Different membrane behaviour and cellular uptake of three basic arginine-rich peptides

Cell penetrating peptides (CPPs) are peptides displaying the ability to cross cell membranes and transport cargo molecules inside cells. Several uptake mechanisms (endocytic or direct translocation through the membrane) are being considered, but the interaction between the CPP and the cell membrane is certainly a preliminary key point to the entry of the peptide into the cell. In this study, we used three basic peptides: RL9 (RRLLRRLRR-NH(2)), RW9 (RRWWRRWRR-NH(2)) and R9 (RRRRRRRRR-NH(2)). While RW9 and R9 were internalised into wild type Chinese Hamster Ovary cells (CHO) and glycosaminoglycan-deficient CHO cells, at 4°C and 37°C, RL9 was not internalised into CHO cells. To better understand the differences between RW9, R9 and RL9 in terms of uptake, we studied the interaction of these peptides with model lipid membranes. The effect of the three peptides on the thermotropic phase behaviour of a zwitterionic lipid (DMPC) and an anionic lipid (DMPG) was investigated with differential scanning calorimetry (DSC). The presence of negative charges on the lipid headgroups appeared to be essential to trigger the peptide/lipid interaction. RW9 and R9 disturbed the main phase transition of DMPG, whereas RL9 did not induce significant effects. Isothermal titration calorimetry (ITC) allowed us to study the binding of these peptides to large unilamellar vesicles (LUVs). RW9 and R9 proved to have about ten fold more affinity for DSPG LUVs than RL9. With circular dichroism (CD) and NMR spectroscopy, the secondary structure of RL9, RW9 and R9 in aqueous buffer or lipid/detergent conditions was investigated. Additionally, we tested the antimicrobial activity of these peptides against Escherichia coli and Staphylococcus aureus, as CPPs and antimicrobial peptides are known to share several common characteristics. Only RW9 was found to be mildly bacteriostatic against E. coli. These studies helped us to get a better understanding as to why R9 and RW9 are able to cross the cell membrane while RL9 remains bound to the surface without entering the cell.     

Vesicle size-dependent translocation of penetratin analogs across lipid membranes

The recent discoveries of serious artifacts associated with the use of cell fixation in studies of the cellular uptake of cell-penetrating peptides (CPPs) have prompted a reevaluation of the current understanding of peptide-mediated cellular delivery. Following a report on the differential cellular uptake of a number of penetratin analogs in unfixed cells, we here investigate their membrane translocation abilities in large and giant unilamellar vesicles (LUVs and GUVs, respectively). Surprisingly, in contrast to the behavior in living cells, all peptides readily entered the giant vesicles (>1 microm) as proved by confocal microscopy, while none of them could cross the membranes of LUVs (100 nm). For determination of the location of the peptides in the LUVs, a new concept was introduced, based on sensitive resonance energy transfer (RET) measurements of the enhanced fluorescence of acceptor fluorophores present solely in the inner leaflet. An easily adopted method to prepare such asymmetrically labeled liposomes is described. The membrane insertion depths of the tryptophan moieties of the peptides were determined by use of brominated lipids and found to be very similar for all of the peptides studied. We also demonstrate that infrared spectroscopy on the lipid carbonyl stretch vibration peak is a convenient technique to determine phospholipid concentration.     

Cell biology meets biophysics to unveil the different mechanisms of penetratin internalization in cells

Although cell-penetrating peptides are widely used as molecular devices to cross membranes and transport molecules or nanoparticles inside cells, the underlying internalization mechanism for such behavior is still studied and discussed. One of the reasons for such a debate is the wide panel of chemically different cell-penetrating peptides or cargo that is used. Indeed the intrinsic physico-chemical properties of CPP and conjugates strongly affect the cell membrane recognition and therefore the internalization pathways. Altogether, the mechanisms described so far should be shared between two general pathways: endocytosis and direct translocation. As it is established now that one cell-penetrating peptide can internalize at the same time by these two different pathways, the balance between the two pathways relies on the binding of the cell-penetrating peptide or conjugate to specific cell membrane components (carbohydrates, lipids). Like endocytosis which includes clathrin- and caveolae-dependent processes and macropinocytosis, different translocation mechanisms could co-exist, an idea that emerges from recent studies. In this review, we will focus solely on penetratin membrane interactions and internalization mechanisms.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from +4 to +5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an alpha-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Enhanced translocation of amphiphilic peptides across membranes by transmembrane proteins

