Interaction Of Melittin Research Articles

Melittin: a possible regulator of cancer proliferation in preclinical cell culture and animal models.

Melittin is a water-soluble cationic peptide derived from bee venom that has been thoroughly studied for the cure of different cancers. However, the unwanted interactions of melittin produce hemolytic and cytotoxic effects that hinder their therapeutic applications. To overcome the shortcomings, numerous research groups have adopted different approaches, including conjugation with tumor-targeting proteins, gene therapy, and encapsulation in nanoparticles, to reduce the non-specific cytotoxic effects and potentiate their anti-cancerous activity. This article aims to provide mechanistic insights into the chemopreventive activity of melittin and its nanoversion in combination with standard anti-cancer drugs for the treatment of cancer. We looked over the pertinent research on melittin's chemopreventive properties in online databases such as PubMed and Scopus. In the present article, the anti-cancerous effects of melittin on different cancers have been discussed very nicely, as have their possible mechanisms of action to act against different tumors. Besides, it interacts with different signal molecules that regulate the diverse pathways of cancerous cells, such as cell cycle arrest, apoptosis, metastasis, angiogenesis, and inflammation. We also discussed the recent progress in the synergistic combination of melittin with standard anti-cancer drugs and a nano-formulated version of melittin for targeted delivery to improve its anticancer potential.

Highly Synergistic Effects of Melittin With Vancomycin and Rifampin Against Vancomycin and Rifampin Resistant Staphylococcus epidermidis.

Methicillin-resistant Staphylococcus epidermidis (MRSE) strains are increasingly emerging as serious pathogens because they can be resistant to many antibiotics called multidrug resistance (MDR) that limit the therapeutic options. In the case of vancomycin- and rifampin-resistant MDR-MRSE, the physicians are not allowed to increase the doses of antibiotics because of severe toxicity. Accordingly, we investigated the synergistic activity of melittin antimicrobial peptide with vancomycin and rifampin against vancomycin-resistant, and rifampin-resistant MDR-MRSE isolates. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), fractional inhibitory concentration index (FICi), and fractional bactericidal concentration index (FBCi) of antimicrobial agents against isolates were determined. Coagulate activities and serum and salt stability as well as melittin cytotoxicity on the human embryonic kidney (HEK) 293 cells and human red blood cells (RBCs) at their synergistic concentrations. MIC and MBC values for melittin were in the range of 0.312–2.5 and 0.312–5, respectively. Results also showed that the interaction of melittin with drugs was highly synergistic in which the geometric means of FICi and FBCi were < 0.5. Induced synergism led to a decrease in melittin, rifampin, and vancomycin concentrations by 8–1,020, 2–16, and 4–16-folds, respectively. This phenomenon caused a reduction in melittin toxicity by which the synergistic concentration of melittin needed to kill bacteria did not show cytotoxicity and hemolytic activity. Besides, no coagulation activity was found for the synergistic and alone concentrations of melittin in both Prothrombin Time (PT) and Partial Thromboplastin Time (PTT). Interestingly, the antibacterial activity of melittin in Mueller Hinton Broth (MHB) containing human serum did no significant differences between MIC and MBC values of melittin in MHB and MHB containing 10% human serum. The present findings showed that the therapeutic index of melittin was improved by 32.08- and 12.82-folds when combined with vancomycin and rifampin, respectively. Taken together, the obtained data show that melittin alone was effective against MDR-MRSE isolates and this antimicrobial peptide showed highly synergistic effects with vancomycin and rifampin without causing toxicity. Therefore, the combination of melittin and traditional antibiotics could be a promising strategy for the treatment of infections caused by MDR-MRSE.

A Supramolecular Antidote to Macromolecular Toxins Prepared through Coassembly of Macrocyclic Amphiphiles.

