Interaction of antimicrobial peptides with biological and model membranes: structural and charge requirements for activity.
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Species right across the evolutionary scale from insects to mammals use peptides as part of their host-defense system to counter microbial infection. The primary structures of a large number of these host-defense peptides have been determined. While there is no primary structure homology, the peptides are characterized by a preponderance of cationic and hydrophobic amino acids. The secondary structures of many of the host-defense peptides have been determined by a variety of techniques. The acyclic peptides tend to adopt helical conformation, especially in media of low dielectric constant, whereas peptides with more than one disulfide bridge adopt beta-structures. Detailed investigations have indicated that a majority of these host-defense peptides exert their action by permeabilizing microbial membranes. In this review, we discuss structural and charge requirements for the interaction of endogenous antimicrobial peptides and short peptides that have been derived from them, with membranes. (+info)
Trachynilysin mediates SNARE-dependent release of catecholamines from chromaffin cells via external and stored Ca2+.
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Trachynilysin, a 159 kDa dimeric protein purified from stonefish (Synanceia trachynis) venom, dramatically increases spontaneous quantal transmitter release at the frog neuromuscular junction, depleting small clear synaptic vesicles, whilst not affecting large dense core vesicles. The basis of this insensitivity of large dense core vesicles exocytosis was examined using a fluorimetric assay to determine whether the toxin could elicit catecholamine release from bovine chromaffin cells. Unlike the case of the motor nerve endings, nanomolar concentrations of trachynilysin evoked sustained Soluble N-ethylmaleimide-sensitive fusion protein Attachment Protein REceptor-dependent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca2+. However, this response to trachynilysin does not rely on Ca2+ influx through voltage-activated Ca2+ channels because the secretion was only slightly affected by blockers of L, N and P/Q types. Instead, trachynilysin elicited a localized increase in intracellular fluorescence monitored with fluo-3/AM, that precisely co-localized with the increase of fluorescence resulting from caffeine-induced release of Ca2+ from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-induced exocytosis. Thus, the observed requirement of external Ca2+ for stimulation of large dense core vesicles exocytosis from chromaffin cells implicates plasma membrane channels that signal efflux of Ca2+ from intracellular stores. This study also suggests that the bases of exocytosis of large dense core vesicles from motor nerve terminals and neuroendocrine cells are distinct. (+info)
Involvement of extracellular signal-regulated kinase (ERK) in pardaxin-induced dopamine release from PC12 cells.
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Pardaxin (PX), an ionophore-peptide neurotoxin isolated from the fish Pardachirus marmoratus, induces neurotransmitter release from neuronal preparations by both calcium-dependent and calcium-independent mechanisms. The aim of the present study was to investigate the role of extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) in pardaxin-induced dopamine (DA) release. The experiments were performed on variants of the PC12 cell line, an established cellular model for investigating DA release. Time course experiments indicated that PX, at nontoxic concentrations, stimulated ERK1 and ERK2 within 5 to 15 min, measured with a dual phospho-ERK antibody. PX stimulation of ERK activity was calcium (Ca(2+))-dependent and followed by ERK translocation to the nucleus. This effect was temporally related to PX-induced exocytosis, and measured by [(3)H]dopamine release as well as by a vesicle fusion-based enzyme-linked immunosorbent assay. Blocking ERK activity with the specific mitogen-activated protein kinase kinase inhibitors PD98059 (50 microM for 45 min) and UO126 (30 microM for 30 min) inhibited PX-induced exocytosis in the presence but not in the absence of extracellular Ca(2+). These results suggest the essential role of ERKs in PX-induced DA release under physiological conditions and support the hypothesis that ERKs are involved in regulating exocytosis. (+info)
Skeletal muscle necrosis and regeneration after injection of Thalassophryne nattereri (niquim) fish venom in mice.