Cell membranes are phospholipid bilayers with a large number of embedded transmembrane proteins. Some of these proteins, such as scramblases, have properties that facilitate lipid flip-flop from one membrane leaflet to another. Scramblases and similar transmembrane proteins could also affect the translocation of other amphiphilic molecules, including cell-penetrating or antimicrobial peptides. We studied the effect of transmembrane proteins on the translocation of amphiphilic peptides through the membrane. Using two very different models, we consistently demonstrate that transmembrane proteins with a hydrophilic patch enhance the translocation of amphiphilic peptides by stabilizing the peptide in the membrane. Moreover, there is an optimum amphiphilicity because the peptide could become overstabilized in the transmembrane state, in which the peptide-protein dissociation is hampered, limiting the peptide translocation. The presence of scramblases and other proteins with similar properties could be exploited for more efficient transport into cells. The described principles could also be utilized in the design of a drug-delivery system by the addition of a translocation-enhancing peptide that would integrate into the membrane.     

Cationic peptide-induced remodelling of model membranes: direct visualization by in situ atomic force microscopy

Our understanding of how antimicrobial and cell-penetrating peptides exert their action at cell membranes would benefit greatly from direct visualization of their modes of action and possible targets within the cell membrane. We previously described how the cationic antimicrobial peptide, indolicidin, interacted with mixed zwitterionic planar lipid bilayers as a function of both peptide concentration and lipid composition [Shaw, J.E. et al., 2006. J. Struct. Biol. 154 (1), 42-58]. In the present report, in situ atomic force microscopy was used to characterize the interactions between three families of cationic peptides: (1) tryptophan-rich antimicrobial peptides--indolicidin and two of its analogues, (2) an amphiphilic alpha-helical membranolytic peptide--melittin, and (3) an arginine-rich cell-penetrating peptide--Tat with phase-separated planar bilayers containing 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC)/1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) or DOPC/N-stearoyl-D-erythro-sphingosylphosphorylcholine (SM)/cholesterol. We found that these cationic peptides all induced remodelling of the model membranes in a concentration, and family-dependent manner. At low peptide concentration, these cationic peptides, despite their different biological roles, all appeared to reduce the interfacial line tension at the domain boundary between the liquid-ordered and liquid-disordered domains. Only at high peptide concentration was the membrane remodelling induced by these peptides morphologically distinct among the three families. While the transformation caused by indolicidin and its analogues were structurally similar, the concentration required to initiate the transformation was strongly dependent on the hydrophobicity of the peptide. Our use of lipid compositions with no net charge minimized the electrostatic interactions between the cationic peptides and the model supported bilayers. These results suggest that peptides within the same functional family have a common mechanism of action, and that membrane insertion of short cationic peptides at low peptide concentration may also alter membrane structure through a common mechanism regardless of the peptide's origin.     

Membrane-active peptides: Binding,translocation, and flux in lipid vesicles

Recently, new and improved methods have been developed to measure translocation of membrane-active peptides (antimicrobial, cytolytic, and amphipathic cell-penetrating peptides) across lipid bilayer membranes. The hypothesis that translocation of membrane-active peptides across a lipid bilayer is determined by the Gibbs energy of insertion of the peptide into the bilayer is re-examined in the light of new experimental tests. The original hypothesis and its motivation are first revisited, examining some of the specific predictions that it generated, followed by the results of the initial tests. Translocation is understood as requiring two previous steps: binding and insertion in the membrane. The problem of peptide binding to membranes, its prediction, measurement, and calculation are addressed. Particular attention is given to understanding the reason for the need for amphipathic structures in the function of membrane-active peptides. Insertion into the membrane is then examined. Hydrophobicity scales are compared, and their influence on calculations is discussed. The relation between translocation and graded or all-or-none peptide-induced flux from or into lipid vesicles is also considered. Finally, the most recent work on translocation is examined, both experimental and from molecular dynamics simulations. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Cationic membrane peptides: atomic-level insight of structure–activity relationships from solid-state NMR

