Biomedical subjects

Y Shai

Publications and source records attributed to Y Shai.

At least 19 recordsLinked to original sources

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↗

PubMed1997

Selective cytotoxicity of dermaseptin S3 toward intraerythrocytic Plasmodium falciparum and the underlying molecular basis.

The antimicrobial activity of various naturally occurring microbicidal peptides was reported to result from their interaction with microbial membrane. In this study, we investigated the cytotoxicity of the hemolytic peptide dermaseptin S4 (DS4) and the nonhemolytic peptide dermaseptin S3 (DS3) toward human erythrocytes infected by the malaria parasite Plasmodium falciparum. Both DS4 and DS3 inhibited the parasite's ability to incorporate [3H]hypoxanthine. However, while DS4 was toxic toward both the parasite and the host erythrocyte, DS3 was toxic only toward the intraerythrocytic parasite. To gain insight into the mechanism of this selective cytotoxicity, we labeled the peptides with fluorescent probes and investigated their organization in solution and in membranes. In Plasmodium-infected cells, rhodamine-labeled peptides interacted directly with the intracellular parasite, in contrast to noninfected cells, where the peptides remained bound to the erythrocyte plasma membrane. Binding experiments to phospholipid membranes revealed that DS3 and DS4 had similar binding characteristics. Membrane permeation studies indicated that the peptides were equally potent in permeating phosphatidylserine/phosphatidylcholine vesicles, whereas DS4 was more permeative with phosphatidylcholine vesicles. In aqueous solutions, DS4 was found to be in a higher aggregation state. Nevertheless, both DS3 and DS4 spontaneously dissociated to monomers upon interaction with vesicles, albeit with different kinetics. In light of these results, we propose a mechanism by which dermaseptins permeate cells and affect intraerythrocytic parasites.

Amino Acid Sequence↗

PubMed1997

Bacillus thuringiensis cytolytic toxin associates specifically with its synthetic helices A and C in the membrane bound state. Implications for the assembly of oligomeric transmembrane pores.

The CytA toxin exerts its activity by the formation of pores within target cell membranes. However, the exact mechanism of pore formation and the structural elements that are involved in the toxic activity are yet to be determined. Recently, the structure of the highly similar CytB toxin was solved (Li et al., 1996), and a beta-barrel was suggested as a possible structure of the pores. Due to the similarity between the toxins, the existence and positioning of alpha-helices and beta-sheets in CytA were predicted from the alignment of the sequences. Here peptides corresponding to beta5, beta6, and beta7 strands, to a conserved nonhelical region of the CytA toxin (P149-170), to helices B and D, and to an analogue of helix A were synthesized, fluorescently labeled, and characterized. We found that, unlike helices A and C (Gazit and Shai, 1993), neither the beta-strand peptides nor helix B could interact with lipid membranes, whereas P149-170 and helix D bind the membrane weakly. Membrane permeation experiments suggested that CytA toxin exerts its activity by aggregation of several monomers. To learn about the structural elements that may mediate CytA oligomerization, the ability of the synthetic peptides to interact with membrane-bound CytA was studied. Helices A and C, but not the beta-strands, helix D, or a control peptide, caused a large increase in the fluorescence of membrane-bound fluorescein-labeled CytA, whereas helix B had only a slight effect. Moreover, the addition of rearranged helix A, a peptide with the same composition as helix A, but with only two pairs of amino acids rearranged, did not affect the fluorescence. The addition of unlabeled CytA also caused an increase in the fluorescence intensity, further demonstrating the interaction between CytA monomers within the membrane. Taken together, our results provide further support for the suggestion that the CytA toxin self-assembles within membrane and that helices A and C are major structural elements involved in the membrane interaction and intermolecular assembly of the toxin.

Amino Acid Sequence↗

PubMed1997

A leucine zipper motif in the ectodomain of Sendai virus fusion protein assembles in solution and in membranes and specifically binds biologically-active peptides and the virus.