Poisoning is a leading cause of admission to medical emergency departments and intensive care units. Supramolecular detoxification, which involves injecting supramolecular receptors that bind with toxins to suppress their biological activity, is an emerging strategy for poisoning treatment; it has few requirements and a broad application scope. However, it is still a formidable challenge to design supramolecular therapeutic materials as an antidote to macromolecular toxins, because the large size, flexible conformation, and presence of multiple and diverse binding sites of biomacromolecules hinder their recognition. Herein, a supramolecular antidote to macromolecular toxins is developed through the coassembly of macrocyclic amphiphiles, relying on heteromultivalent recognition between the coassembled components and toxic macromolecules. The coassembly of amphiphilic cyclodextrin and calixarene strongly and selectively captures melittin, a toxin studied herein; this imparts various therapeutic effects such as inhibiting the interactions of melittin with cell membranes, alleviating melittin cytotoxicity and hemolytic toxicity, reducing the mortality rate of melittin-poisoned mice, and mitigating damage to major organs. The use of the proposed antidote overcomes the limitation of supramolecular detoxification applicability to only small-molecular toxins. The antidote can also detoxify other macromolecular toxins as long as selective and strong binding is achieved because of the coassembling tunability.

Mixed Silane‐based Self‐assembled Monolayers Deposited on Fluorine Doped Tin Oxide as Model System for Development of Biosensors for Toxin Detection

AbstractIn this work mixed hybrid phospholipid bilayers (mhBLM) were deposited on fluorine doped tin oxide (FTO) films. Two component silane‐based self‐assembled monolayers (SAMs) formed on FTO surface trigger vesicle fusion and formation of mhBLMs which are stable, can be easily regenerated, and therefore, used for multiple experiments. We found that certain chemical and physical conditions under which mixed SAMs are fabricated translate into functional properties of mhBLMs. In all cases we observed interaction of melittin with mhBLMs demonstrating biological relevance of these biomimetic surface constructs and their possible application in biosensors for toxin detection.

Revisiting the Interaction of Melittin with Phospholipid Bilayers: The Effects of Concentration and Ionic Strength.

Melittin is an anti-microbial peptide (AMP) and one of the most studied membrane-disrupting peptides. There is, however, a lack of accurate measurements of the concentration-dependent kinetics and affinity of binding of melittin to phospholipid membranes. In this study, we used surface plasmon resonance spectroscopy to determine the concentration-dependent effect on the binding of melittin to 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayers in vesicles. Three concentration ranges were considered, and when combined, covered two orders of magnitudes (0.04 µM to 8 µM), corresponding to concentrations relevant to the membrane-disrupting and anti-microbial activities of melittin. Binding kinetics data were analysed using a 1:1 Langmuir-binding model and a two-state reaction model. Using in-depth quantitative analysis, we characterised the effect of peptide concentration, the addition of NaCl at physiological ionic strength and the choice of kinetic binding model on the reliability of the calculated kinetics and affinity of binding parameters. The apparent binding affinity of melittin for POPC bilayers was observed to decrease with increasing peptide/lipid (P/L) ratio, primarily due to the marked decrease in the association rate. At all concentration ranges, the two-state reaction model provided a better fit to the data and, thus, a more reliable estimate of binding affinity. Addition of NaCl significantly reduced the signal response during the association phase; however, no substantial effect on the binding affinity of melittin to the POPC bilayers was observed. These findings based on POPC bilayers could have important implications for our understanding of the mechanism of action of melittin on more complex model cell membranes of higher physiological relevance.

How Do the Properties of Amphiphilic Polymer Membranes Influence the Functional Insertion of Peptide Pores?

Pore-forming peptides are of high biological relevance particularly as cytotoxic agents, but their properties are also applicable for the permeabilization of lipid membranes for biotechnological applications, which can then be translated to the more stable and versatile polymeric membranes. However, their interactions with synthetic membranes leading to pore formation are still poorly understood, hampering the development of peptide-based nanotechnological applications, such as biosensors or catalytic compartments. To elucidate these interactions, we chose the model peptide melittin, the main component of bee venom. Here, we present our systematic investigation on how melittin interacts with and inserts into synthetic membranes, based on amphiphilic block copolymers, to induce pore formation in three different setups (planar membranes and micrometric and nanometric vesicles). By varying selected molecular properties of block copolymers and resulting membranes (e.g., hydrophilic to hydrophobic block ratio, membrane thickness, surface roughness, and membrane curvature) and the stage of melittin addition to the synthetic membranes, we gained a deeper understanding of melittin insertion requirements. In the case of solid-supported planar membranes, melittin interaction was favored by membrane roughness and thickness, but its insertion and pore formation were hindered when the membrane was excessively thick. The additional property provided by micrometric vesicles, curvature, increased the functional insertion of melittin, which was evidenced by the even more curved nanometric vesicles. Using nanometric vesicles allowed us to estimate the pore size and density, and by changing the stage of melittin addition, we overcame the limitations of peptide-polymer membrane interaction. Mirroring the functionality assay of planar membranes, we produced glucose-sensing vesicles. The design of synthetic membranes permeabilized with melittin opens a new path toward the development of biosensors and catalytic compartments based on pore-forming peptides functionally inserted in synthetic planar or three-dimensional membranes.

