RESUMO
Anionic lipid membrane electrostatic potential and solution pH can influence cationic peptide adsorption to these bilayers, especially those containing simultaneously acid and basic residues. Here, we investigate the effects of the pH solution on MP1 (IDWKKLLDAAKQIL-NH2) adsorption to anionic (7POPC:3POPG) lipid vesicles in comparison to its analog H-MP1, with histidines substituting lysines. We used the association of adsorption isotherms and constant pH molecular dynamic simulations (CpHMD) to explore the effects of membrane potential and pH on peptides' adsorption on this lipid membrane. We analyzed the fluorescence and zeta potential adsorption isotherms using the Gouy-Chapman theory. In CpHMD simulations for the peptides in solution and adsorbed on the lipid bilayer, we used the conformations obtained by conventional MD simulations at a µs timescale. Non-equilibrium Monte Carlo simulations provided the protonation states of acidic and basic residues. CpHMD showed average pKa shifts of two to three units, resulting in a higher net charge for the analog than for MP1, strongly modulating the peptide adsorption. The fractions of the protonation of acidic and basic residues and the peptides' net charges obtained from the analysis of the adsorption isotherms were in reasonable agreement with those from CpHMD. MP1 adsorption was almost insensitive to solution pH. H-MP1 was much more sensitive to partitioning, at acidic pH, with an affinity ten times higher than in neutral ones.
RESUMO
Antimicrobial peptides (AMPs) are part of the innate immune system of many species. AMPs are short sequences rich in charged and non-polar residues. They act on the lipid phase of the plasma membrane without requiring membrane receptors. Polybia-MP1 (MP1), extracted from a native wasp, is a broad-spectrum bactericide, an inhibitor of cancer cell proliferation being non-hemolytic and non-cytotoxic. MP1 mechanism of action and its adsorption mode is not yet completely known. Its adsorption to lipid bilayer and lytic activity is most likely dependent on the ionization state of its two acidic and three basic residues and consequently on the bulk pH. Here we investigated the effect of bulk acidic (pH 5.5) and neutral pH (7.4) solution on the adsorption, insertion, and lytic activity of MP1 and its analog H-MP1 to anionic (7POPC:3POPG) model membrane. H-MP1 is a synthetic analog of MP1 with lysines replaced by histidines. Bulk pH changes could modulate this peptide efficiency. The combination of different experimental techniques and molecular dynamics (MD) simulations showed that the adsorption, insertion, and lytic activity of H-MP1 are highly sensitive to bulk pH in opposition to MP1. The atomistic details, provided by MD simulations, showed peptides contact their N-termini to the bilayer before the insertion and then lay parallel to the bilayer. Their hydrophobic faces inserted into the acyl chain phase disturb the lipid-packing.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Bicamadas Lipídicas/química , Venenos de Vespas/química , Adsorção , Animais , Histidina/análise , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , VespasRESUMO
Polybia-MP1 is an antimicrobial peptide that shows a decreased activity in membranes with cholesterol (CHO). Since it is now accepted that hopanoids act as sterol-surrogates in some sterol-lacking bacteria, we here inquire about the impact of Polybia-MP1 on membranes containing the hopanoid diplopterol (DP) in comparison to membranes with CHO. We found that, despite the properties induced on lipid membranes by DP are similar to those induced by CHO, the effect of Polybia-MP1 on membranes with CHO or DP was significantly different. DP did not prevent dye release from LUVs, nor the insertion of Polybia-MP1 into monolayers, and peptide-membrane affinity was higher for those with DP than with CHO. Zeta potentials ( ζ ) for DP-containing LUVs showed a complex behavior at increasing peptide concentration. The effect of the peptide on membrane elasticity, investigated by nanotube retraction experiments, showed that peptide addition softened all membrane compositions, but membranes with DP got stiffer at long times. Considering this, and the ζ results, we propose that peptides accumulate at the interface adopting different arrangements, leading to a non-monotonic behavior. Possible correlations with cell membranes were inquired testing the antimicrobial activity of Polybia-MP1 against hopanoid-lacking bacteria pre-incubated with DP or CHO. The fraction of surviving cells was lower in cultures incubated with DP compared to those incubated with CHO. We propose that the higher activity of Polybia-MP1 against some bacteria compared to mammalian cells is not only related to membrane electrostatics, but also the composition of neutral lipids, particularly the hopanoids, could be important.
RESUMO
Solitary wasps use their stinging venoms for paralyzing insect or spider prey and feeding them to their larvae. We have surveyed bioactive substances in solitary wasp venoms, and found antimicrobial peptides together with some other bioactive peptides. Eumenine mastoparan-AF (EMP-AF) was the first to be found from the venom of the solitary eumenine wasp Anterhynchium flavomarginatum micado, showing antimicrobial, histamine-releasing, and hemolytic activities, and adopting an α-helical secondary structure under appropriate conditions. Further survey of solitary wasp venom components revealed that eumenine wasp venoms contained such antimicrobial α-helical peptides as the major peptide component. This review summarizes the results obtained from the studies of these peptides in solitary wasp venoms and some analogs from the viewpoint of (1) chemical and biological characterization; (2) physicochemical properties and secondary structure; and (3) channel-like pore-forming properties.
