Localization of the anti‐cancer peptide EGFR‐lytic hybrid peptide in human pancreatic cancer BxPC‐3 cells by immunocytochemistry

Cationic lytic‐type peptides have been studied for clinical application in various infections and cancers, but their functional cellular mechanisms remain unclear. We generated anti‐cancer epithelial growth factor receptor (EGFR)‐lytic hybrid peptide, a 32‐amino‐acid peptide composed of an EGFR‐binding sequence and lytic sequence. In this study, we investigated the distribution of EGFR‐lytic hybrid peptide in BxPC‐3 human pancreatic cancer cells by an immunocytochemical (ICC) method. Distribution of EGFR protein expression was unchanged after treatment with EGFR‐lytic peptide compared with non‐treated cells. In confocal laser scanning microscopy, immunostaining of EGFR‐lytic peptide was observed in the cytoplasm, mostly in the form of granules. Some staining was also localized on the mitochondrial membrane. At the ultrastructure level, cells treated with EGFR‐lytic peptide had a low electron density, disappearance of microvilli, and swollen mitochondria. Fragments of cell membrane were also observed in the proximity of the membrane. In immunoelectron microscopy, EGFR‐lytic peptide was observed in the cell membrane and cytoplasm. A number of granules were considered swollen mitochondria. Activation of the caspase pathway as a result of mitochondrial dysfunction was also examined to determine the cytotoxic activity of EGFR‐lytic peptide; however, no effect on cell death after EGFR‐lytic treatment was observed, and moreover, apoptosis was not found to play a critical role in the cell death mechanism. These results suggest that EGFR‐lytic peptide is localized on cell and mitochondrial membranes, with disintegration of the cell membrane contributing mainly to cell death. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Membrane lysis by gramicidin S visualized in red blood cells and giant vesicles

The cationic amphiphilic antimicrobial peptide gramicidin S (GS) is an effective antibiotic. Its applicability is however restricted to topical infections due to its hemolytic activity. In this study, the process of GS induced hemolysis was investigated in detail for the first time. The morphological changes of red blood cells (RBCs) inflicted by GS were visualized and explained in terms of a physical model. The observed fast rupture events were further investigated with giant unilamellar vesicles (GUVs) as model systems for RBCs. Measurements of membrane fluctuations in GUVs revealed that the membrane surface tension was increased after incubation with GS. These findings are in agreement with the hypothesis that amphiphilic peptides induce membrane rupture by an increase in membrane tension.     

Antifungal mechanism of an antimicrobial peptide, HP (2--20), derived from N-terminus of Helicobacter pylori ribosomal protein L1 against Candida albicans

The antifungal activity and mechanism of HP (2-20), a peptide derived from the N-terminus sequence of Helicobacter pylori Ribosomal Protein L1 were investigated. HP (2--20) displayed a strong antifungal activity against various fungi, and the antifungal activity was inhibited by Ca(2+) and Mg(2+) ions. In order to investigate the antifungal mechanism(s) of HP (2-20), fluorescence activated flow cytometry was performed. As determined by propidium iodide staining, Candida albicans treated with HP (2-20) showed a higher fluorescence intensity than untreated cells and was similar to melittin-treated cells. The effect on fungal cell membranes was examined by investigating the change in membrane dynamics of C. albicans using 1,6-diphenyl-1,3,5-hexatriene as a membrane probe and by testing the membrane disrupting activity using liposome (PC/PS; 3:1, w/w) and by treating protoplasts of C. albicans with the peptide. The action of peptide against fungal cell membrane was further examined by the potassium-release test, and HP (2-20) was able to increase the amount of K(+) released from the cells. The result suggests that HP (2-20) may exert its antifungal activity by disrupting the structure of cell membrane via pore formation or directly interacts with the lipid bilayers in a salt-dependent manner.     