Oligopeptide-side chained alginate nanocarrier for melittin-targeted chemotherapy

A melittin-targeting drug carrier was successfully synthesized by the grafting of sodium alginate to an oligopeptide via an amidation method at different oligopeptide:alginate unit molar ratios. The average sizes of the oligopeptide–alginate nanoparticles formed in the presence of 1 mM CaCl2 decreased with increasing oligopeptide contents, indicating intramolecular interactions between oligopeptide-side chains. While the doxorubicin-loading efficiency on nanoparticles (0.1:1) was similar to that of alginate nanoparticles, the melittin-loading onto oligopeptide–alginate nanoparticles (0.1:1) was more than double that onto alginate nanoparticles, suggesting the specific interaction of melittin with the oligopeptide-side chain in the oligopeptide–alginate nanoparticles. While 2.5 μM free melittin caused almost no damage to Caco-2 cells, more than 80% of cells did not survive under the dose of 2.5 μM melittin-loaded oligopeptide–alginate nanoparticles. The results confirm that the derivation of an oligopeptide-side chain in alginate offers a specific binding site for melittin and effectively works in cancer chemotherapy. A melittin-targeting drug carrier was synthesized by the grafting of sodium alginate to an oligopeptide. The melittin-loading onto oligopeptide–alginate nanoparticles at 0.1:1 unit ratio was more than double that onto alginate nanoparticles, suggesting the specific interaction of melittin with the oligopeptide-side chain in the oligopeptide–alginate nanoparticles. More than 80% of Caco-2 cells did not survive under the dose of 2.5 μM melittin-loaded oligopeptide–alginate nanoparticles. The results confirm that the derivation of an oligopeptide-side chain in alginate offers a specific binding site for melittin and effectively works in cancer chemotherapy.

Filling gaps in the knowledge of melittin on lipid membranes

Melittin (ML) is a small peptide of 26 residues rich in arginine (Arg) and lysine (Lys). Several studies have been done to understand the mechanism of interaction with neutral and negatively charged lipids. However, it is not known with certainty how this interaction depends on the electrostatic or hydrophobic forces according to the composition of the membrane, nor with the different organization of lipids on the membrane, such as the microdomains that could take place in it. Therefore, comparative studies of the interaction and the effect of ML with respect to cationic peptides (Arg-7 and Lys-5) were conducted to get a deeper insight of the ML interaction mechanism with membranes. In this regard, measurements of zeta potential of different model membranes (DOPC, DMPC and DMPE liposomes) in the presence of the peptides, and molecular dynamics simulations were performed. In the special case of DMPC, we worked in its gel like-ripple phase, in order to analyzed defects of packing that potentially expose hydrocarbon regions.In relation with experimental results, molecular analysis of ML interaction with zwiterionic lipid membrane in its ripple phase was performed by unbiased molecular dynamics simulations.The results allow us to remark that ML penetration is favored in the gel-liquid crystalline phase transition in zwitterionic lipids. The importance of this study lies in the understanding of the first stages of action of the ML in eukaryotic membranes, in model systems of its main lipid composition.

