Biomedical subjects

Y Shai

Publications and source records attributed to Y Shai.

At least 37 records · Page 2Linked to original sources

PubMed2000

Inhibition of HIV-1 entry before gp41 folds into its fusion-active conformation.

HIV-1 entry into its host cell is modulated by its transmembrane envelope glycoprotein (gp41). The core of the activated conformation of gp41 consists of a trimer of heterodimers comprising a leucine/isoleucine zipper sequence (represented here by the synthetic peptide N36 or by the longer N51 peptide) and a C-terminal highly conserved region (represented here by C34). A correlation was found between the action of DP178, which is a potent inhibitor of HIV-1 entry into its host cell, and its ability to interact with the leucine/isoleucine zipper sequence. This correlation was further tested and confirmed by circular dichroism spectroscopy. We found that whereas DP178 perturbs the partial alpha-helix nature of peptides corresponding to the leucine/isoleucine zipper sequence (N36 or N51), it cannot perturb the trimer of heterodimers conformation, modeled by the complex of N36 or N51 with C34. Therefore, we suggest that the already formed trimer of heterodimers is not the target of inhibition by DP178. Our results are consistent with a model in which DP178 acquires its inhibitory activity by binding to an earlier intermediate of gp41, in which the N and C peptide regions are not yet associated, thus allowing DP178 to bind to the leucine/isoleucine zipper sequence and consequently to inhibit transition to the fusion-active conformation.

Amino Acid Sequence↗

PubMed2000

Functional domains within fusion proteins: prospectives for development of peptide inhibitors of viral cell fusion.

The entry of enveloped viruses into host cells is accomplished by fusion of the viral envelope and target plasma membrane and is mediated by fusion proteins. Recently, several functional domains within fusion proteins from different viral families were identified. Some are directly involved in conformational changes after receptor binding, as suggested by the recent release of crystallographically determined structures of a highly stable core structure of the fusion proteins in the absence of membranes. However, in the presence of membranes, this core binds strongly to the membrane's surface and dissociates therein. Other regions, besides the N-terminal fusion peptide, which include the core region and an internal fusion peptide in paramyxoviruses, are directly involved in the actual membrane fusion event, suggesting an "umbrella" like model for the membrane induced conformational change of fusion proteins. Peptides resembling these regions have been shown to have specific antiviral activity, presumably because they interfere with the corresponding domains within the viruses. Overall, these studies shed light into the molecular mechanism of membrane fusion induced by envelope glycoproteins and suggest that fusion proteins from different viral families share common structural and functional motifs.

Antiviral Agents↗

PubMed2000

Rapid entry of bitter and sweet tastants into liposomes and taste cells: implications for signal transduction.

Some amphipathic bitter tastants and non-sugar sweeteners are direct activators of G proteins and stimulate transduction pathways in cells not related to taste. We demonstrate that the amphipathic bitter tastants quinine and cyclo(Leu-Trp) and the non-sugar sweetener saccharin translocate rapidly through multilamellar liposomes. Furthermore, when rat circumvallate (CV) taste buds were incubated with the above tastants for 30 s, their intracellular concentrations increased by 3.5- to 7-fold relative to their extracellular concentrations. The time course of this dramatic accumulation was also monitored in situ in rat single CV taste buds under a confocal laser-scanning microscope. Tastants were clearly localized to the taste cell cytosol. It is proposed that, due to their rapid permeation into taste cells, these amphipathic tastants may be available for activation of signal transduction components (e. g., G proteins) directly within the time course of taste sensation. Such activation may occur in addition to the action of these tastants on putative G protein-coupled receptors. This phenomenon may be related to the slow taste onset and lingering aftertaste typically produced by many bitter tastants and non-sugar sweeteners.

Animals↗

PubMed1999

Structure and organization of hemolytic and nonhemolytic diastereomers of antimicrobial peptides in membranes.