Peptide ligand‐mediated endocytosis of nanoparticles to cancer cells: Cell receptor‐binding‐ versus cell membrane‐penetrating peptides

The endocytosis‐mediating performances of two types of peptide ligands, cell receptor binding peptide (CRBP) and cell membrane penetrating peptide (CMPP), were analyzed and compared using a common carrier of peptide ligands‐human ferritin heavy chain (hFTH) nanoparticle. Twenty‐four copies of a CMPP(human immunodeficiency virus‐derived TAT peptide) and/or a CRBP (peptide ligand with strong and specific affinity for either human integrin(α_vβ₃) or epidermal growth factor receptor I (EGFR) that is overexpressed on various cancer cells) were genetically presented on the surface of each hFTH nanopariticle. The quantitative level of endocytosis and intracellular localization of fluorescence dye‐labeled CRBP‐ and CMPP‐presenting nanoparticles were estimated in the in vitro cultures of integrin‐ and EGFR‐overexpressing cancer and human dermal fibroblast cells(control). From the cancer cell cultures treated with the CMPP‐ and CRBP‐presenting nanoparticles, it was notable that CRBPs resulted in quantitatively higher level of endocytosis than CMPP (TAT) and successfully transported the nanoparticles to the cytosol of cancer cells depending on concentration and treatment period of time, whereas TAT‐mediated endocytosis localized most of the nanoparticles within endosomal vesicles under the same conditions. These novel findings provide highly useful informations to many researchers both in academia and in industry who are interested in developing anticancer drug delivery systems/carriers.     

Antinematodal effect of antimicrobial peptide, PMAP-23, isolated from porcine myeloid against Caenorhabditis elegans.

The antinematodal activity and mechanism of a 23-mer antimicrobial peptide, PMAP-23, derived from pig myeloid was investigated. PMAP-23 displayed a strong antinematodal activity against the eggs and worms of Caenorhabditis elegans. To investigate the antinematodal mechanism of PMAP-23, fluorescence activated flow cytometry and confocal laser scanning microscopy were performed. C. elegans treated with PMAP-23 showed higher fluorescence intensity by propidium iodide (PI) staining than normal cells. Confocal microscopy showed that the peptide was localized in the egg's shell and cell membrane. The action of the peptide against C. elegans membranes was examined by testing the membrane disrupting activity using liposome (PC/PS; 3:1, w/w). The result suggests that PMAP-23 may exert its antinematodal activity by disrupting the structure of the cell membrane via pore formation or via direct interaction with the lipid bilayers.     

Effect of N-terminal acetylation on lytic activity and lipid-packing perturbation induced in model membranes by a mastoparan-like peptide

L1A (IDGLKAIWKKVADLLKNT-NH2) is a peptide that displays a selective antibacterial activity to Gram-negative bacteria without being hemolytic. Its lytic activity in anionic lipid vesicles was strongly enhanced when its N-terminus was acetylated (ac-L1A). This modification seems to favor the perturbation of the lipid core of the bilayer by the peptide, resulting in higher membrane lysis. In the present study, we used lipid monolayers and bilayers as membrane model systems to explore the impact of acetylation on the L1A lytic activity and its correlation with lipid-packing perturbation. The lytic activity investigated in giant unilamellar vesicles (GUVs) revealed that the acetylated peptide permeated the membrane at higher rates compared with L1A, and modified the membrane's mechanical properties, promoting shape changes. The peptide secondary structure and the changes in the environment of the tryptophan upon adsorption to large unilamellar vesicles (LUVs) were monitored by circular dichroism (CD) and red-edge excitation shift experiments (REES), respectively. These experiments showed that the N-terminus acetylation has an important effect on both, peptide secondary structure and peptide insertion into the bilayer. This was also confirmed by experiments of insertion into lipid monolayers. Compression isotherms for peptide/lipid mixed films revealed that ac-L1A dragged lipid molecules to the more disordered phase, generating a more favorable environment and preventing the lipid molecules from forming stiff films. Enthalpy changes in the main phase transition of the lipid membrane upon peptide insertion suggested that the acetylated peptide induced higher impact than the non-acetylated one on the thermotropic behavior of anionic vesicles.     