Melittin-Induced Permeabilization, Re-sealing, and Re-permeabilization of E. coli Membranes

The permeabilization of model lipid bilayers by cationic peptides has been studied extensively over decades, with the bee-sting toxin melittin perhaps serving as the canonical example. However, the relevance of these studies to the permeabilization of real bacterial membranes by antimicrobial peptides remains uncertain. Here, we employ single-cell fluorescence microscopy in a detailed study of the interactions of melittin with the outer membrane (OM) and the cytoplasmic membrane (CM) of live Escherichia coli. Using periplasmic green fluorescent protein (GFP) as a probe, we find that melittin at twice the minimum inhibitory concentration first induces abrupt cell shrinkage and permeabilization of the OM to GFP. Within ∼4 s of OM permeabilization, the CM invaginates to form inward facing “periplasmic bubbles.” Seconds later the bubbles begin to leak periplasmic GFP into the cytoplasm. Permeabilization is localized, consistent with possible formation of toroidal pores. Within ∼20 s, first the OM and then the CM re-seals to GFP. Some 2–20 min later, both CM and OM are re-permeabilized to GFP. We invoke a mechanism based on curvature stress concepts derived from model bilayer studies. The permeabilization and re-sealing events involve sequential, time-dependent build-up of melittin density within the outer and inner leaflets of each bilayer. We also propose a mechanical explanation for the early cell shrinkage event induced by melittin and a variety of other cationic peptides. As peptides gain access to the periplasm, they bind to the anionic peptido-crosslinks of the lipopolysaccharide layer, increasing its longitudinal elastic modulus. The cell wall shrinks because it can withstand the same turgor pressure with smaller overall extension. Shrinkage in turn induces invagination of the CM, preserving its surface area. We conclude by comparing the behavior of different peptides.

Liquid Crystal Unveiled Interactions between Melittin and Phospholipids at Aqueous-Liquid Crystal Interface

The interaction of honeybee apitoxin (melittin) with phospholipid membrane is very crucial to unveil the mechanistic details of the interaction of peptide with phospholipid membrane. In this regard, herein, our paper advances the design of a liquid crystal (LC)-based sensor for precise and quantitative imaging of melittin–phospholipid interaction through interfacial ordering transition. Micrometer-thick films of nematic 4-cyano-4’-pentylbiphenyl (5CB) LC undergo easily visualized ordering transitions in response to melittin at 1,2-didodecanoyl-sn-glycero-3-phosphocholine (DLPC) laden aqueous-LC interfaces. This observation implies strong interaction of DLPC with melittin at those interfaces. Our experiments also reveal that Ca2+ plays a crucial role for faster dynamic response of the LC during the interaction. The presence of Ca2+ towards kinetics of melittin-DLPC interaction has been found to be highly specific compared to other alkaline earth metal ions. In addition, these interactions were conveniently quantified by measurement of both light intensity (transmitted through the LC under crossed polars) and optical retardance of the LC. Finally, we have investigated the conformational behavior of the melittin in solution as well as in presence of DLPC vesicles using circular dichroism (CD) and vibrational circular dichroism (VCD) spectroscopy. This sensing system significantly outweighs the conventional biological and sepctrscopic assays in terms of robustness, sensitivity, easy sample preparation and clear optical read out. Overall, this paper demonstrates a promising approach in designing a simple LC based biosensor to study and quantify the interaction of melittin with phospholipid membrane at aqueous-LC interfaces.

Supported binary liposome vesicle-gold nanoparticle for enhanced label free DNA and protein sensing

Supported binary liposome mixture of cationic liposome N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium propane (DOTAP) and the zwitterionic liposome 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE) were tethered on thiol monolayers in the absence and presence of gold nanoparticle to enhance sensor stability and sensitivity for label free DNA and protein sensing for the first time. Cysteamine hydrochloride (Cyst), 3-Mercaptopropionic acid (MPA), 11-Mercaptoundecanoic acid (MUDA) and 11-amino-1-undecane thiol (AUT) monolayers were used as tethers on gold surfaces. Electrochemical studies in the presence of [Fe(CN)6]3-/4- indicate that the presence of both DOPE and AuNP decreases the electrostatic interaction between DOTAP and MPA layer during the formation of DOPE-DOTAP-AuNP (DDA) whereas they enhance the repulsive force on the Cyst and AUT monolayers. In the thiol monolayer supported DDA, the gelation of neutral lipid DOPE by the AuNP is disfavored which inturn promotes stability of vesicle structure. The membrane protein melittin's interaction with the DDA indicates the presence of intact vesicle by showing decreased charge transfer for the MUDA and AUT in the presence of [Fe(CN)6]3-/4-. On the contrary, the presence of the bilayer and semi circled DDA on the MPA and cysteamine layers were confirmed by the increased redox reaction. Atomic Force Microscopic (AFM) and Transmission Electron Microscopic (TEM) images support the presence of an array like semi circled DDA on the MPA and well separated DDA vesicles on the MUDA with variable sizes. Dynamic Light Scattering (DLS) and Fourier Transform Infrared spectroscopy (FTIR) suggest effective coordination between DOPE, DOTAP and AuNP. Label free DNA hybridization sensing in presence of the negatively charged [Fe(CN)6]3-/4- indicates the lowest DNA detection limit of 1×10−14M with linearity range 1×10–13 to 1×10−9M. Similarly, streptavidin sensing shows the lowest detection of 1ngml−1 with a linear range 100ng to 1µg due to the increased reactive sites and distance.

