowed to bind to host cells (HEp-2, BALB-3T3) at 4 degrees C. The effect of lipophosphoglycan (LPG), isolated from Leishmania donovani, on virus fusion was investigated by incorporation of LPG (0, 5, 10 or 20 microM) into the host cell membrane (HEp-2) before addition of SV. LPG did not affect the number of SV bound per cell. After incubation at 37 degrees C for 15 min without LPG, CLSM revealed a redistribution of NBD-NH-C18 from the SV envelope to the host cell membrane and an increase in average fluorescence intensity, indicating dequenching. ICS analysis of images obtained after incubation at 37 degrees C showed an increased mean cluster density to 260% of the value at 4 degrees C, reflecting the disappearance of labeled SV from the cell surface and diffusion of NBD-NH-C18 into the host cell membrane. Preincubation of the cells with LPG inhibited the temperature-induced redistribution and dequenching of NBD-NH-C18 in a concentration-dependent manner, with a total inhibition of fusion at 20 microM LPG. Together, the results demonstrate that CLSM combined with ICS is a powerful tool for studies of fusion of enveloped viruses with individual host cells and that LPG inhibits the fusion process at or before the hemifusion (lipid mixing) stage of SV interaction with cells.

4-Chloro-7-nitrobenzofurazan↗

PubMed1998

The chirality of phosphatidylserine and the activation of protein kinase C.

The properties of phosphatidyl-L-serine (L-PS) and phosphatidyl-D-serine (D-PS) were compared. The two forms of PS have similar but nonidentical L to L phase transition temperatures. Mixtures of phosphatidylserine with phosphatidylethanolamine and cholesterol (molar ratio 1:1:2) show polymorphic behavior at higher temperatures and in the presence of Ca2+. Mixtures with L-PS undergo conversion to nonlamellar phases at lower temperatures than do similar mixtures with D-PS. The aggregation of vesicles upon addition of histones is greater for L-PS than for D-PS. With fluorescence digital imaging microscopy we could show differences in the extent of formation of histone-induced domains enriched in PS or in diacylglycerol. The most enriched domains were induced with histone in membranes containing L-PS. The MARCKS peptide showed no differences in domain formation between L-PS and D-PS. The maximal activity of protein kinase C was greater in the presence of L-PS when histone, which could form more enriched domains, was the substrate. However, with a MARCKS peptide substrate, which formed domains of equal enrichment with L-PS and D-PS, the maximal activity of protein kinase C was the same with D-PS and with L-PS. These observations demonstrate that L-PS and D-PS have different physical properties. These differences likely contribute to the greater ability of L-PS to activate protein kinase C.

Calorimetry, Differential Scanning↗

PubMed1998

Relationship of membrane curvature to the formation of pores by magainin 2.

Magainin 2, an antimicrobial peptide from the Xenopus skin, kills bacteria by permeabilizing the cell membranes. We have proposed that the peptide preferentially interacts with acidic phospholipids to form a peptide-lipid supramolecular complex pore, which allows mutually coupled transbilayer traffic of ions, lipids, and peptides, thus simultaneously dissipating transmembrane potential and lipid asymmetry [Matsuzaki, K., Murase, O., Fujii, N., and Miyajima, K. (1996) Biochemistry 35, 11361-11368]. In this paper, we examined the effect of membrane curvature strain on pore formation. Magainin effectively forms the pore only in phosphatidylglycerol bilayers at low peptide-to-lipid ratios, well below 1/100. In contrast, the permeabilization of phosphatidylserine, phosphatidic acid, or cardiolipin bilayers occurred at much higher peptide-to-lipid ratios (1/50 to 1/10) with some morphological change of the vesicles. The latter three classes of phospholipids are known to form hexagonal II structures under conditions of reduced interlipid electrostatic repulsions. Incorporation of phosphatidylethanolamine also inhibited the magainin-induced pore formation in the inhibitory order of dioleoylphosphatidylethanolamine > dielaidoylphosphatidylethanolamine. Addition of a small amount of palmitoyllysophosphatidylcholine enhanced the peptide-induced permeabilization of phosphatidylglycerol bilayers. Magainin greatly raised the bilayer to hexagonal II phase transition temperature of dipalmitoleoylphosphatidylethanolamine. These results suggest that the peptide imposes positive curvature strain, facilitating the formation of a torus-type pore, and that the presence of negative curvature-inducing lipids inhibits pore formation.