Characterization of endocytic uptake of MK2‐inhibitor peptides

Cell penetrating peptides (CPP) have been widely used to increase the cellular delivery of their associated cargo. Multiple modes of uptake have been identified; however, they cannot be predicted a priori. Elucidating these mechanisms is important for understanding peptide function as well as further optimizing cellular delivery. We have developed a class of mitogen activated protein kinase activated protein kinase 2 (MK2) inhibitor peptides, named FAK and YARA that utilize CPP domains to gain cellular access. In this study, we investigate the mechanism of endocytosis of these MK2 inhibitors by examining the uptake of fluorescently labeled peptide in human monocyte (THP‐1) and mesothelial cells, and looking for colocalization with known markers of endocytosis. Our results indicate that uptake of the MK2 inhibitors was minimally enhanced by the addition of the fluorescent label, and that the type of endocytosis used by the inhibitor depends on several factors including concentration, cell type, and which CPP was used. We found that in THP‐1 cells, the uptake of YARA occurred primarily via macropinocytosis, whereas FAK entered via all three mechanisms of endocytosis examined in this study. In mesothelial cells, uptake of YARA occurred via caveolae‐mediated endocytosis, but became less specific at higher concentrations; whereas uptake of FAK occurred through clathrin‐mediated endocytosis. In all cases, the delivery resulted in active inhibition of MK2. In summary, the results support endocytic uptake of fluorescently labeled FAK and YARA in two different cell lines, with the mechanism of uptake dependent on extracellular concentration, cell type, and choice of CPP. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Direct visualization of membrane leakage induced by the antibiotic peptides: maculatin, citropin, and aurein

Membrane lysis caused by antibiotic peptides is often rationalized by means of two different models: the so-called carpet model and the pore-forming model. We report here on the lytic activity of antibiotic peptides from Australian tree frogs, maculatin 1.1, citropin 1.1, and aurein 1.2, on POPC or POPC/POPG model membranes. Leakage experiments using fluorescence spectroscopy indicated that the peptide/lipid mol ratio necessary to induce 50% of probe leakage was smaller for maculatin compared with aurein or citropin, regardless of lipid membrane composition. To gain further insight into the lytic mechanism of these peptides we performed single vesicle experiments using confocal fluorescence microscopy. In these experiments, the time course of leakage for different molecular weight (water soluble) fluorescent markers incorporated inside of single giant unilamellar vesicles is observed after peptide exposure. We conclude that maculatin and its related peptides demonstrate a pore-forming mechanism (differential leakage of small fluorescent probe compared with high molecular weight markers). Conversely, citropin and aurein provoke a total membrane destabilization with vesicle burst without sequential probe leakage, an effect that can be assigned to a carpeting mechanism of lytic action. Additionally, to study the relevance of the proline residue on the membrane-action properties of maculatin, the same experimental approach was used for maculatin-Ala and maculatin-Gly (Pro-15 was replaced by Ala or Gly, respectively). Although a similar peptide/lipid mol ratio was necessary to induce 50% of leakage for POPC membranes, the lytic activity of maculatin-Ala and maculatin-Gly decreased in POPC/POPG (1:1 mol) membranes compared with that observed for the naturally occurring maculatin sequence. As observed for maculatin, the lytic action of Maculatin-Ala and maculatin-Gly is in keeping with the formation of pore-like structures at the membrane independently of lipid composition.     

Membrane translocation of penetratin and its derivatives in different cell lines

The third helix of the homeodomain of the Antennapedia homeoprotein can translocate through the cell membrane into the nucleus and can be used as an intracellular vehicle for the delivery of oligopeptides and oligonucleotides. A 16-amino acid-long peptide fragment, called penetratin, is internalized by the cells in a specific, non-receptor-mediated manner. For a better understanding of the mechanism of the transfer, penetratin and two analogs were synthesized:The conformation of penetratin peptides 1-3 was examined in both extracellular matrix-mimetic and membrane-mimetic environments. (1)H-NMR and CD spectroscopic measurements were performed in mixtures of TFE/water with different ratios. Peptides 1-3 were labeled by reacting their N-terminal free amino group with fluorescein isothiocyanate (FITC). Membrane translocation of the labelled peptides was studied with cell cultures [WEHI 164 murine fibrosarcoma cells (WC/1); chicken fibroblast cells (CEC-32); chicken monocytic cells (HD-11); human fibroblast (SV 80) and human monocytic cells (MonoMac-6)]. Confocal laser scanning microscopy and flow cytometry assay were used to study membrane translocation. Amphiphilicity was calculated for each peptide. In our experiments all the penetratin peptides penetrated into the cells. Helical conformation and membrane translocation ability showed little correlation: substitution of the two Trp with Phe increased the stability of helical conformation but decreased membrane translocation activity. The results of fluorescence microscopy and flow cytometry show that penetratin can be translocated into the cells by two mechanisms: endocytosis and direct transport through the cell membrane.     