Dynamic Structure and Orientation of Melittin Bound to Acidic Lipid Bilayers, As Revealed by Solid-State NMR and Molecular Dynamics Simulation.

Melittin is a venom peptide that disrupts lipid bilayers at temperatures below the liquid-crystalline to gel phase transition temperature (Tc). Notably, the ability of melittin to disrupt acidic dimyristoylphosphatidylglycerol (DMPG) bilayers was weaker than its ability to disrupt neutral dimyristoylphosphatidylcholine bilayers. The structure and orientation of melittin bound to DMPG bilayers were revealed by analyzing the 13C chemical shift anisotropy of [1-13C]-labeled melittin obtained from solid-state 13C NMR spectra. 13C chemical shift anisotropy showed oscillatory shifts with the index number of residues. Analysis of the chemical shift oscillation properties indicated that melittin bound to a DMPG membrane adopts a bent α-helical structure with tilt angles for the N- and C-terminal helices of -32 and +30°, respectively. The transmembrane melittin in DMPG bilayers indicates that the peptide protrudes toward the C-terminal direction from the core region of the lipid bilayer to show a pseudotransmembrane bent α-helix. Molecular dynamics simulation was performed to characterize the structure and interaction of melittin with lipid molecules in DMPG bilayers. The simulation results indicate that basic amino acid residues in melittin interact strongly with lipid head groups to generate a pseudo-transmembrane alignment. The N-terminus is located within the lipid core region and disturbs the lower surface of the lipid bilayer.

Interaction of Melittin with Negatively Charged Lipid Bilayers Supported on Gold Electrodes

ABSTRACT The interactions of melittin, a cationic antimicrobial peptide, with model lipid membranes consisting of negatively charged phospholipids: 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) or 1,2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS) were investigated using electrochemical techniques and atomic force microscopy. Lipid bilayers were deposited on gold electrodes using a combination of Langmuir-Blodgett and Langmuir-Schaefer methods and the resulting membranes established a barrier for electron transfer between the electrode and the redox probe in the solution. After exposure to melittin, the blocking properties of the membranes were monitored using cyclic voltammetry and electrochemical impedance spectroscopy. It was observed that after treatment with peptide, the charge transfer through lipid bilayer is initially strongly inhibited. However, after longer exposure to melittin, the structure of the lipid film becomes less compact and the electrode reactions are facilitated due to the presence of numerous defect sites exposing bare substrate. We have assumed that such behavior reflects initial adsorption of melittin on top of the membrane and its further insertion which leads to formation of the pores or partial micellization of the lipid film. AFM imaging revealed that the exposure to 10 μM melittin solution induces significant structural changes in DMPG and DMPS membranes. However, melittin seems to affect their organization in a different manner. DMPG film appears to be more susceptible to peptide action compared with DMPS bilayer. In the latter case, long-time exposure to melittin does not result in the rupture of the membrane but rather leads to formation of pore-like defects. This observation is explained in terms of different nanomechanical properties of DMPG and DMPS films and different barrier for the reorientation and insertion of the peptide molecules into the membranes.