We have detected a leucine zipper-like motif in the ectodomain of the Sendai virus fusion protein (aa 269-307) which is extremely conserved in the family of Sendai viruses. To find a possible role for this motif, we synthesized SV-269, a 39 amino acid peptide corresponding to this domain, and a mutant peptide, MuSV-269, with an amino acid pair interchanged their positions. The peptides were labeled with fluorescent probes at their N-terminal amino acid and functionally and structurally characterized. The data show that SV-269, but not MuSV-269, specifically binds Sendai virus. Expectedly, SV-269 is more active than the mutant MuSV-269 in inhibiting Sendai virus-mediated hemolysis. Fluorescence studies reveal that SV-269 assembles in aqueous solution, binds to zwitterionic PC and negatively-charged PS/PC vesicles, and assembles therein. Although MuSV-269 similarly binds to both types of vesicles, it only slightly assembles in solution and not at all in membranes. Moreover, SV-269, but not MuSV-269, coassembles with the biologically-active heptad repeats SV-150 and SV-473 (Rapaport et al. , 1995) in solution as revealed by fluorescence and circular dichroism (CD) spectroscopy, and with SV-150 within negatively-charged PS/PC and zwitterionic PC vesicles. Despite these differences, both SV-269 and MuSV-269 adopt similar secondary structures in 40% TFE and 1% SDS as revealed by CD spectroscopy, and disrupt the packing of the lipid bilayers to the same extent, as shown by the dissipation of diffusion potential. The role of this leucine zipper motif is discussed in terms of the assembly of the Sendai virus fusion protein in solution and within membranes. Since most of the heptadic leucines are also conserved in the corresponding domains of other paramyxoviruses such as rinderpest, measles, SV5, and parainfluenza, it may indicate a similar role of this domain in these viruses as well.

Amino Acid Sequence↗

PubMed1997

A repertoire of novel antibacterial diastereomeric peptides with selective cytolytic activity.

The increase in infectious diseases and bacterial resistance to antibiotics has resulted in intensive studies focusing on the use of linear, alpha-helical, cytolytic peptides from insects and mammals as potential drugs for new target sites in bacteria. Recent studies with diastereomers of the highly potent cytolytic peptides, pardaxin and melittin, indicate that alpha-helical structure is required for mammalian cells lysis but is not necessary for antibacterial activity. Thus, hydrophobicity and net positive charge of the polypeptide might confer selective antibacterial lytic activity. To test this hypothesis, a series of diastereomeric model peptides (12 amino acids long) composed of varying ratios of leucine and lysine were synthesized, and their structure and biological function were investigated. Peptide length and the position of D-amino acids were such that short peptides with stretches of only 1-3 consecutive L-amino acids that cannot form an alpha-helical structure were constructed. Circular dichroism spectroscopy showed that the peptides do not retain any detectable secondary structure in a hydrophobic environment. This enabled examination of the sole effect of hydrophobicity and positive charge on activity. The data reveal that modulating hydrophobicity and positive charge is sufficient to confer antibacterial activity and cell selectivity. A highly hydrophobic diastereomer that permeated both zwitterionic and negatively charged phospholipid vesicles, lysed eukaryotic and prokaryotic cells. In contrast, a highly positively charged diastereomer that only permeated slightly negatively charged phospholipid vesicles had low antibacterial activity and could not lyse eukaryotic cells. In the boundary between high hydrophobicity and high positive charge, the diastereomers acquired selective and potent antibacterial activity. Furthermore, they were completely resistant to human serum inactivation, which dramatically reduces the activity of native antibacterial peptides. In addition, a strong synergistic effect was observed at nonlethal concentrations of the peptides with the antibiotic tetracycline on resistant bacteria. The results are discussed in terms of proposed mechanisms of antibacterial activity, as well as a new strategy for the design of a repertoire of short, simple, and easily manipulated antibacterial peptides as potential drugs in the treatment of infectious diseases.

Amino Acid Sequence↗

PubMed1997

Fusion peptides derived from the HIV type 1 glycoprotein 41 associate within phospholipid membranes and inhibit cell-cell Fusion. Structure-function study.