Recently, we reported on a new group of diastereomers of short-model peptides (12 amino acids long) composed of leucine and lysine with varying ratios, possessing several properties that make them potentially better than native or de novo-designed all L-amino acid antimicrobial peptides. Preliminary studies have revealed that modulating the hydrophobicity and positive charges of these diastereomers is sufficient to confer antibacterial activity and cell selectivity. However, the relationship between their biological function, structure, and mode of action was not investigated. Here we synthesized and investigated three types of linear model diastereomers (12 amino acids long) with varying lysine:leucine (or tryptophan) ratios (i.e., K(3)L(8)W, K(5)L(6)W, and K(7)L(4)W), which confer different levels of lytic activities. For each K:L ratio, tryptophan was introduced in the middle or the N- or C-terminus of the peptides, as an intrinsic fluorescent probe. Only the hemolytic peptide K(3)L(8)W binds to both negatively charged and zwitterionic phospholipid membranes. K(5)L(6)W and K(7)L(4)W bind similarly, but only to negatively charged membranes, despite the fact that K(5)L(6)W is substantially more lytic to bacteria than K(7)L(4)W. Interestingly, although K(3)L(8)W contains 33% D-amino acids, ATR-FTIR spectroscopy revealed a structure of approximately 90% alpha-helix in both types of membranes. In addition, K(5)L(6)W contains approximately 40% 3(10)-helix and K(7)L(4)W is predominantly a random coil in membranes. Polarized ATR-FTIR and tryptophan-quenching experiments, using brominated phospholipids, revealed a similar depth of penetration and an orientation that was parallel to the membrane surface for all the peptides, but with K(3)L(8)W affecting the lipid order more than the others. The results provide insight into the mode of action of this group of diastereomeric peptides, and the effect of hydrophobicity and positive charges on their membrane structure, function, and cell selectivity. Moreover, this research should assist in the development of suitable diastereomeric peptide antibiotics for therapeutic use that would overcome the problem the increasing resistance of bacteria to conventional antibiotics.

Acinetobacter calcoaceticus↗

PubMed1999

Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides.

Permeation of the cell membrane leading to cell death is a mechanism used by a large number of membrane-lytic peptides. Some are linear, mostly helical, and others contain one or more disulfide bonds forming beta-sheet or both beta-sheet and alpha-helix structures. They are all soluble in solution but when they reach the target membrane, conformational changes occur which let them associate with and lyse the membrane. Some lytic peptides are not cell-selective and lyse different microorganisms and normal mammalian cells, while others are specific to either type of cells. Despite extensive studies, the mode of action of membrane-lytic peptides is not fully understood and the basis for their selectivity towards specific target cells is not known. Many studies have shown that peptide-lipid interactions leading to membrane permeation play a major role in their activity. Membrane permeation by amphipathic alpha-helical peptides has been proposed to occur via one of two general mechanisms: (i) transmembrane pore formation via a 'barrel-stave' mechanism; and (ii) membrane destruction/solubilization via a 'carpet' mechanism. This review, which is focused on the different stages of membrane permeation induced by representatives of amphipathic alpha-helical antimicrobial and cell non-selective lytic peptides distinguishes between the 'carpet' mechanism, which holds for antimicrobial peptides versus the 'barrel-stave' mechanism, which holds for cell non-selective lytic peptides.

Amphibian Proteins↗

PubMed1999

2D-NMR and ATR-FTIR study of the structure of a cell-selective diastereomer of melittin and its orientation in phospholipids.

Melittin, a 26 residue, non-cell-selective cytolytic peptide, is the major component of the venom of the honey bee Apis mellifera. In a previous study, a diastereomer ([D]-V(5,8),I(17),K(21)-melittin, D-amino acids at positions V(5,8),I(17),K(21)) of melittin was synthesized and its function was investigated [Oren, Z., and Shai, Y. (1997) Biochemistry 36, 1826-1835]. [D]-V(5,8),I(17),K(21)-melittin lost its cytotoxic effects on mammalian cells; however, it retained antibacterial activity. Furthermore, [D]-V(5,8),I(17),K(21)-melittin binds strongly and destabilizes only negatively charged phospholipid vesicles, in contrast to native melittin, which binds strongly also zwitterionic phospholipids. To understand the differences in the properties of melittin and its diastereomer, 2D-NMR experiments were carried out with [D]-V(5,8),I(17),K(21)-melittin, and polarized attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy experiments were done with both melittin and [D]-V(5,8), I(17),K(21)-melittin. The structure of the diastereomer was characterized by NMR in water, as well as in three different membrane-mimicking environment, 40% 2,2,2-trifluoroethanol (TFE)/water, methanol, and dodecylphosphocholine/phosphatidylglycerol (DPC/DMPG) micelles. The NMR data revealed an amphipathic alpha-helix only in the C-terminal region of the diastereomer in TFE/water and methanol solutions and in DPC/DMPG micelles. ATR-FTIR experiments revealed that melittin and [D]-V(5,8),I(17),K(21)-melittin are oriented parallel to the membrane surface. This study indicates the role of secondary structure formation in selective cytolytic activity of [D]-V(5,8), I(17),K(21)-melittin. While the N-terminal helical structure is not required for the cytolytic activity toward negatively charged membranes and bacterial cells, it appears to be a crucial structural element for binding and insertion into zwitterionic membranes and for hemolytic activity.