Melittin-induced cholesterol reorganization in lipid bilayer membranes

The peptide melittin, a 26 amino acid, cationic peptide from honey bee (Apis mellifera) venom, disrupts lipid bilayer membranes in a concentration-dependent manner. Rather than interacting with a specific receptor, the peptide interacts directly with the lipid matrix of the membrane in a manner dependent on the lipid composition. Here, a small-angle neutron scattering study of the interaction of melittin with lipid bilayers made of mixtures of dimyristoylphosphatidylcholine (DMPC) and cholesterol (Chol) is presented. Through the use of deuterium-labeled DMPC, changes in the distribution of the lipid and cholesterol in unilamellar vesicles were observed for peptide concentrations below those that cause pores to form. In addition to disrupting the in-plane organization of Chol, melittin produces vesicles having inner and outer leaflet compositions that depend on the lipid–Chol molar ratio and on the peptide concentration. The changes seen at high cholesterol and low peptide concentration are similar to those produced by alamethicin (Qian, S. et al., J. Phys. Chem. B 2014, 118, 11200–11208), which points to an underlying physical mechanism driving the redistribution of Chol, but melittin displays an additional effect not seen with alamethicin. A model for how the peptide drives the redistribution of Chol is proposed. The results suggest that redistribution of the lipids in a target cell membrane by membrane active peptides takes places as a prelude to the lysis of the cell.

Site of fluorescent label modifies interaction of melittin with live cells and model membranes

The mechanism of membrane disruption by melittin (MLT) of giant unilamellar vesicles (GUVs) and live cells was studied using fluorescence microscopy and two fluorescent synthetic analogues of MLT. The N-terminus of one of these was acylated with thiopropionic acid to enable labeling with maleimido-AlexaFluor 430 to study the interaction of MLT with live cells. It was compared with a second analogue labeled at P14C. The results indicated that the fluorescent peptides adhered to the membrane bilayer of phosphatidylcholine GUVs and inserted into the plasma membrane of HeLa cells. Fluorescence and light microscopy revealed changes in cell morphology after exposure to MLT peptides and showed bleb formation in the plasma membrane of HeLa cells. However, the membrane disruptive effect was dependent upon the location of the fluorescent label on the peptide and was greater when MLT was labeled at the N-terminus. Proline at position 14 appeared to be important for antimicrobial activity, hemolysis and cytotoxicity, but not essential for cell membrane disruption.

Interaction of bee venom toxin melittin with ganglioside GM1 bicelle

Melittin is a bee venom toxin that can act as antimicrobial peptide. Gangliosides are glycosphingolipids that help maintain membrane structure and organization as well as act as anchors for lectins, toxins, pathogens and antimicrobial peptides. Here we investigate interaction of melittin with fast tumbling isotropic control DMPC/CHAPS bicelles and ganglioside doped DMPC/CHAPS/GM1 bicelles. DOSY result shows that larger percentage of peptide binds to GM1 containing bicelles than that of the control PC bicelles. Bound peptide induces leakage of the bicelles entrapped carboxyfluorescein. Percentage of leakage is higher from control PC bicelles than that of the GM1 containing bicelles. In the presence of control PC bicelles melittin acquired fully α-helical structure. But in the presence of GM1 containing bicelles the peptide is not fully α-helical i.e., some random coil structure is present in this folded form. The present study shows that GM1 has an effect on membrane active antimicrobial peptide melittin.

Vesicle deposition and subsequent membrane–melittin interactions on different substrates: A QCM-D experiment

Quartz crystal microbalance with dissipation (QCM-D) technique is one of the most effective methods to monitor the dynamic behaviors of a layer on a solid surface. Moreover, it has been reported recently that it is able to provide a fingerprint for the peptide–membrane interactions. In this work, QCM-D technique combined with computer simulations was employed to investigate the deposition and transformation of vesicles, as well as the subsequent membrane–melittin interactions on different substrates. A range of substrate surfaces, i.e. naked SiO2 without or with Au/polyelectrolyte coating, were produced. The nature of the substrate determined whether the adsorbed vesicles were present as a high-quality supported bilayer or an assembled vesicle matrix, which consequently influenced the membrane–melittin interactions. It was indicated by the related computer simulations that the lipid packing state of the membrane was a key factor to determine the mechanism of membrane–peptide interactions. Furthermore, this work might be a good example of the application of QCM-D for the exploration of membrane-active peptides.