Identification and structural characterization of a new pro-apoptotic cyclic octapeptide cyclosaplin from somatic seedlings of Santalum album L.

Small cyclic peptides exhibiting potent biological activity have great potential for anticancer therapy. An antiproliferative cyclic octapeptide, cyclosaplin was purified from somatic seedlings of Santalum album L. (sandalwood) using gel filtration and RP-HPLC separation process. The molecular mass of purified peptide was found to be 858 Da and the sequence was determined by MALDI-ToF-PSD-MS as ‘RLGDGCTR’ (cyclic). The cytotoxic activity of the peptide was tested against human breast cancer (MDA-MB-231) cell line in a dose and time-dependent manner. The purified peptide exhibited significant antiproliferative activity with an IC₅₀ 2.06 μg/mL. In a mechanistic approach, apoptosis was observed in differential microscopic studies for peptide treated MDA-MB-231 cells, which was further confirmed by mitochondrial membrane potential, DNA fragmentation assay, cell cycle analysis and caspase 3 activities. The modeling and docking experiments revealed strong affinity (kcal/mol) of peptide toward EGFR and procaspase 3. The co-localization studies revealed that the peptide sensitizes MDA-MB-231 cells by possibly binding to EGFR and induces apoptosis. This unique cyclic octapeptide revealed to be a favorable candidate for development of anticancer agents.     

Single giant unilamellar vesicle method reveals effect of antimicrobial peptide magainin 2 on membrane permeability

It is thought that magainin 2, an antimicrobial peptide, acts by binding to lipid membranes. Recent studies using a suspension of large unilamellar vesicles (LUVs) indicate that magainin 2 causes gradual leakage from LUVs containing negatively charged lipids. However, the details of the characteristics of the membrane permeability and the mechanism of pore formation remain unclear. In this report, we investigated the interaction of magainin 2 with single giant unilamellar vesicles (GUVs) composed of a dioleoylphosphatidylcholine and dioleoylphosphatidylglycerol mixture (50% DOPG/50% DOPC GUVs) containing the fluorescent dye, calcein, by phase contrast, fluorescence microscopy using the single GUV method. Low concentrations (3-10 microM) of magainin 2 caused the rapid leakage of calcein from single GUVs but did not disrupt the liposomes or change the membrane structure, showing directly that magainin 2 forms membrane pores through which calcein leaked. The rapid leakage of calcein from a GUV started stochastically, and once it began, the complete leakage occurred rapidly (6-60 s). The fraction of completely leaked GUV, P(L), increased with time and also with an increase in magainin 2 concentration. Shape changes in these GUVs occurred prior to the pore formation and also at lower concentrations of magainin 2, which could not induce the pore formation. Their analysis indicates that binding of magainin 2 to the external monolayer of the GUV increases its membrane area, thereby raising its surface pressure. The addition of lysophosphatidylcholine into the external monolayer of GUVs increased P(L). On the basis of these results, we propose the two-state transition model for the pore formation.     

Giant unilamellar vesicles electroformed from native membranes and organic lipid mixtures under physiological conditions

In recent years, giant unilamellar vesicles (GUVs) have become objects of intense scrutiny by chemists, biologists, and physicists who are interested in the many aspects of biological membranes. In particular, this "cell size" model system allows direct visualization of particular membrane-related phenomena at the level of single vesicles using fluorescence microscopy-related techniques. However, this model system lacks two relevant features with respect to biological membranes: 1), the conventional preparation of GUVs currently requires very low salt concentration, thus precluding experimentation under physiological conditions, and 2), the model system lacks membrane compositional asymmetry. Here we show for first time that GUVs can be prepared using a new protocol based on the electroformation method either from native membranes or organic lipid mixtures at physiological ionic strength. Additionally, for the GUVs composed of native membranes, we show that membrane proteins and glycosphingolipids preserve their natural orientation after electroformation. We anticipate our result to be important to revisit a vast variety of findings performed with GUVs under low- or no-salt conditions. These studies, which include results on artificial cell assembly, membrane mechanical properties, lipid domain formation, partition of membrane proteins into lipid domains, DNA-lipid interactions, and activity of interfacial enzymes, are likely to be affected by the amount of salt present in the solution.     