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Stings by Thalassophryne nattereri are responsible for envenomation of fishermen in north-eastern Brazil. Its venom induces prominent local tissue damage, characterized by pain, oedema and necrosis. The pathogenesis of acute muscle damage induced by T. nattereri venom was studied in mice. Intramuscular injection induced myonecrosis within the first hours. Some muscle cells presented a hypercontracted morphology, but most necrotic fibres were not hypercontracted, being instead characterized by a disorganization of myofibrils, with Z line loss, mitochondrial swelling and sarcolemmal disruption. In addition, thrombosis was observed histologically in venules and veins, together with vascular congestion and stasis, evidenced by intravital microscopy. Venom induced a rapid increment in serum creatine kinase (CK) levels, concomitant with a reduction in gastrocnemius muscle CK activity, whereas no increments in muscle lactic acid were detected. A rapid cytolytic effect was induced by the venom on C2C12 murine myoblasts in culture. The inflammatory reaction in affected muscle was characterized by oedema and scarce cellular infiltrate of polymorphonuclear leucocytes and macrophages, with a consequent delay in the removal of necrotic material. Skeletal muscle regeneration was partially impaired, as evidenced by the presence of regenerating fibres of variable size and by the increase of fibrotic tissue in endomysium and perimysium. It is suggested that T. nattereri venom affects muscle fibres by a direct cytotoxic effect, and that the vascular alterations described preclude a successful regenerative process. (+info)
Insertion and pore formation driven by adsorption of proteins onto lipid bilayer membrane-water interfaces.
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We describe the binding of proteins to lipid bilayers in the case for which binding can occur either by adsorption to the lipid bilayer membrane-water interface or by direct insertion into the bilayer itself. We examine in particular the case when the insertion and pore formation are driven by the adsorption process using scaled particle theory. The adsorbed proteins form a two-dimensional "surface gas" at the lipid bilayer membrane-water interface that exerts a lateral pressure on the lipid bilayer membrane. Under conditions of strong intrinsic binding and a high degree of interfacial converge, this pressure can become high enough to overcome the energy barrier for protein insertion. Under these conditions, a subtle equilibrium exists between the adsorbed and inserted proteins. We propose that this provides a control mechanism for reversible insertion and pore formation of proteins such as melittin and magainin. Next, we discuss experimental data for the binding isotherms of cytochrome c to charged lipid membranes in the light of our theory and predict that cytochrome c inserts into charged lipid bilayers at low ionic strength. This prediction is supported by titration calorimetry results that are reported here. We were furthermore able to describe the observed binding isotherms of the pore-forming peptides endotoxin (alpha 5-helix) and of pardaxin to zwitterionic vesicles from our theory by assuming adsorption/insertion equilibrium. (+info)
Giant miniature EPSCs at the hippocampal mossy fiber to CA3 pyramidal cell synapse are monoquantal.
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The mechanisms generating giant miniature excitatory postsynaptic currents (mEPSCs) were investigated at the hippocampal mossy fiber (MF) to CA3 pyramidal cell synapse in vitro. These giant mEPSCs have peak amplitudes as large as 1,700 pA (13.6 nS) with a mean maximal mEPSC amplitude of 366 +/- 20 pA (mean +/- SD; 5 nS; n = 25 cells). This is compared with maximal mEPSC amplitudes of <100 pA typically observed at other cortical synapses. We tested the hypothesis that giant mEPSCs are due to synchronized release of multiple vesicles across the release sites of single MF boutons by directly inducing vesicular release using secretagogues. If giant mEPSCs result from simultaneous multivesicular release, then secretagogues should increase the frequency of small mEPSCs selectively. We found that hypertonic sucrose and spermine increased the frequency of both small and giant mEPSCs. The peptide toxin secretagogues alpha-latrotoxin and pardaxin failed to increase the frequency of giant mEPSCs, but the possible lack of tissue penetration of the toxins make these results equivocal. Because a multiquantal release mechanism is likely to be mediated by a spontaneous increase in presynaptic calcium concentration, a monoquantal mechanism is further supported by results that giant mEPSCs were not affected by manipulations of extracellular or intracellular calcium concentrations. In addition, reducing the temperature of the bath to 15 degrees C failed to desynchronize the rising phases of giant mEPSCs. Together these data suggest that the giant mEPSCs are generated via a monovesicular mechanism. Three-dimensional analysis through serial electron microscopy of the MF boutons revealed large clear vesicles (50 to 160 nm diam) docked presynaptically at the MF synapse in sufficient numbers to account for the amplitude and frequency of giant mEPSCs recorded electrophysiologically. It is concluded that release of the contents of a single large clear vesicle generates giant mEPSCs at the MF to CA3 pyramidal cell synapse. (+info)
Pardaxin stimulation of phospholipases A2 and their involvement in exocytosis in PC-12 cells.