Assuntos
Anti-Infecciosos/química , Conformação Proteica em alfa-Hélice , Venenos de Vespas/análise , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Venenos de Vespas/química , Venenos de Vespas/farmacologiaRESUMO
Peptide sequences containing acidic and basic residues could potentially have their net charges modulated by bulk pH with a possible influence on their lytic activity in lipid vesicles. The present study reports on a biophysical investigation of these modulatory effects on the synthetic mastoparan-like peptide L1A (IDGLKAIWKKVADLLKNT-NH2). At pH 10.0 L1A was 6 times more efficient in lysing large anionic (1-palmitoyl-oleoyl-sn-glycero-3-phosphocholine (POPC):1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG)/(8:2)) unilamellar vesicles (LUVs) than at pH 4.0. Despite the reduction of 60% in the L1A net charge in basic pH its affinity for this vesicle was almost insensitive to pH. On the other hand, L1A insertion into monolayers was dramatically influenced by subphase condition, showing that, in the neutral and basic subphases, the peptide induced surface pressure changes that surpassed the membrane lateral pressure, being able to destabilize a bilayer structure. In addition, in the basic subphase, visualization of the compression isotherms of co-spread 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC):POPG (8:2) + 4.8 mol% L1A showed that the peptide induced significant changes in solid lipid domains, indicating its capability in perturbing lipid-packing. An insight into L1A lytic activity was also obtained in giant unilamellar vesicles (GUVs) using phase contrast microscopy. The suppression of L1A lytic activity at acidic pH is in keeping with its lower insertion capability and ability to disturb the lipid monolayer. The lytic activity observed under neutral and basic conditions showed a quick and stochastic leakage following a lag-time. The permeability and the leakage-time averaged over at least 14 single GUVs were dependent on the bulk condition. At basic pH, permeability is higher and quicker than in a neutral medium in good accordance with the lipid-packing perturbation.
Assuntos
Peptídeos/síntese química , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Venenos de Vespas/síntese química , Ânions/síntese química , Ânions/química , Concentração de Íons de Hidrogênio , Peptídeos e Proteínas de Sinalização Intercelular , Tamanho da Partícula , Peptídeos/química , Propriedades de Superfície , Venenos de Vespas/químicaRESUMO
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.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos/química , Processamento de Proteína Pós-Traducional , Venenos de Vespas/química , Acetilação , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Dicroísmo Circular , Peptídeos e Proteínas de Sinalização Intercelular , Bicamadas Lipídicas , Fluidez de Membrana , Lipídeos de Membrana/química , Proteínas de Membrana/química , Permeabilidade , Fosfolipídeos/química , Estrutura Secundária de Proteína/efeitos dos fármacos , Espectrometria de Fluorescência , Temperatura , Triptofano/química , Lipossomas UnilamelaresRESUMO
Polybia-MP1 or simply MP1 (IDWKKLLDAAKQIL-NH2) is a peptide with broad-spectrum bactericidal activity and a strong inhibitory effect against cancer cells. The aim of this work was to evaluate the effect of biophysical properties such as membrane texture and film thickness on MP1 interaction with neutral and anionic lipid membranes. For this purpose, we first explored the peptide's surface behavior. MP1 showed high surface activity, adsorbing onto bare air/aqueous interfaces up to higher surface pressures than the collapse pressure of MP1 Langmuir films. The MP1-lipid membrane interaction was studied using Langmuir phosphatidylcholine and phosphatidylserine (PS) monolayers as model membrane systems. PS was chosen since this negatively charged lipid was found predominantly on the outer leaflet of tumor cells, and it enhances MP1 activity for PS-containing membranes to a greater extent than for other negatively charged lipids. MP1 incorporated into anionic PS monolayers, which show a liquid-expanded (LE) phase or LE-liquid-condensed (LC) phase coexistence, up to lipid-packing densities higher than those of cell membranes. The mixed lipid/MP1 films were explored by Brewster angle microscopy and atomic force microscopy. MP1 partitioned preferentially into the LE phase state of PS films, and were thus excluded from the coexisting LC phase. This interaction had strong electrostatic bases: in pure water, the lipid-peptide interaction was strong enough to induce formation of reversible lipid-peptide 3D structures associated with the interface. MP1 incorporation into the LE phase was accompanied by a shift of the phase transition pressure to higher values and a thinning of the lipid film. These results showed a clear correlation between peptide penetration capacity and the presence or induction of the thin LE phase. This capacity to regulate membrane physical properties may be of relevance in the binding, incorporation and membrane selectivity of this promising antitumor peptide.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Fosfolipídeos/química , Lipossomas Unilamelares/metabolismo , Venenos de Vespas/química , Venenos de Vespas/metabolismo , Ânions/química , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Microscopia de Força Atômica , Concentração Osmolar , Propriedades de Superfície , Lipossomas Unilamelares/químicaRESUMO