Characterization of antilytic peptide antibody: application for the detection of lytic‐based hybrid peptide in serum samples

We previously reported that a novel targeted drug termed hybrid epidermal growth factor receptor (EGFR)‐lytic peptide, made by chemical conjugation of targeted binding peptide and cell‐killing, lytic‐peptide components, has selective cytotoxic activity that allows it to discriminate between normal and cancer cells. In addition, in vivo analysis revealed that this hybrid peptide displays significant antitumor activity in a xenograft model of human breast and pancreatic cancer in mice. Here, we characterized antilytic peptide antibody, which was raised from rabbit serum using the antigen of lytic peptide conjugated with keyhole limpet hemocyanin. It was found that antilytic peptide antibody is specific to the lytic peptide as assessed by both ELISA and surface plasmon resonance analysis and can also bind to EGFR‐lytic peptide. Epitope mapping analysis using Biacore showed that two successive lysine regions in the lytic‐peptide sequence are significant for recognition by this antibody. In addition, it was shown that this antibody can detect lytic‐based hybrid peptide in serum samples from mouse blood and also in cultured breast cancer MDA‐MB‐231 cell samples by immunocytochemical staining experiments. It was found that the maximum concentrations of this peptide in serum were reached within 15–30 min of i.v. administration of EGFR‐lytic peptide to mice. These results indicate that this antibody will be a useful tool for the detection of lytic‐based peptides to investigate their in vivo stability and pharmacokinetics. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Fungicidal effect of antimicrobial peptide, PMAP-23, isolated from porcine myeloid against Candida albicans

The antifungal activity and mechanism of a 23-mer peptide, PMAP-23, derived from pig myeloid was investigated. PMAP-23 displayed strong antifungal activity against yeast and mold. To investigate the antifungal mechanism of PMAP-23, fluorescence activated flow cytometry and confocal laser scanning microscopy were performed. Candida albicans treated with PMAP-23 showed higher fluorescence intensity by propidium iodide(PI) staining, which was similar to that of Melittin than untreated cells. Confocal microscopy showed that the peptide was located in the plasma membrane. The action of peptides against fungal cell membranes was examined by treating prepared protoplasts of C. albicans with the peptide and lipid vesicle titration test. The result showed that the peptide prevented the regeneration of fungal cell walls and induced release of the fluorescent dye trapped in the artificial membrane vesicles, indicating that the peptide exerts its antifungal activity by acting on the plasma lipid membrane.     

The human prothrombin kringle-2 derived peptide, NSA9, is internalized into bovine capillary endothelial cells through endocytosis and energy-dependent pathways

Human prothrombin kringle-2 and its partial peptide, NSA9 (NSAVQLVEN), have been reported to have potent anti-angiogenic activities. Here, the internalization mechanism of NSA9 into bovine capillary endothelial (BCE) cells was examined using lactate dehydrogenase (LDH) release assay, fluorescence microscopy, and flow cytometry. LDH release assay results suggested that the integrity of the BCE cell membrane was unaffected by NSA9. Fluorescence microscopy indicated that internalized NSA9 was localized in the cytoplasm around the nucleus, and showed a punctuated fluorescence pattern, which is indicative of endocytic vesicles. Also, the cellular internalization of NSA9 is significantly inhibited by depletion of the cellular ATP pool, endocytosis inhibitors such as chloroquine and nocodazole, and incubation at low temperature (4 degrees C). In addition, the anti-proliferative activity of NSA9 against BCE cells was diminished in the presence of endocytosis or metabolic inhibitors. In conclusion, these results strongly suggest that NSA9 might exert its anti-proliferative activity through internalization into BCE cells by endocytosis and energy-dependent pathways.     