Amino Acid Sequence↗

PubMed1999

Direct evidence that the N-terminal heptad repeat of Sendai virus fusion protein participates in membrane fusion.

Recent studies have demonstrated the importance of heptad repeat regions within envelope proteins of viruses in mediating conformational changes at various stages of viral infection. However, it is not clear if heptad repeats have a direct role in the actual fusion event. Here we have synthesized, fluorescently labeled and functionally and structurally characterized a wild-type 70 residue peptide (SV-117) composed of both the fusion peptide and the N-terminal heptad repeat of Sendai virus fusion protein, two of its mutants, as well as the fusion peptide and heptad repeat separately. One mutation was introduced in the fusion peptide (G119K) and another in the heptad repeat region (I154K). Similar mutations have been shown to drastically reduce the fusogenic ability of the homologous fusion protein of Newcastle disease virus. We found that only SV-117 was active in inducing lipid mixing of egg phosphatidylcholine/phosphatidyiglycerol (PC/PG) large unilamellar vesicles (LUV), and not the mutants nor the mixture of the fusion peptide and the heptad repeat. Functional characterization revealed that SV-117, and to a lesser extent its two mutants, were potent inhibitors of Sendai virus-mediated hemolysis of red blood cells, while the fusion peptide and SV-150 were negligibly active alone or in a mixture. Hemagglutinin assays revealed that none of the peptides disturb the binding of virions to red blood cells. Further studies revealed that SV-117 and its mutants oligomerize similarly in solution and in membrane, and have similar potency in inducing vesicle aggregation. Circular dichroism and FTIR spectroscopy revealed a higher helical content for SV-117 compared to its mutants in 40 % tifluorethanol and in PC/PG multibilayer membranes, respectively, ATR-FTIR studies indicated that SV-117 lies more parallel with the surface of the membrane than its mutants. These observations suggest a direct role for the N-terminal heptad repeat in assisting the fusion peptide in mediating membrane fusion.

Amino Acid Sequence↗

PubMed1999

Effect of nonpolar substitutions of the conserved Phe11 in the fusion peptide of HIV-1 gp41 on its function, structure, and organization in membranes.

The fusion domain of the HIV-1 envelope glycoprotein (gp120-gp41) is a conserved hydrophobic region located at the N-terminus of the transmembrane subunit (gp41). A prominent feature of this domain is a conserved five-residue "FLGFL" sequence at positions 8-12. Mutation of the highly conserved Phe(11) to Val (F11V), presumed not to significantly affect the hydrophobicity and the structure of this region, has been shown to decrease the level of syncytium formation and virus infectivity. Here we show that the substitution of Gly for Phe(11) (F11G) reduces cell-cell fusion activity by 80-90%. To determine the effect of these mutations on the properties of the fusion peptide, a 33-residue peptide (WT) identical to the extended fusion domain and its F11V and F11G mutants were synthesized, fluorescently labeled, and studied with respect to their function, structure, and organization in phospholipid membranes. The WT peptide alone induced fusion of both zwitterionic (PC/Chol) and negatively charged (PS/PC/Chol and POPG) vesicles, in contrast to a 23-mer fusion peptide lacking the C-terminal domain which has been shown to be inactive with PC vesicles but able to induce fusion of POPG vesicles which had been preaggragated with Ca(2+) or Mg(2+). The F11V peptide preserved 50% activity, and the F11G peptide was virtually inactive. ATR-FTIR spectroscopy indicated similar secondary structure of the peptides in multibilayers that was independent of membrane composition. Furthermore, all the peptides increased the extent of lipid disorder to a similar extent, but the kinetics of amide II H to D exchange was in the following order: F11G > F11V > WT. Fluorescence studies in the presence of membranes, as well as SDS-PAGE, revealed that the WT and F11V peptides self-associate to similar levels while F11G exhibited a decreased level of self-association. The data suggest that the FLGFL motif contributes to the functional organization of the HIV-1 fusion peptide and that the C-terminal domain following the fusion peptide contributes to the membrane fusion process.

