The effect of counterions on melittin aggregation.
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ABSTRACT:
SUBMITTER: Tatham AS
PROVIDER: S-EPMC1154414 | biostudies-other | 1983 Jun
REPOSITORIES: biostudies-other
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Project description:We introduce a new mixed resolution, all-atom/coarse-grained approach (AACG), for modeling peptides in aqueous solution and apply it to characterizing the aggregation of melittin. All of the atoms in peptidic components are represented, while a single site is used for each water molecule. With the full flexibility of the peptide retained, our AACG method achieves speedups by a factor of 3-4 for CPU time reduction and another factor of roughly 7 for diffusion. An Ewald treatment permits the inclusion of long-range electrostatic interactions. These characteristics fit well with the requirements for studying peptide association and aggregation, where the system sizes and time scales require considerable computational resources with all-atom models. In particular, AACG is well suited for biologics since changes in peptide shape and long-range electrostatics may play an important role. The application of AACG to melittin, a 26-residue peptide with a well-known propensity to aggregate in solution, serves as an initial demonstration of this technology for studying peptide aggregation. We observed the formation of melittin aggregates during our simulations and characterized the time-evolution of aggregate size distribution, buried surface areas, and residue contacts. Key interactions including π-cation and π-stacking involving TRP19 were also examined. Our AACG simulations demonstrated a clear salt effect and a moderate temperature effect on aggregation and support the molten globule model of melittin aggregates. As a showcase, this work illustrates the useful role for AACG in investigations of peptide aggregation and its potential to guide formulation and design of biologics.
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Project description:The 25 residue presequence (p25) for subunit IV of yeast cytochrome oxidase had previously been shown to possess structural and behavioural characteristics in common with the bee venom polypeptide, melittin. The present study extends the results of leakage experiments on model-membrane systems to the haemolysis of human erythrocytes, which both peptides are shown to accomplish in a manner sensitive to membrane potential. In addition, the laser flash-induced transient dichroism technique for measuring protein rotational diffusion has been used to show that both peptides aggregate band 3, the major integral membrane protein of the erythrocyte. Aggregation cannot be reversed by high ionic strength; this serves to differentiate these peptides from other positively charged species such as polylysine that aggregate band 3 at low ionic strength. These results suggest that aggregation of membrane proteins may possibly prove to be a feature of the interaction of p25 signal peptide with mitochondrial membranes.
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Project description:The cytolytic activity of the bee venom toxin, melittin, is abolished on permethylation of the ammonium groups into quaternary trimethylammonium groups. The loss of activity in permethylated melittin may result partly from the absence of the hydrogen bonding potential and partly from steric effects involving the bulky trimethylammonium groups. Displacing the trimethylammonium groups away from the backbone to relieve steric effects (by acylating melittin with glycine or 5-aminopentanoic acid followed by permethylation) restored moderate activity at 5-fold increase in concentration.
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Project description:The molecular mechanism by which HFIP stabilizes the alpha-helical structure of peptides is not well understood. In the present study, we use melittin as a model to gain insight into the details of the atomistic interactions of HFIP with the peptide. We have performed extensive comparative molecular dynamics simulations (up to 100 nsec) in the absence and in the presence of HFIP. In agreement with recent NMR experiments, the simulations show rapid loss of tertiary structure in water at pH 2 but much higher helicity in 35% HFIP. The MD simulations also indicate that melittin adopts a highly dynamic global structure in 35% HFIP solution with two alpha-helical segments sampling a wide range of angular orientations. The analysis of the HFIP distribution shows the tendency of HFIP to aggregate around the peptide, increasing the local cosolvent concentration to more than two times that in the bulk concentration. The correlation of local peptide structure with HFIP coating suggests that displacement of water at the peptide surface is the main contribution of HFIP in stabilizing the secondary structure of melittin. Finally, a stabilizing effect promoted by the presence of counter-ions was also observed in the simulations.