The fusion domain of human immunodeficiency virus (HIV-1) envelope glycoprotein (gp120-gp41) is a conserved hydrophobic region located at the N terminus of the transmembrane glycoprotein (gp41). A V2E mutant has been shown to dominantly interfere with wild-type envelope-mediated syncytium formation and virus infectivity. To understand this phenomenon, a 33-residue peptide (wild type, WT) identical to the N-terminal segment of gp41 and its V2E mutant were synthesized, fluorescently labeled, and characterized. Both peptides inhibited HIV-1 envelope-mediated cell-cell fusion and had similar alpha-helical content in membrane mimetic environments. Studies with fluorescently labeled peptide analogues revealed that both peptides have high affinity for phospholipid membranes, are susceptible to digestion by proteinase-K in their membrane-bound state, and tend to self- and coassemble in the membranes. In SDS-polyacrylamide gel electrophoresis the WT peptide formed dimers as well as higher order oligomers, whereas the V2E mutant only formed dimers. The WT, but not the V2E mutant, induced liposome aggregation, destabilization, and fusion. Moreover, the V2E mutant inhibited vesicle fusion induced by the WT peptide, probably by forming inactive heteroaggregates. These data form the basis for an explanation of the mechanism by which the gp41 V2E mutant inhibits HIV-1 infectivity in cells when co-expressed with WT gp41.

Amino Acid Sequence↗

PubMed1997

A leucine zipper-like sequence from the cytoplasmic tail of the HIV-1 envelope glycoprotein binds and perturbs lipid bilayers.

HIV-1 transmembrane envelope glycoprotein (gp41) has an unusually long cytoplasmic domain that has secondary associations with the inner leaflet of the membrane. Two highly amphiphatic alpha-helices in the cytoplasmic domain of gp41 have previously been shown to interact with lipid bilayers. We have detected a highly conserved leucine zipper-like sequence between the two alpha-helices. A peptide corresponding to this segment (residues 789-815, LLP-3) aggregates in aqueous solution, but spontaneously inserts into phospholipid membranes and dissociates into alpha-helical monomers. The peptide perturbs the bilayer structure resulting in the formation of micelles and other non-bilayer structures. Tryptophan fluorescence quenching experiments using brominated phospholipids revealed that the peptide penetrates deeply into the hydrophobic milieu of the membrane bilayer. The peptide interacts equally with zwitterionic and negatively-charged phospholipid membranes and is protected from proteolytic digestion in its membrane-bound state. Polarized attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy showed that the LLP-3 alpha-helix axis is about 70 degrees from the normal to the membrane plane. The ATR-FTIR CH2-stretching dichroic ratio increases when the peptide is incorporated into pure phospholipid membranes, further indicating that the peptide can deeply penetrate and perturb the bilayer structure. Integrating these data with what is already known about the membrane-associating features of adjacent segments, we propose a revised structural model in which a large portion of the cytoplasmic tail of the HIV-1 envelope glycoprotein is associated with the membrane.

Amino Acid Sequence↗

PubMed1997

Selective lysis of bacteria but not mammalian cells by diastereomers of melittin: structure-function study.

Studies on lipid-peptide interactions of cytolytic polypeptides tend to emphasize the importance of the amphipathic alpha-helical structure for their cytolytic activity. In this study, diasetereomers of the bee venom melittin (26 a.a.), a non-cell-selective cytolysin, were synthesized and investigated for their structure and cytolytic activity toward bacteria and mammalian cells. Similarly to the findings with the diastereomers of the less cytolytic peptide pardaxin (33 a.a.) (Shai & Oren. 1996), the melittin diastereomer, lest their alpha-helical structure, which abrogated their hemolytic activity toward human erythrocytes. However, they retained their antibacterial activity and completely lysed both Gram-positive and Gram-negative bacteria, as revealed by transmission electron microscopy. To understand the molecular mechanism underlying this selectivity, binding experiments utilizing the intrinsic tryptophan of melittin, tryptophan quenching experiments using brominated phospholipids, and membrane destabilization studies were done. The data revealed that the melittin diastereomers bound to and destabilized only negatively-charged phospholipid vesicles, in contrast to native melittin, which binds strongly to both negatively-charged and zwitterionic phospholipids. However, the partition coefficient, the depth of penetration into the membrane, and the membrane-permeating activity of the diastereomers with negatively-charged phospholipids were similar to those obtained with melittin. The results obtained do not support the formation of transmembrane pores as the mode of action of the diastereomers, but rather suggest that these peptides bind to the surface of the bacterial membrane, cover it in a "carpet-like" manner, and dissolve it like a detergent. The results presented here together with those obtained with the cytolytic peptide pardaxin suggest that the combination of hydrophobicity and net positive charge may be sufficient in the design of potent diastereomers of antibacterial polypeptides for the treatment of infectious diseases.