Amino Acid Sequence↗

PubMed1999

Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity.

The antimicrobial peptide LL-37 belongs to the cathelicidin family and is the first amphipathic alpha-helical peptide isolated from human. LL-37 is considered to play an important role in the first line of defence against local infection and systemic invasion of pathogens at sites of inflammation and wounds. Understanding its mode of action may assist in the development of antimicrobial agents mimicking those of the human immune system. In vitro studies revealed that LL-37 is cytotoxic to both bacterial and normal eukaryotic cells. To gain insight into the mechanism of its non-cell-selective cytotoxicity, we synthesized and structurally and functionally characterized LL-37, its N-terminal truncated form FF-33, and their fluorescent derivatives (which retained structure and activity). The results showed several differences, between LL-37 and other native antimicrobial peptides, that may shed light on its in vivo activities. Most interestingly, LL-37 exists in equilibrium between monomers and oligomers in solution at very low concentrations. Also, it is significantly resistant to proteolytic degradation in solution, and when bound to both zwitterionic (mimicking mammalian membranes) and negatively charged membranes (mimicking bacterial membranes). The results also showed a role for the N-terminus in proteolytic resistance and haemolytic activity, but not in antimicrobial activity. The LL-37 mode of action with negatively charged membranes suggests a detergent-like effect via a 'carpet-like' mechanism. However, the ability of LL-37 to oligomerize in zwitterionic membranes might suggest the formation of a transmembrane pore in normal eukaryotic cells. To examine this possibility we used polarized attenuated total reflectance Fourier-transform infrared spectroscopy and found that the peptide is predominantly alpha-helical and oriented nearly parallel with the surface of zwitterionic-lipid membranes. This result does not support the channel-forming hypothesis, but rather it supports the detergent-like effect.

Amino Acid Sequence↗

PubMed1999

Peptide-bilayer interactions: simulations of dermaseptin B, an antimicrobial peptide.

Dermaseptins, a family of antimicrobial peptides, are believed to act by forming amphipathic alpha-helices which associate with the cell membrane, leading to its permeabilisation and disruption. A simple mean field method is described for simulation of the interactions of peptides with lipid bilayers which includes an approximate representation of the electrostatic effects of the head-group region of the bilayer. Starting from an atomistic model of a PC phospholipid bilayer we calculate an average electrostatic potential along the bilayer normal. By combining the interaction of the peptide with this electrostatic potential and with the hydrophobic core of the membrane we arrive at a more complete description of peptide-bilayer energetics than would be obtained using sidechain hydrophobicities alone. Using this interaction potential in MD simulations of the frog skin peptide dermaseptin B reveals that the lipid bilayer stabilises the alpha-helical conformation of the peptide. This is in agreement with FTIR data. A surface associated orientation thus appears to be the most stable arrangement of the peptide, at least at zero ionic strength and without taking account of possible peptide-peptide interactions.

Algorithms↗

PubMed1999

Membrane-induced step in the activation of Sendai virus fusion protein.