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Pardaxin (PX) is a voltage-dependent ionophore that stimulates catecholamine exocytosis from PC-12 pheochromocytoma cells both in the presence and absence of extracellular calcium. Using a battery of phospholipase A(2) inhibitors we show that PX stimulation of phospholipase A(2) (PLA(2)) enzymes is coupled with induction of exocytosis. We investigated the relationship between PX-induced PLA(2) activity and neurotransmitter release by measuring the levels of arachidonic acid (AA), prostaglandin E(2) (PGE(2)), and dopamine release. In the presence of extracellular calcium, the cytosolic PLA(2) inhibitor arachidonyl trifluoromethyl ketone (AACOCF(3)) inhibited by 100, 70, and 73%, respectively, the release of AA, PGE(2), and dopamine induced by PX. The mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor 2'-amino-3'-methoxyflavone (PD98059) reduced by 100 and 82%, respectively, the release of AA and PGE(2) induced by PX. In the absence of extracellular calcium, the calcium-independent PLA(2) (iPLA(2)) inhibitors methyl arachidonyl fluorophosphonate, AACOCF(3), and bromoenol lactone (BEL) inhibited by 80 to 90% PX stimulation of AA release, by 65 to 85% PX stimulation of PGE(2) release, and by 80 to 90% PX-induced dopamine release. Using vesicle fusion-based enzyme-linked immunosorbent assay we found similar levels of inhibition of PX-induced exocytosis by these inhibitors. Also, PX induced the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor complexes, an effect that was augmented by N-methylmaleimide. This complex formation was completely inhibited by BEL. Botulinum toxins type C1 and F significantly inhibited the release of AA, PGE(2), and dopamine induced by PX. Our data suggest that PX stimulates exocytosis by activating cystolic PLA(2) and iPLA(2), leading to the generation of AA and eicosanoids, which, in turn, stimulate vesicle competence for fusion and neurotransmitter release. (+info)
Membrane composition determines pardaxin's mechanism of lipid bilayer disruption.
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Pardaxin is a membrane-lysing peptide originally isolated from the fish Pardachirus marmoratus. The effect of the carboxy-amide of pardaxin (P1a) on bilayers of varying composition was studied using (15)N and (31)P solid-state NMR of mechanically aligned samples and differential scanning calorimetry (DSC). (15)N NMR spectroscopy of [(15)N-Leu(19)]P1a found that the orientation of the peptide's C-terminal helix depends on membrane composition. It is located on the surface of lipid bilayers composed of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and is inserted in lipid bilayers composed of 1,2-dimyristoyl-phosphatidylcholine (DMPC). The former suggests a carpet mechanism for bilayer disruption whereas the latter is consistent with a barrel-stave mechanism. The (31)P chemical shift NMR spectra showed that the peptide significantly disrupts lipid bilayers composed solely of zwitterionic lipids, particularly bilayers composed of POPC, in agreement with a carpet mechanism. P1a caused the formation of an isotropic phase in 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) lipid bilayers. This, combined with DSC data that found P1a reduced the fluid lamellar-to-inverted hexagonal phase transition temperature at very low concentrations (1:50,000), is interpreted as the formation of a cubic phase and not micellization of the membrane. Experiments exploring the effect of P1a on lipid bilayers composed of 4:1 POPC:cholesterol, 4:1 POPE:cholesterol, 3:1 POPC:1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), and 3:1 POPE:POPG were also conducted, and the presence of anionic lipids or cholesterol was found to reduce the peptide's ability to disrupt bilayers. Considered together, these data demonstrate that the mechanism of P1a is dependent on membrane composition. (+info)