Acinetobacter calcoaceticus↗

PubMed1997

The structure and organization of synthetic putative membranous segments of ROMK1 channel in phospholipid membranes.

The hydropathy plot of ROMK1, an inwardly rectifying K+ channel, suggests that the channel contains two transmembrane domains (M1 and M2) and a linker between them with significant homology to the H5 pore region of voltage-gated K+ channels. To gain structural information on the pore region of the ROMK1 channel, we used a spectrofluorimetric approach and characterized the structure, the organization state, and the ability of the putative membranous domains of the ROMK1 channel to self-assemble and coassemble within lipid membranes. Circular dichroism (CD) spectroscopy revealed that M1 and M2 adopt high alpha-helical structures in egg phosphatidylcholine small unilamellar vesicles and 40% trifluoroethanol (TFE)/water, whereas H5 is not alpha-helical in either egg phosphatidylcholine small unilamellar vesicles or 40% TFE/water. Binding experiments with 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled peptide demonstrated that all of the peptides bind to zwitterionic phospholipid membranes with partition coefficients on the order of 10(5) M-1. Tryptophan quenching experiments using brominated phospholipids revealed that M1 is dipped into the hydrophobic core of the membrane. Resonance energy transfer (RET) measurements between fluorescently labeled pairs of donor (NBD)/acceptor (rhodamine) peptides revealed that H5 and M2 can self-associate in their membrane-bound state, but M1 cannot. Moreover, the membrane-associated nonhelical H5 serving as a donor can coassemble with the alpha-helical M2 but not with M1, and M1 can coassemble with M2. No coassembly was observed between any of the segments and a membrane-embedded alpha-helical control peptide, pardaxin. The results are discussed in terms of their relevance to the proposed topology of the ROMK1 channel, and to general aspects of molecular recognition between membrane-bound polypeptides.

4-Chloro-7-nitrobenzofurazan↗

PubMed1996

Coassembly of synthetic segments of shaker K+ channel within phospholipid membranes.

Increasing evidence suggests that membrane-embedded hydrophobic segments can interact within the phospholipid milieu of the membrane with varying degrees of specificity and thus contribute to the folding and oligomerization of proteins. We have used synthetic peptides corresponding to segments from the hydrophobic core of the Shaker potassium channel as a model system to study interactions between membrane-embedded segments. Three synthetic segments of the Shaker K+ channel, comprising the hydrophobic S2, S3, and S4 sequences, were used, and their secondary structure, their interactions with, and orientation within phospholipid membranes were examined. Secondary structure studies revealed that though S3 and S4 both adopt certain fractions of alpha-helical structures in membrane mimetic environments, the alpha-helical content of S3 is lower. Both S3 and S4 bind strongly to zwitterionic phospholipids, with partition coefficients in the order of 10(4) and 10(5) M-1. ATR-FTIR studies showed that while the S4 peptide is oriented parallel to the membrane surface, S3 tends to a more transmembranal orientation. Enzymatic cleavage experiments demonstrated that the presence of S3 induces some change in the proteolytic accessibility of the S4 segment. Resonance energy transfer measurements, done in high lipid/peptide molar ratios, revealed that S3 and S4 cannot self-associate in zwitterionic phospholipid vesicles but can associate with each other and with the S2 segment of the channel. Furthermore, S3 does not interact with the homologous S4 region from the first repeat of the eel sodium channel, demonstrating specificity in the interactions. These results are in line with data indicating that functionally important interactions indeed exist between the negatively charged S2 and S3 regions and the positively charged S4 region [Papazian, D. M., et al (1995) Neuron 14, 1293-1301; Planells-Cases, R., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 92, 9422-9426]. From a broader point of view, these results provide further support to the notion that interactions (either specific or nonspecific) may exist between transmembrane segments of integral membrane proteins and therefore can contribute to their assembly and organization.