Peptides derived from conserved heptad-repeat regions of several viruses have been shown recently to inhibit virus-cell fusion. To find out their possible role in the fusion process, two biologically active heptad-repeat segments of the fusion protein (F) of Sendai virus, SV-150 (residues 150-186), and SV-473 (residues 473-495) were synthesized, fluorescently labeled and spectroscopically characterized for their structure and organization in solution and within the membrane. SV-150 was found to be 50-fold less active than SV-473 in inhibiting Sendai virus-cell fusion. Circular dichroism (CD) spectroscopy revealed that in aqueous solution, the peptides are self-associated and adopt low alpha-helical structure. However, when the two peptides are mixed together, their alpha-helical content significantly increases. Fluorescence studies, CD, and polarized attenuated total reflection infrared (ATR-FTIR) spectroscopy showed that both peptides, alone or as a complex, bind strongly to negatively charged and zwitterionic phospholipid membranes, dissociate therein into alpha-helical monomers, but do not perturb the lipid packing of the membrane. The ability of the peptides to interact with each other in solution may be correlated with antiviral activity, whereas their ability to interact with the membrane, together with their location near the fusion peptide and the transmembrane domain, suggests a revision to the currently accepted model for viral-induced membrane fusion. In the revised model, in the sequence of events associated with viral entry, the two heptad-repeat sequences may assist in bringing the viral and cellular membranes closer, thus facilitating membrane fusion.

Amino Acid Sequence↗

PubMed1999

A comparative study on the structure and function of a cytolytic alpha-helical peptide and its antimicrobial beta-sheet diastereomer.

Antimicrobial peptides which adopt mainly or only beta-sheet structures have two or more disulfide bonds stabilizing their structure. The disruption of the disulfide bonds results in most cases in a large decrease in their antimicrobial activity. In the present study we examined the effect of d-amino acids incorporation on the structure and function of a cytolytic alpha-helical peptide which acts on erythrocytes and bacteria. The influence of a single or double d-amino acid replacement in alpha-helical peptides on their structure was reported previously in 50% 2,2,2, trifluoroethanol/water [Krause et al. (1995) Anal. Chem. 67, 252-258]. Here we used Attenuated Total Reflectance Fourier-Transform Infrared (ATR-FTIR) spectroscopy and found that the predominant structure of the wild-type peptide is alpha-helix in phospholipid membranes, whereas the structure of the diastereomer is beta-sheet. However, the linear, beta-sheet diastereomer preserved its cytolytic activity on bacteria but not on erythrocytes. Previous studies have shown that the ability of antimicrobial peptides to lyse bacteria but not normal mammalian cells correlated with their ability to disintegrate preferentially negatively charged, but not zwitterionic phospholipid membranes. In contrast, the diastereomer described here disrupts zwitterionic and negatively charged vesicles with similar potencies to those of the hemolytic wild-type peptide. Interestingly, whereas addition of a positive charge to the N-terminus of the wild-type peptide (which caused a minor effect on its structure) increased activity only towards some of the bacteria tested, similar modification in the diastereomer increased activity towards all of them. Furthermore, the modified wild-type peptide preserved its potency to destabilize zwitterionic and negatively charged vesicles, whereas the modified diastereomer had a reduced potency on zwitterionic vesicles but increased potency on negatively charged vesicles. Overall our results suggest that this new class of antimicrobial diastereomeric peptides bind to the membrane in 'carpet-like' manner followed by membrane disruption and breakdown, rather than forming a transmembrane pore which interfere with the bacteria potential. These studies also open a way to design new broad-spectrum antibacterial peptides.

Amino Acid Sequence↗

PubMed1998

Structure-function study of a heptad repeat positioned near the transmembrane domain of Sendai virus fusion protein which blocks virus-cell fusion.

A synthetic heptad repeat, SV-473, derived from Sendai virus fusion protein is a potent inhibitor of virus-cell fusion. In order to understand the mechanism of the inhibitory effect, we synthesized and fluorescently labeled SV-465, an extended version of SV-473 by one more heptad, its mutant peptide A17,24-SV-465, in which two heptadic leucines were substituted with two alanines, and its enatiomer D-SV-465, composed entirely of Damino acids. Similar mutations in the homologous fusion protein of the Newcastle disease virus drastically reduced its activity. The data revealed that SV-465, but not A17,24-SV-465 or its enantiomer, is highly active in inhibiting Sendai virus-induced hemolysis of red blood cells. None of the peptides interfere with the binding of virions to the target red blood cells as demonstrated by hemagglutinin assay. Fluorescence and circular dichroism (CD) spectroscopy indicated that: (i) only SV-465 could self-assemble in aqueous environment; (ii) only SV-465 could co-assemble with two other biologically active heptad repeats derived from Sendai virus fusion protein; (iii) SV-465 has a higher helical content than A17,24-SV-465 in solution, and (iv) all the peptides bind strongly to zwitterionic and negatively charged phospholipids. Polarized attenuated total reflection infrared spectroscopy revealed that they bound as monomers onto the surface of zwitterionic membranes with predominantly alpha-helical structures. The functional role of the amino acid 465-497 domain in Sendai virus-mediated membrane fusion is discussed in light of these findings.