Antimicrobial peptide magainin 2-induced rupture of single giant unilamellar vesicles comprising E. coli polar lipids

Most antimicrobial peptides (AMPs) damage the cell membrane of bacterial cells and induce rapid leakage of the internal cell contents, which is a main cause of their bactericidal activity. One of the AMPs, magainin 2 (Mag), forms nanopores in giant unilamellar vesicles (GUVs) comprising phosphatidylcholine (PC) and phosphatidylglycerol (PG), inducing leakage of fluorescent probes. In this study, to elucidate the Mag-induced pore formation in lipid bilayer region in E. coli cell membrane, we examined the interaction of Mag with single GUVs comprising E. coli polar lipids (E. coli-lipid-GUVs). First, we investigated the Mag-induced leakage of a fluorescent probe AF488 from single E. coli-lipid-GUVs, and found that Mag caused rupture of GUVs, inducing rapid AF488 leakage. The rate constant of Mag-induced GUV rupture increased with the Mag concentration. Using fluorescence microscopy with a time resolution of 5 ms, we revealed the GUV rupture process: first, a small micropore was observed in the GUV membrane, then the pore radius increased within 50 ms without changing the GUV diameter, the thickness of the membrane at the pore rim concomitantly increased, and eventually membrane aggregates were formed. Mag bound to only the outer monolayer of the GUV before GUV rupture, which increased the area of the GUV bilayer. We also examined the physical properties of E. coli-lipid-GUVs themselves. We found that the rate constant of the constant tension-induced rupture of E. coli-lipid-GUVs was higher than that of PG/PC-GUVs. Based on these results, we discussed the Mag-induced rupture of E. coli-lipid-GUVs and its mechanism.     

A comprehensive model for the cellular uptake of cationic cell-penetrating peptides

The plasma membrane represents an impermeable barrier for most macromolecules. Still some proteins and so-called cell-penetrating peptides enter cells efficiently. It has been shown that endocytosis contributes to the import of these molecules. However, conflicting results have been obtained concerning the nature of the endocytic process. In addition, there have been new findings for an endocytosis-independent cellular entry. In this study, we provide evidence that the Antennapedia-homeodomain-derived antennapedia (Antp) peptide, nona-arginine and the HIV-1 Tat-protein-derived Tat peptide simultaneously use three endocytic pathways: macropinocytosis, clathrin-mediated endocytosis and caveolae/lipid-raft-mediated endocytosis. Antennapedia differs from Tat and R9 by the extent by which the different import mechanisms contribute to uptake. Moreover, at higher concentrations, uptake occurs by a mechanism that originates from spatially restricted sites of the plasma membrane and leads to a rapid cytoplasmic distribution of the peptides. Endocytic vesicles could not be detected, suggesting an endocytosis-independent mode of uptake. Heparinase treatment of cells negatively affects this import, as does the protein kinase C inhibitor rottlerin, expression of dominant-negative dynamin and chlorpromazine. This mechanism of uptake was observed for a panel of different cell lines. For Antp, significantly higher peptide concentrations and inhibition of endocytosis were required to induce its uptake. The relevance of these findings for import of biologically active cargos is shown.     

Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies

The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires α-helical conformation, the helix spanning residues 3–11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide:lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential (ζ) of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. The mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content. While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events – large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism.     

Role of membranotropic sequences from herpes simplex virus type I glycoproteins B and H in the fusion process

The entry of enveloped viruses involves attachment followed by close apposition of the viral and plasma membranes. Then, either on the cell surface or in an endocytotic vesicle, the two membranes fuse by an energetically unfavourable process requiring the destabilisation of membrane microenvironment in order to release the viral nucleocapsid into the cytoplasm. The core fusion machinery, conserved throughout the herpesvirus family, involves glycoprotein B (gB) and the non-covalently associated complex of glycoproteins H and L (gH/gL). Both gB and gH possess several hydrophobic domains necessary for efficient induction of fusion, and synthetic peptides corresponding to these regions are able to associate to membranes and induce fusion of artificial liposomes. Here, we describe the first application of surface plasmon resonance (SPR) to the study of the interaction of viral membranotropic peptides with model membranes in order to enhance our molecular understanding of the mechanism of membrane fusion. SPR spectroscopy data are supported by tryptophan fluorescence, circular dichroism and electron spin resonance spectroscopy (ESR). We selected peptides from gB and gH and also analysed the behaviour of HIV gp41 fusion peptide and the cationic antimicrobial peptide melittin. The combined results of SPR and ESR showed a marked difference between the mode of action of the HSV peptides and the HIV fusion peptide compared to melittin, suggesting that viral-derived membrane interacting peptides all act via a similar mechanism, which is substantially different from that of the non-cell selective lytic peptide melittin.