The indiscriminate use of conventional antibiotics is leading to an increase in the number of resistant bacterial strains, motivating the search for new compounds to overcome this challenging problem. Antimicrobial peptides, acting only in the lipid phase of membranes without requiring specific membrane receptors as do conventional antibiotics, have shown great potential as possible substituents of these drugs. These peptides are in general rich in basic and hydrophobic residues forming an amphipathic structure when in contact with membranes. The outer leaflet of the prokaryotic cell membrane is rich in anionic lipids, while the surface of the eukaryotic cell is zwitterionic. Due to their positive net charge, many of these peptides are selective to the prokaryotic membrane. Notwithstanding this preference for anionic membranes, some of them can also act on neutral ones, hampering their therapeutic use. In addition to the electrostatic interaction driving peptide adsorption by the membrane, the ability of the peptide to perturb lipid packing is of paramount importance in their capacity to induce cell lysis, which is strongly dependent on electrostatic and hydrophobic interactions. In the present research, we revised the adsorption of antimicrobial peptides by model membranes as well as the perturbation that they induce in lipid packing. In particular, we focused on some peptides that have simultaneously acidic and basic residues. The net charges of these peptides are modulated by pH changes and the lipid composition of model membranes. We discuss the experimental approaches used to explore these aspects of lipid membranes using lipid vesicles and lipid monolayer as model membranes.
RESUMO
Polybia-MP1 (IDWKKLLDAAKQIL-NH2) is a lytic peptide from the Brazilian wasp venom with known anti-cancer properties. Previous evidence indicates that phosphatidylserine (PS) lipids are relevant for the lytic activity of MP1. In agreement with this requirement, phosphatidylserine lipids are translocated to the outer leaflet of cells, and are available for MP1 binding, depending on the presence of liquid-ordered domains. Here, we investigated the effect of PS on MP1 activity when this lipid is reconstituted in membranes of giant or large liposomes with different lipid-phase states. By monitoring the membrane and soluble luminal content of giant unilamellar vesicles (GUVs), using fluorescence confocal microscopy, we were able to determine that MP1 has a pore-forming activity at the membrane level. Liquid-ordered domains, which were phase-separated within the membrane of GUVs, influenced the pore-forming activity of MP1. Experiments evaluating the membrane-binding and lytic activity of MP1 on large unilamellar vesicles (LUVs), with the same lipid composition as GUVs, demonstrated that there was synergy between liquid-ordered domains and PS, which enhanced both activities. Based on our findings, we propose that the physicochemical properties of cancer cell membranes, which possess a much higher concentration of PS than normal cells, renders them susceptible to MP1 binding and lytic pore formation. These results can be correlated with MP1's potent and selective anti-cancer activity and pave the way for future research to develop cancer therapies that harness and exploit the properties of MP1.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Membrana Celular/metabolismo , Lipídeos de Membrana/metabolismo , Fosfatidilserinas/metabolismo , Lipossomas Unilamelares/metabolismo , Venenos de Vespas/metabolismo , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Transporte Biológico , Linhagem Celular Tumoral , Membrana Celular/química , Colesterol/química , Colesterol/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Cinética , Lipídeos de Membrana/química , Especificidade de Órgãos , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/química , Porosidade , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Lipossomas Unilamelares/química , Venenos de Vespas/química , VespasRESUMO
We investigate the effect of the N-terminus modification of the L1A, a synthetic octadecapeptide, on its helical content, affinity and lytic action in model membranes and on its hemolytic and antibacterial activities. L1A and its acetylated analog displayed a selective antibacterial activity to Gram-negative bacteria without being hemolytic. The covalently linked 2-aminobezoic acid to the N-terminus impaired the antibacterial efficacy and increased hemolysis. Despite their lower net charge (+2), N-terminus modifications resulted in enhanced affinity and improved lytic efficiency in anionic vesicles. The analogs also showed higher helical content and consequently higher amphipathicity in these vesicles. The conformational analysis by molecular dynamics simulations in 30 % of TFE/water showed that the hydrophobic faces of the peptides are in close contact with CF3 groups of TFE while the hydrophilic faces with water molecules. Due to the loss of the amino charge, the N-termini of the analogs are buried in TFE molecules. The analysis of the pair distribution functions, obtained for the center of mass of the charged groups, has evidenced that the state of the N-terminus has influenced the possibility of different ion-pairing. The higher complexity of the bacterial cells compared with anionic vesicles hampers to establish correlations structure-function for the analogs.