Amino Acid Sequence↗

PubMed1998

The structure and organization within the membrane of the helices composing the pore-forming domain of Bacillus thuringiensis delta-endotoxin are consistent with an "umbrella-like" structure of the pore.

The aim of this study was to elucidate the mechanism of membrane insertion and the structural organization of pores formed by Bacillus thuringiensis delta-endotoxin. We determined the relative affinities for membranes of peptides corresponding to the seven helices that compose the toxin pore-forming domain, their modes of membrane interaction, their structures within membranes, and their orientations relative to the membrane normal. In addition, we used resonance energy transfer measurements of all possible combinatorial pairs of membrane-bound helices to map the network of interactions between helices in their membrane-bound state. The interaction of the helices with the bilayer membrane was also probed by a Monte Carlo simulation protocol to determine lowest-energy orientations. Our results are consistent with a situation in which helices alpha4 and alpha5 insert into the membrane as a helical hairpin in an antiparallel manner, while the other helices lie on the membrane surface like the ribs of an umbrella (the "umbrella model"). Our results also support the suggestion that alpha7 may serve as a binding sensor to initiate the structural rearrangement of the pore-forming domain.

Amino Acid Sequence↗

PubMed1998

A synthetic all D-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 recognizes the wild-type fusion peptide in the membrane and inhibits HIV-1 envelope glycoprotein-mediated cell fusion.

Recent studies demonstrated that a synthetic fusion peptide of HIV-1 self-associates in phospholipid membranes and inhibits HIV-1 envelope glycoprotein-mediated cell fusion, presumably by interacting with the N-terminal domain of gp41 and forming inactive heteroaggregates [Kliger, Y., Aharoni, A., Rapaport, D., Jones, P., Blumenthal, R. & Shai, Y. (1997) J. Biol. Chem. 272, 13496-13505]. Here, we show that a synthetic all D-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 (D-WT) of HIV-1 associates with its enantiomeric wild-type fusion (WT) peptide in the membrane and inhibits cell fusion mediated by the HIV-1 envelope glycoprotein. D-WT does not inhibit cell fusion mediated by the HIV-2 envelope glycoprotein. WT and D-WT are equally potent in inducing membrane fusion. D-WT peptide but not WT peptide is resistant to proteolytic digestion. Structural analysis showed that the CD spectra of D-WT in trifluoroethanol/water is a mirror image of that of WT, and attenuated total reflectance-fourier transform infrared spectroscopy revealed similar structures and orientation for the two enantiomers in the membrane. The results reveal that the chirality of the synthetic peptide corresponding to the HIV-1 gp41 N-terminal sequence does not play a role in liposome fusion and that the peptides' chirality is not necessarily required for peptide-peptide interaction within the membrane environment. Furthermore, studies along these lines may provide criteria to design protease-resistant therapeutic agents against HIV and other viruses.

Amino Acid Sequence↗

PubMed1998

A peptide derived from a conserved domain of Sendai virus fusion protein inhibits virus-cell fusion. A plausible mode of action.