Amino Acid Sequence↗

PubMed1998

Evidence for a regulatory binding site for arginine-rich peptides on protein kinase C.

The peptides N-biotinyl-RRRCLRRL and N-biotinyl-RKRCLRRL covalently modify protein kinase C (PKC) through reaction of the Cys sulfhydryl group with the active site of the enzyme. The labeling of PKC occurs only in the presence of the cofactors phosphatidylserine, diacylglycerol, and Ca2+ but not in their absence. Low concentrations of the Arg-rich substrate, R4YGSR6Y greatly increase the extent of the reaction of these biotinylated peptides with PKC in the presence of lipid cofactors but in the absence of calcium. This effect can be observed at 50 nM R4YGSR6Y and suggests the presence of a high-affinity binding site for Arg-rich peptides which is separate from the active site but which enhances accessibility of the active site. The study also demonstrates the utility of the biotinylated peptides as active site labels which can detect the conformational change accompanying the activation of PKC.

Affinity Labels↗

PubMed1998

Properties of lipoamino acids incorporated into membrane bilayers.

Several lipoamino acids were synthesized in which palmitic acid was coupled with the alpha-amino group of an amino acid. These lipoamino acids were tested for their inhibitory action against Sendai virus fusion to liposomes composed of egg phosphatidylethanolamine and 5 mol% of the ganglioside GD1a. A commonly employed viral fusion assay based on the dilution of the fluorescent probe octadecylrhodamine (R18) exhibited an additional complication in the presence of Nalpha-palmitoyl tryptophan (palm-Trp). At higher mol fraction of palm-Trp it was observed that there was an increase in R18 quenching. Studies on the dependence of the emission wavelength of palm-Trp on excitation wavelength demonstrated that the presence of R18 alters the environment of the indole. The results illustrate one of the complexities of viral fusion assays using the R18 probe. Despite this complication it was possible to demonstrate that several of the lipoamino acids are effective at inhibiting the fusion of Sendai virus to liposomes as measured by the R18 assay. One of the most effective inhibitors of this process is palm-Trp which, at a concentration of 4 mol% in liposomes, markedly reduces the apparent rate of fusion. At pH 5.0 this amphiphile is also an inhibitor of Sendai virus fusion, indicating that the ionization of the carboxyl group of this amphiphile is not required for its antiviral activity. The inhibitory action of palm-Trp against Sendai virus was confirmed by demonstrating inhibition of Sendai-mediated cytopathic effects studied in tissue culture. A property associated with antiviral activity is the ability of amphiphiles to raise the bilayer to hexagonal phase transition temperature of dielaidoyl phosphatidylethanolamine. All of these lipoamino acids were found to possess this property, but a quantitative relationship with inhibition of viral fusion was not found.

Amino Acids↗

PubMed1998

Interfacial membrane properties modulate protein kinase C activation: role of the position of acyl chain unsaturation.

We studied the effects of the addition of a series of 1, 2-dioctadecenoyl-sn-glycerol-3-phosphoethanolamines to vesicles composed of 1-palmitoyl-2-oleoylphosphatidylserine and 1-palmitoyl-2-oleoylphosphatidylcholine on the activity and membrane binding of protein kinase C (PKC). The three phosphatidylethanolamines (PEs) were dipetroselinoyl-PE, dioleoyl-PE, and divaccenoyl-PE, which have double bonds in positions 6, 9, and 11, respectively. These lipids represent a group of structurally homologous compounds whose physical properties have been compared. We also used a fluorescent probe, 4-[(n-dodecylthio)methyl]-7-(N, N-dimethylamino)coumarin to measure the relative interfacial polarities of LUVs containing each of the three PEs. We find dipetroselinoyl-PE allows the least access of the fluorescent probe to the membrane. This is also the lipid that shows the lowest activation of PKC. The activity of PKC was found to correlate best with the interfacial properties of the three PEs rather than with the curvature energy of the membrane. The results show the sensitivity of the activity of PKC to small changes in lipid structure.

Animals↗