Many membrane-active peptides, such as cationic cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs), conduct their biological functions by interacting with the cell membrane. The interactions of charged residues with lipids and water facilitate membrane insertion, translocation or disruption of these highly hydrophobic species. In this review, we will summarize high-resolution structural and dynamic findings towards the understanding of the structure–activity relationship of lipid membrane-bound CPPs and AMPs, as examples of the current development of solid-state NMR (SSNMR) techniques for studying membrane peptides. We will present the most recent atomic-resolution structure of the guanidinium-phosphate complex, as constrained from experimentally measured site-specific distances. These SSNMR results will be valuable specifically for understanding the intracellular translocation pathway of CPPs and antimicrobial mechanism of AMPs, and more generally broaden our insight into how cationic macromolecules interact with and cross the lipid membrane.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from + 4 to + 5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an α-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Enhancing membrane disruption by targeting and multivalent presentation of antimicrobial peptides

In order to enhance the membrane disruption of antimicrobial peptides both targeting and multivalent presentation approaches were explored. The antimicrobial peptides anoplin and temporin L were conjugated via click chemistry to vancomycin and to di- and tetravalent dendrimers. The vancomycin unit led to enhanced membrane disruption of large unilamellar vesicles (LUVs) displaying the vancomycin target lipid II, but only for temporin L and not for anoplin. The multivalent presentation led to enhanced LUV membrane disruption in the case of anoplin but not for temporin L.     

Cell-penetrating peptide induces leaky fusion of liposomes containing late endosome-specific anionic lipid

Cationic cell-penetrating peptides (CPPs) are a promising vehicle for the delivery of macromolecular drugs. Although many studies have indicated that CPPs enter cells by endocytosis, the mechanisms by which they cross endosomal membranes remain elusive. On the basis of experiments with liposomes, we propose that CPP escape into the cytosol is based on leaky fusion (i.e., fusion associated with the permeabilization of membranes) of the bis(monoacylglycero)phosphate (BMP)-enriched membranes of late endosomes. In our experiments, prototypic CPP HIV-1 TAT peptide did not interact with liposomes mimicking the outer leaflet of the plasma membrane, but it did induce lipid mixing and membrane leakage as it translocated into liposomes mimicking the lipid composition of late endosome. Both membrane leakage and lipid mixing depended on the BMP content and were promoted at acidic pH, which is characteristic of late endosomes. Substitution of BMP with its structural isomer, phosphatidylglycerol (PG), significantly reduced both leakage of the aqueous probe from liposomes and lipid mixing between liposomes. Although affinity of binding to TAT was similar for BMP and PG, BMP exhibited a higher tendency to support the inverted hexagonal phase than PG. Finally, membrane leakage and peptide translocation were both inhibited by inhibitors of lipid mixing, further substantiating the hypothesis that cationic peptides cross BMP-enriched membranes by inducing leaky fusion between them.     

S4(13)-PV cell-penetrating peptide induces physical and morphological changes in membrane-mimetic lipid systems and cell membranes: implications for cell internalization

The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.     

Translocation of analogues of the antimicrobial peptides magainin and buforin across human cell membranes

Cationic antimicrobial peptides play important roles in innate immunity. Compared with extensive studies on peptide-bacteria interactions, little is known about peptide-human cell interactions. Using human cervical carcinoma HeLa and fibroblastic TM12 cells, we investigated the cellular uptake of fluorescent analogues of the two representative antimicrobial peptides magainin 2 and buforin 2 in comparison with the representative Arg-rich cell-penetrating Tat-(47-57) peptide (YGRKKRRQRRR). The dose, time, temperature, and energy dependence of translocation suggested that the three peptides cross cell membranes through different mechanisms. The magainin peptide was internalized within a time scale of tens of minutes. The cooperative concentration dependence of uptake suggested that the peptide forms a pore as an intermediate similar to the observations in model membranes. Furthermore, the translocation was coupled with cytotoxicity, which was larger for tumor HeLa cells. In contrast, the buforin peptide translocated within 10 min by a temperature-independent, less concentration-dependent passive mechanism without showing any significant cytotoxicity at the highest concentration investigated (100 microm). The uptake of the Tat peptide was proportional to the peptide concentration, and the concentration dependence was lost upon ATP depletion. The peptide exhibited a moderate cytotoxicity at higher concentrations. The time course did not show saturation even after 120 min. The buforin peptide, covalently attached to the 28-kDa green fluorescent protein, also entered cells, suggesting a potency of the peptide as a vector for macromolecular delivery into cells. However, the mechanism appeared to be different from that of the parent peptide.     