Amino Acid Sequence↗

PubMed1996

Structure and orientation of the mammalian antibacterial peptide cecropin P1 within phospholipid membranes.

Cecropins are positively charged antibacterial peptides that act by permeating the membrane of susceptible bacteria. To gain insight into the mechanism of membrane permeation, the secondary structure and the orientation within phospholipid membranes of the mammalian cecropin P1 (CecP) was studied using attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy and molecular dynamics simulations. The shape and frequency of the amide I and II absorption peaks of CecP within acidic PE/PG multibilayers (phosphatidylethanolamine/phosphatidylglycerol) in a 7:3 (w/w) ratio (a phospholipid composition similar to that of many bacterial membranes), indicated that the peptide is predominantly alpha-helical. Polarized ATR-FTIR spectroscopy was used to determine the orientation of the peptide relative to the bilayer normal of phospholipid multibilayers. The ATR dichroic ratio of the amide I band of CecP peptide reconstituted into oriented PE/PG phospholipid membranes indicated that the peptide is preferentially oriented nearly parallel to the surface of the lipid membranes. A similar secondary structure and orientation were found when zwitterionic phosphatidylcholine phospholipids were used. The incorporation of CecP did not significantly change the order parameters of the acyl chains of the multibilayer, further suggesting that CecP does not penetrate the hydrocarbon core of the membranes. Molecular dynamics simulations were used to gain insight into possible effects of transmembrane potential on the orientation of CecP relative to the membrane. The simulations appear to confirm that CecP adopts an orientation parallel to the membrane surface and does not insert into the bilayer in response to a cis positive transmembrane voltage difference. Taken together, the results further support a "carpet-like" mechanism, rather than the formation of transmembrane pores, as the mode of action of CecP. According to this model, formation of a layer of peptide monomers on the membrane surface destablizes the phospholipid packing of the membrane leading to its eventual disintegration.

Amino Acid Sequence↗

PubMed1996

Cytoplasmic and extracellular IsK peptides activate endogenous K+ and Cl- channels in Xenopus oocytes. Evidence for regulatory function.

IsK is a 14.5-kDa type III membrane glycoprotein which induces slowly activating K+ and Cl- currents when expressed in Xenopus oocytes and HEK 293 cells. Recently, mutagenesis experiments identified amino- and carboxyl-terminal domains of IsK as critical for induction of Cl- and K+ currents, respectively. This hypothesis was tested by examining effects of synthetic IsK hydrophilic peptides on untreated Xenopus oocytes. In agreement with IsK membrane topology, we show here that peptides derived from carboxyl and amino termini are sufficient to activate slow K+ and Cl- channels whose biophysical and pharmacological characteristics are similar to those exhibited by the native IsK protein. That data provide further evidence that IsK is a regulatory subunit of pre-existing silent channel complexes rather than a channel per se.

Amino Acid Sequence↗

PubMed1996

A class of highly potent antibacterial peptides derived from pardaxin, a pore-forming peptide isolated from Moses sole fish Pardachirus marmoratus.