Molecular Response and Cooperative Behavior during the Interactions of Melittin with a Membrane: Dissipative Quartz Crystal Microbalance Experiments and Simulations

The molecular-level interactions of an antimicrobial peptide melittin with supported membrane were studied by the combination of dissipative quartz crystal microbalance (QCM-D) experiments and computer simulations. We found the response behavior of lipids upon peptide adsorption greatly influence their interactions. The perturbance and reorientation of the lipid in liquid phase facilitate the insertion of melittin in a trans-membrane way, but in solid phase, asymmetrical membrane disruption happens. Apart from the lipid state, the local peptide-to-lipid ratio also affects the insertion capacity of melittin. When the local peptide number density is high, adjacent peptides can cooperatively penetrate into the membrane. This observation explains the occurrence of the conventional "carpet" mechanism.

Consequences of Alteration in Leucine Zipper Sequence of Melittin in Its Neutralization of Lipopolysaccharide-induced Proinflammatory Response in Macrophage Cells and Interaction with Lipopolysaccharide

The bee venom antimicrobial peptide, melittin, besides showing versatile activity against microorganisms also neutralizes lipopolysaccharide (LPS)-induced proinflammatory responses in macrophage cells. However, how the amino acid sequence of melittin contributes in its anti-inflammatory properties is mostly unknown. To determine the importance of the leucine zipper sequence of melittin in its neutralization of LPS-induced inflammatory responses in macrophages and interaction with LPS, anti-inflammatory properties of melittin and its three analogues and their interactions with LPS were studied in detail. Two of these analogues, namely melittin Mut-1 (MM-1) and melittin Mut-2 (MM-2), possess leucine to alanine substitutions in the single and double heptadic leucine residue(s) of melittin, respectively, whereas the third analogue is a scrambled peptide (Mel-SCR) that contains the amino acid composition of melittin with minor rearrangement in its leucine zipper sequence. Although MM-1 partly inhibited the production of proinflammatory cytokines in RAW 264.7 and rat primary macrophage cells in the presence of LPS, MM-2 and Mel-SCR were negligibly active. A progressive decrease in interaction of melittin with LPS, aggregation in LPS, and dissociation of LPS aggregates with alteration in the leucine zipper sequence of melittin was observed. Furthermore, with alteration in the leucine zipper sequence of melittin, these analogues failed to exhibit cellular responses associated with neutralization of LPS-induced inflammatory responses in macrophage cells by melittin. The data indicated a probable important role of the leucine zipper sequence of melittin in neutralizing LPS-induced proinflammatory responses in macrophage cells as well as in its interaction with LPS.

Interaction of Melittin Peptides with Perfluorocarbon Nanoemulsion Particles

Melittin, an antimicrobial peptide, forms pores in biological membranes and triggers cell death. Therefore, it has potential as an anticancer therapy. However, until recently, the therapeutic application of melittin has been impractical because a suitable platform for delivery was not available. Recently, we showed that phospholipid-stabilized perfluorooctyl bromide based nanoemulsion particles (PFOB-NEPs) were resistant to destruction by melittin and enabled specific delivery of melittin to tumor cells, killing them and reducing tumor growth. Earlier, prior work also showed that melittin adsorbed onto the stabilizing phospholipid monolayer of PFOB-NEP but did not disrupt the phospholipid monolayer or produce "cracking" of the PFOB-NEPs. The present work identifies the important structural motifs for melittin binding to PFOB-NEPs through a series of atomistic molecular dynamics simulations. The conformational ensemble of melittin bound to PFOB-NEP lipid monolayer was compared to structure from a control simulation of melittin bound to a lipid bilayer to identify several differences in melittin-lipid interactions between the two systems. First, melittin was deeply buried in the hydrophobic tail region of bilayer, while its depth was attenuated in the PFOB-NEP monolayer. Second, a helical conformation was the major secondary structure in the bilayer, but the fraction of helix was reduced in the PFOB-NEP. Finally, the overall pattern for the direct interaction of melittin with surrounding lipids was similar between liposome and PFOB-NEP, but the level of interaction was slightly decreased in the PFOB-NEP. These results suggest that melittin interacts with the monolayer of PFOB-NEP in a way that is similar way to its interaction with bilayers but that deeper penetration into the hydrophobic interior is inhibited.