A novel amphipathic cell-penetrating peptide based on the N-terminal glycosaminoglycan binding region of human apolipoprotein E

In the direct cell membrane penetration, arginine-rich cell-penetrating peptides are thought to penetrate into cells across the hydrophobic lipid membranes. To investigate the effect of the amphipathic property of arginine-rich peptide on the cell-penetrating ability, we designed a novel amphipathic cell-penetrating peptide, A2-17, and its derivative, A2-17KR, in which all lysine residues are substituted with arginine residues, based on the glycosaminoglycan binding region in the N-terminal α-helix bundle of human apolipoprotein E. Isothermal titration calorimetry showed that A2-17 variants have a strong ability to bind to heparin with high affinity. Circular dichroism and tryptophan fluorescence measurements demonstrated that A2-17 variants bind to lipid vesicles with a structural change from random coil to amphipathic α-helix, being inserted into the hydrophobic membrane interiors. Flow cytometric analysis and confocal laser scanning microscopy demonstrated the great cell penetration efficiency of A2-17 variants into CHO-K1 cells when incubated at low peptide concentrations (2 μM or less), suggesting that the increased amphipathicity with α-helix formation enhances the cell membrane penetration ability of arginine-rich peptides. Interestingly, A2-17KR exhibited lower efficiency of cell membrane penetration compared to A2-17 despite of their similar binding affinity to lipid membranes. Since high peptide concentrations (typically >10 μM) are usually prerequisite for efficient cell penetration of arginine-rich peptides, A2-17 is a unique amphipathic cell-penetrating peptide that exhibits an efficient cell penetration ability even at low peptide concentrations.     

Effect of lipid composition on buforin II structure and membrane entry

Buforin II is a 21-amino acid polycationic antimicrobial peptide derived from a peptide originally isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans. It is hypothesized to target a wide range of bacteria by translocating into cells without membrane permeabilization and binding to nucleic acids. Previous research found that the structure and membrane interactions of buforin II are related to lipid composition. In this study, we used molecular dynamics (MD) simulations along with lipid vesicle experiments to gain insight into how buforin II interacts differently with phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) lipids. Fluorescent spectroscopic measurements agreed with the previous assertion that buforin II does not interact with pure PC vesicles. Nonetheless, the reduced entry of the peptide into anionic PG membranes versus neutral PC membranes during simulations correlates with the experimentally observed reduction in BF2 translocation through pure PG membranes. Simulations showing membrane entry into PC also provide insight into how buforin II may initially penetrate cell membranes. Our MD simulations also allowed us to consider how neutral PE lipids affect the peptide differently than PC. In particular, the peptide had a more helical secondary structure in simulations with PE lipids. A change in structure was also apparent in circular dichroism measurements. PE also reduced membrane entry in simulations, which correlates with decreased translocation in the presence of PE observed in previous studies. Together, these results provide molecular-level insight into how lipid composition can affect buforin II structure and function and will be useful in efforts to design peptides with desired antimicrobial and cell-penetrating properties.     

Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing

Laurdan and di-4-ANEPPDHQ are used as probes for membrane order, with a blue shift in emission for membranes in liquid-ordered (lo) phase relative to membranes in liquid-disordered (ld) phase. Their use as membrane order probes requires that their spectral shifts are unaffected by membrane proteins, which we have examined by using membrane inserting peptides and large unilamellar vesicles (LUVs). The transmembrane polypeptides, mastoparan and bovine prion protein-derived peptide (bPrPp), were added to LUVs of either lo or ld phase, up to 1:10 peptide/total lipid ratio. The excitation and emission spectra of laurdan and di-4-ANEPPDHQ in both lipid phases were unaltered by peptide addition. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic light scattering. The insertion efficiency of the polypeptides into LUVs was determined by measuring their secondary structure by circular dichroism. Mastoparan had an α-helical and bPrPp a β-strand conformation compatible with insertion into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ, supporting that the dyes are appropriate probes for assessing lipid order in cells.     

How to address CPP and AMP translocation? Methods to detect and quantify peptide internalization in vitro and in vivo (Review)

Membrane translocation is a crucial issue when addressing the activity of both cell-penetrating and antimicrobial peptides. Translocation is responsible for the therapeutic potential of cell-penetrating peptides as drug carriers and can dictate the killing mechanisms, selectivity and efficiency of antimicrobial peptides. It is essential to evaluate if the internalization of cell-penetrating peptides is mediated by endocytosis and if it is able to internalize attached cargoes. The mode of action of an antimicrobial peptide cannot be fully understood if it is not known whether the peptide acts exclusively at the membrane level or also at the cytoplasm. Therefore, experimental methods to evaluate and quantify translocation processes are of first importance. In this work, over 20 methods described in the literature for the assessment of peptide translocation in vivo and in vitro, with and without attached macromolecular cargoes, are discussed and their applicability, advantages and disadvantages reviewed. In addition, a classification of these methods is proposed, based on common approaches to detect translocation.     