Pardaxin, a 33-amino-acid pore-forming polypeptide toxin isolated from the Red Sea Moses sole Pardachirus marmoratus, has a helix-hinge-helix structure. This is a common structural motif found both in antibacterial peptides that can act selectively on bacterial membranes (e.g., cecropin), and in cytotoxic peptides that can lyse both mammalian and bacterial cells (e.g., melittin). Herein we show that pardaxin possesses a high antibacterial activity with a significantly reduced hemolytic activity towards human red blood cells (hRBC), compared with melittin. Its potency is comparable to that of other known native antibacterial peptides such as magainin, cecropins and dermaseptins. To determine the structural features responsible for the selective hemolytic and antibacterial activities, and the structural requirements for a high antibacterial activity, 8 truncated and modified pardaxin analogues were synthesized and structurally and functionally characterized. Each peptide was synthesized with a free carboxylate or amino group (i.e., aminated form) at its C-terminus. The aminated form of pardaxin has both high hemolytic and antibacterial activity. A truncated analogue, with 11 amino acids removed from the C-terminal domain, had dramatically reduced hemolytic activity. However, the aminated form of this analogue was significantly more potent that pardaxin against most bacteria tested, suggesting that the C-terminal tail of pardaxin is responsible for non-selective activity against erythrocytes and bacteria. Furthermore, a positive charge added to its N-terminus significantly increased its antibacterial activity and abolished its low hemolytic activity. The 22-amino-acid C-terminal domain and the short 11-amino-acid N-terminal domain were, in their aminated forms, active only against gram-positive bacteria. Secondary-structure determination using circular dichroism spectroscopy revealed that all the aminated analogues had 25-80% more alpha-helical content in 40% CF3CH2OH/water than their non-aminated forms. Using model phospholipid membranes it was found that all the analogues that were less hemolytic but had retained antibacterial activity could permeate acidicly charged phospholipid vesicles better than zwitterionic phospholipid vesicles, a property characteristics of all the native antibacterial peptides tested so far (e.g., cecropins, magainins and dermaseptins). Pardaxin and its analogues therefore represent a new class of antibacterial peptides that can serve as a basis for the design of therapeutic agents. Furthermore, negative-staining electron microscopy revealed that total inhibition of bacterial growth was due to total lysis of the bacterial wall. Therefore, it might be more difficult for bacteria to develop resistance to such a destructive mechanism, compared with the more specific mechanisms of the currently used antibiotics.

Amino Acid Sequence↗

PubMed1996

Diastereoisomers of cytolysins, a novel class of potent antibacterial peptides.

An amphipathic alpha-helical structure is considered to be a prerequisite for the lytic activity of most short linear cytolytic polypeptides that act on both mammalian cells and bacteria. This structure allows them also to exert diverse pathological and pharmacological effects, presumably by mimicking protein components that are involved in membrane-related events. In this study D-amino acid-incorporated analogues (diastereomers) of the cytolysin pardaxin, which is active against mammalian cells and bacteria, were synthesized and structurally and functionally characterized. We demonstrate that the diastereomers do not retain the alpha-helical structure, which in turn abolishes their cytotoxic effects on mammalian cells. However, they retain a high antibacterial activity, which is expressed in a complete lysis of the bacteria, as revealed by negative staining electron microscopy. The disruption of the alpha-helical structure should prevent the diastereomer analogues from permeating the bacterial wall by forming transmembrane pores but rather by dissolving the membrane as a detergent. These findings open the way for a new strategy in developing a novel class of highly potent antibacterial polypeptides for the treatment of infectious diseases, due to the increasing resistance of bacteria to the available antibacterial drugs.

Acinetobacter calcoaceticus↗

PubMed1996

Broad spectrum antibiotic activity of the skin-PYY.

Neuropeptide Y (NPY) and polypeptide YY (PYY) are two ubiquitous neuropeptides, found in brain and intestines, respectively, where they exert important regulatory functions. In this study, a new member of the YY family recently isolated from amphibian skin, skin-PYY (SPYY), is reported to inhibit irreversibly the proliferation of a broad spectrum of pathogenic microorganisms. NPY and PYY are shown to be endowed with the same activity. Their potency is similar to that of other antibacterial peptides which have been shown to exert their function by disintegrating the bacterial membrane. These findings and the fact that the C-terminal alpha-helical domain SPYY14-36, which is highly conserved among family members, was responsible for killing microorganisms and for permeation of phospholipid vesicles, suggested that the antibiotic activity may emerge via a membrane permeation mechanism. These findings also raise the question whether NPY and PYY exert in vivo a similar function in mammals.

Amino Acid Sequence↗

PubMed1996

Secondary structure, membrane localization, and coassembly within phospholipid membranes of synthetic segments derived from the N- and C-termini regions of the ROMK1 K+ channel.