SV-201, a peptide derived from a conserved and potentially amphipathic region (amino acids 201-229) in the Sendai virus ectodomain, specifically inhibited virus-mediated hemolysis only when added to virions prior to their attachment to red blood cells. Sendai virus-mediated hemagglutinin assay in the presence of SV-201 demonstrated that the peptide does not disturb the binding of virions to the target red blood cells. A mutated peptide with 2 amino acids substitution, rendering the peptide neutral, was biologically inactive. A second mutant with 7 amino acids randomized at the N terminus keeping the hydrophobicity of the peptide unaltered was only slightly active. A hydrophobic peptide corresponding to the fusion peptide domain was also inactive. SV-201, the two mutants, and the fusion peptide bind similarly with high affinity to both negatively charged phosphatidylserine/phosphatidylcholine and zwitterionic phosphatidylcholine lipid vesicles, suggesting that the inhibitory effect is not due merely to membrane modulation. Fluorescence studies with rhodamine-labeled peptides and SV-201-induced inhibition assays, demonstrated that the SV-201 binding site is most probably located in the region corresponding to amino acids 201-229 of the Sendai virus fusion protein. The data presented here suggest that SV-201 disturbs a functional domain in the Sendai virus fusion protein, which is most probably associated with the assembly of the fusion protein and/or membrane apposition. The existence of homologous SV-201 regions in other viruses suggests that these regions may have a similar role, and their synthetic counterparts may act as inhibitors for the corresponding viruses.

Amino Acid Sequence↗

PubMed1998

Mode of action of linear amphipathic alpha-helical antimicrobial peptides.

The increasing resistance of bacteria to conventional antibiotics resulted in a strong effort to develop antimicrobial compounds with new mechanisms of action. Antimicrobial peptides seem to be a promising solution to this problem. Many studies aimed at understanding their mode of action were described in the past few years. The most studied group includes the linear, mostly alpha-helical peptides. Although the exact mechanism by which they kill bacteria is not clearly understood, it has been shown that peptide-lipid interactions leading to membrane permeation play a role in their activity. Membrane permeation by amphipathic alpha-helical peptides can proceed via either one of the two mechanisms: (a) transmembrane pore formation via a "barrel-stave" mechanism; and (b) membrane destruction/solubilization via a "carpet-like" mechanism. The purpose of this review is to summarize recent studies aimed at understanding the mode of action of linear alpha-helical antimicrobial peptides. This review, which is focused on magainins, cecropins, and dermaseptins as representatives of the amphipathic alpha-helical antimicrobial peptides, supports the carpet-like rather the barrel-stave mechanism. That these peptides vary with regard to their length, amino acid composition, and next positive charge, but act via a common mechanism, may imply that other linear antimicrobial peptides that share the same properties also share the same mechanism.

Amino Acid Sequence↗

PubMed1998

Pardaxin, a new pharmacological tool to stimulate the arachidonic acid cascade in PC12 cells.

The effect of Pardaxin, a neurotoxin that induces neurotransmitter release from neurons, on the arachidonic acid (AA) cascade was studied in PC12 cells. Both native and the synthetic Pardaxin selectively stimulated phospholipase A2 (PLA2) activity (measured by [3H]AA release) in the presence as well as in the absence of extracellular calcium. Pardaxin-stimulated PLA2 activity was also evident in the increased formation of lysophosphatidylcholine. Pardaxin analogs, lacking the alpha-helical structure that is essential for insertion into the plasma membrane, were ineffective in stimulating the AA cascade in PC12 cells. Pardaxin stimulation of PLA2 was markedly inhibited by the nonselective PLA2 inhibitors bromophenacyl bromide and mepacrine, by methyl arachidonyl fluorophosphonate, a dual inhibitor of calcium-dependent cytosolic PLA2 and the calcium-independent PLA2 and by bromoenol lactone[(E)-6-(bromoethylene)tetrahydro-3-(1-naphthalenyl-2H-pyran -2- one], a highly specific inhibitor of calcium-independent PLA2. After Pardaxin treatment, there was increased release of AA metabolites produced by the cyclooxygenase pathway as expressed in an 8-fold increase of PGE2 release. The release of other eicosanoids, such as 6-keto-PGF1alpha and thromboxane B2, was also augmented. Pardaxin-induced PGE2 release was observed in calcium-free medium and in the absence of any increase in cytosolic calcium. Dexamethasone partially inhibited Pardaxin-induced PGE2 release. This effect was reversed by the type II corticosteroid receptor antagonist RU-38486. Our results indicate that Pardaxin stimulates release of AA and eicosanoids, independently of calcium, and suggest that calcium-independent PLA2 plays an important role in Pardaxin stimulation of the AA cascade.

Amino Acid Sequence↗