Effects of hemagglutinin fusion peptide on poly(ethylene glycol)-mediated fusion of phosphatidylcholine vesicles.

The effects of hemagglutinin (HA) fusion peptide (X-31) on poly(ethylene glycol)- (PEG-) mediated vesicle fusion in three different vesicle systems have been compared: dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV) and palmitoyloleoylphosphatidylcholine (POPC) large unilamellar perturbed vesicles (pert. LUV). POPC LUVs were asymmetrically perturbed by hydrolyzing 2.5% of the outer leaflet lipid with phospholipase A(2) and removing hydrolysis products with BSA. The mixing of vesicle contents showed that these perturbed vesicles fused in the presence of PEG as did DOPC SUV, but unperturbed LUV did not. Fusion peptide had different effects on the fusion of these different types of vesicles: fusion was not induced in the absence of PEG or in unperturbed DOPC LUV even in the presence of PEG. Fusion was enhanced in DOPC SUV at low peptide surface occupancy but hindered at high surface occupancy. Finally, fusion was hindered in proportion to peptide concentration in perturbed POPC LUV. Contents leakage assays demonstrated that the peptide enhanced leakage in all vesicles. The peptide enhanced lipid transfer between both fusogenic and nonfusogenic vesicles. Peptide binding was detected in terms of enhanced tryptophan fluorescence or through transfer of tryptophan excited-state energy to membrane-bound diphenylhexatriene (DPH). The peptide had a higher affinity for vesicles with packing defects (SUV and perturbed LUV). Quasi-elastic light scattering (QELS) indicated that the peptide caused vesicles to aggregate. We conclude that binding of the fusion peptide to vesicle membranes has a significant effect on membrane properties but does not induce fusion. Indeed, the fusion peptide inhibited fusion of perturbed LUV. It can, however, enhance fusion between highly curved membranes that normally fuse when brought into close contact by PEG.     

The application of pH-sensitive fluorescent dyes in lactic acid bacteria reveals distinct extrusion systems for unmodified and conjugated dyes

Membrane translocation is a crucial issue when addressing the activity of both cell-penetrating and antimicrobial peptides. Translocation is responsible for the therapeutic potential of cell-penetrating peptides as drug carriers and can dictate the killing mechanisms, selectivity and efficiency of antimicrobial peptides. It is essential to evaluate if the internalization of cell-penetrating peptides is mediated by endocytosis and if it is able to internalize attached cargoes. The mode of action of an antimicrobial peptide cannot be fully understood if it is not known whether the peptide acts exclusively at the membrane level or also at the cytoplasm. Therefore, experimental methods to evaluate and quantify translocation processes are of first importance. In this work, over 20 methods described in the literature for the assessment of peptide translocation in vivo and in vitro, with and without attached macromolecular cargoes, are discussed and their applicability, advantages and disadvantages reviewed. In addition, a classification of these methods is proposed, based on common approaches to detect translocation.     

Interaction of cationic antimicrobial peptides with model membranes

A series of natural and synthetic cationic antimicrobial peptides from various structural classes, including alpha-helical, beta-sheet, extended, and cyclic, were examined for their ability to interact with model membranes, assessing penetration of phospholipid monolayers and induction of lipid flip-flop, membrane leakiness, and peptide translocation across the bilayer of large unilamellar liposomes, at a range of peptide/lipid ratios. All peptides were able to penetrate into monolayers made with negatively charged phospholipids, but only two interacted weakly with neutral lipids. Peptide-mediated lipid flip-flop generally occurred at peptide concentrations that were 3- to 5-fold lower than those causing leakage of calcein across the membrane, regardless of peptide structure. With the exception of two alpha-helical peptides V681(n) and V25(p,) the extent of peptide-induced calcein release from large unilamellar liposomes was generally low at peptide/lipid molar ratios below 1:50. Peptide translocation across bilayers was found to be higher for the beta-sheet peptide polyphemusin, intermediate for alpha-helical peptides, and low for extended peptides. Overall, whereas all studied cationic antimicrobial peptides interacted with membranes, they were quite heterogeneous in their impact on these membranes.