The hydropathy plot of the inwardly rectifying ROMK1 K+ channel, which reveals two transmembrane and a pore region domains, also reveals areas of intermediate hydrophobicity in the N terminus (M0) and in the C terminus (post-M2). Peptides that correspond to M0, post-M2, and a control peptide, pre-M0, were synthesized and characterized for their structure, affinity to phospholipid membranes, organizational state in membranes, and ability to self-assemble and coassemble in the membrane-bound state. CD spectroscopy revealed that both M0 and post-M2 adopt highly alpha-helical structures in 1% SDS and 40% TFE/water, whereas pre-M0 is not alpha-helical in either 1% SDS or 40% TFE/water. Binding experiments with NBD-labeled peptides demonstrated that both M0 and post-M2, but not pre-M0, bind to zwitterionic phospholipid membranes with partition coefficients of 10(3)-10(5) M-1. A surface localization for both post-M2 and M0 was indicated by NBD shift, tryptophan quenching experiments with brominated phospholipids, and enzymatic cleavage. Resonance energy transfer measurements between fluorescently labeled pairs of donor (NBD)/ acceptor (rhodamine) peptides revealed that M0 and post-M2 can coassemble in their membrane-bound state, but cannot self-associate when membrane-bound. The results are in agreement with recent data indicating that amino acids in the carboxy terminus of inwardly rectifying K+ channels have a major role in specifying the pore properties of the channels (Taglialatela M, Wible BA, Caporaso R, Brown AM, 1994 Science 264:844-847; Pessia M, Bond CT, Kavanaugh MP, Adelman JP, 1995, Neuron 14:1039-1045). The relevance of the results presented herein to the suggested model for the structure of the ROMK1 channel and to general aspects of molecular recognition between membrane-bound polypeptides are also discussed.

Amino Acid Sequence↗

PubMed1996

Reversible surface aggregation in pore formation by pardaxin.

The mechanism of leakage induced by surface active peptides is not yet fully understood. To gain insight into the molecular events underlying this process, the leakage induced by the peptide pardaxin from phosphatidylcholine/ phosphatidylserine/cholesterol large unilamellar vesicles was studied by monitoring the rate and extent of dye release and by theoretical modeling. The leakage occurred by an all-or-none mechanism: vesicles either leaked or retained all of their contents. We further developed a mathematical model that includes the assumption that certain peptides become incorporated into the vesicle bilayer and aggregate to form a pore. The current experimental results can be explained by the model only if the surface aggregation of the peptide is reversible. Considering this reversibility, the model can explain the final extents of calcein leakage for lipid/peptide ratios of > 2000:1 to 25:1 by assuming that only a fraction of the bound peptide forms pores consisting of M = 6 +/- 3 peptides. Interestingly, less leakage occurred at 43 degrees C, than at 30 degrees C, although peptide partitioning into the bilayer was enhanced upon elevation of the temperature. We deduced that the increased leakage at 30 degrees C was due to an increase in the extent of reversible surface aggregation at the lower temperature. Experiments employing fluorescein-labeled pardaxin demonstrated reversible aggregation of the peptide in suspension and within the membrane, and exchange of the peptide between liposomes. In summary, our experimental and theoretical results support reversible surface aggregation as the mechanism of pore formation by pardaxin.

Amino Acid Sequence↗

PubMed1995

A synthetic peptide corresponding to a conserved heptad repeat domain is a potent inhibitor of Sendai virus-cell fusion: an emerging similarity with functional domains of other viruses.

A series of peptides derived from three domains within the fusion protein of Sendai virus was synthesized and examined for their potential to inhibit the fusion of the virus with human red blood cells. These domains include the 'fusion peptide' and two heptad repeats, one adjacent to the fusion peptide (SV-163) and the other to the transmembrane domain (SV-473). Of all the peptides tested, only SV-473 was highly inhibitive. Using fluorescently-labelled peptides, the mechanism through which the SV-473 peptide inhibits the haemolytic activity of the virus was investigated. The results suggest that interactions of the active peptide with virion elements and lipid membranes are involved. Since it has recently been found that synthetic peptides corresponding to putative coiled-coil domains of the human immunodeficiency virus (HIV) type 1 transmembrane protein gp41 are potent inhibitors of HIV, we discuss the general property of virus-derived coiled-coil peptides as inhibitors of viral infection.

Amino Acid Sequence↗