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Project description:Melittin, the major active peptide of honeybee venom (BV), has potential for use in adjuvant immunotherapy. The immune system response to different stimuli depends on the secretion of different metabolites from macrophages. One potent stimulus is lipopolysaccharide (LPS), a component isolated from gram-negative bacteria, which induces the secretion of pro-inflammatory cytokines in macrophage cell cultures. This secretion is amplified when LPS is combined with melittin. In the present study, pure melittin was isolated from whole BV by flash chromatography to obtain pure melittin. The ability of melittin to enhance the release of tumour necrosis factor-α (TNF-α), Interleukin (IL-1β, IL-6, and IL-10) cytokines from a macrophage cell line (THP-1) was then assessed. The response to melittin and LPS, applied alone or in combination, was characterised by metabolic profiling, and the metabolomics results were used to evaluate the potential of melittin as an immune adjuvant therapy. The addition of melittin enhanced the release of inflammatory cytokines induced by LPS. Effective chromatographic separation of metabolites was obtained by liquid chromatography-mass spectrometry (LC-MS) using a ZIC-pHILIC column and an ACE C4 column. The levels of 108 polar and non-polar metabolites were significantly changed (p ˂ 0.05) following cell activation by the combination of LPS and melittin when compared to untreated control cells. Overall, the findings of this study suggested that melittin might have a potential application as a vaccine adjuvant.
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Project description:Melittin is an extensively studied, 26-residue toxic peptide from honey bee venom. Because of its versatility in adopting a variety of secondary (helix or coil) and quaternary (monomer or tetramer) structures in various environments, melittin has been the focus of numerous investigations as a model peptide in protein folding studies as well as in studies involving binding to proteins, lipids, and polysaccharides. A significant body of evidence supports the view that melittin binds to these macromolecules in a predominantly helical conformation, but detailed structural knowledge of this conformation is lacking. In this report, we present nuclear magnetic resonance (NMR)-based structural insights into the helix formation of recombinant melittin in the presence of trifluoroethanol (TFE): a known secondary structure inducer in peptides. These studies were performed at neutral pH, with micromolar amounts of the peptide. Using nuclear Overhauser effect (NOE)-derived distance restraints from three-dimensional NMR spectra, we determined the atomic resolution solution NMR structure of recombinant melittin bearing a TFE-stabilized helix. To circumvent the complications with structure determination of small peptides with high conformational flexibility, we developed a workflow for enhancing proton NOEs by increasing the viscosity of the medium. In the TFE-containing medium, recombinant monomeric melittin forms a long, continuous helical structure, which consists of the N- and C-terminal α-helices and the noncanonical 310-helix in the middle. The noncanonical 310-helix is missing in the previously solved X-ray structure of tetrameric melittin and the NMR structure of melittin in methanol. Melittin's structure in TFE-containing medium provides insights into melittin's conformational transitions, which are relevant to the peptide's interactions with its biological targets.
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Project description:In previous studies, we developed a novel fusion protein named "melittin-MIL-2" which exhibited more anti-tumor activity. However, it remains unclear whether melittin-MIL-2 possesses antitumor immune effect on lung adenocarcinoma. In this study, the immune effect and mechanism of melittin-MIL-2 inhibiting the growth and invasion of lung adenocarcinoma will be investigated, in order to provide novel perspectives for the immunotherapy of lung cancer. The results indicated that melittin-MIL-2 promoted T cell proliferation, enhanced NK cell cytotoxicity, and boosted IFN-γ secretion in PBMCs. After melittin-MIL-2 stimulation, perforin expression and LAK/NK-like killing activities of human PBMCs and NK cells were significantly enhanced. Melittin-MIL-2 is capable of hampering the development and proliferation of lung adenocarcinoma cell A549. ICAM-1 and Fas expression in A549 cells exposed to melittin-MIL-2 rose significantly. The expression levels of TLR8 and VEGF in A549 cells decreased significantly after melittin-MIL-2 stimulation. In vivo, melittin-MIL-2 substantially impeded the growth of lung adenocarcinoma and formed an immune-stimulating microenvironment locally in tumor tissues. In conclusion, the novel fusion protein melittin-MIL-2 exhibits strong anti-tumor immune effect in lung adenocarcinoma cell A549 via activating the LFA-1/ICAM-1 and Fas/FasL pathways to enhance cytolytic activity, upregulating the secretion of IFN-γ and perforin, and boosting LAK/NK-like killing activities. Immuno-effector cells and their secreted cytokines can form immune stimulation microenvironment locally in lung adenocarcinoma Lewis mice tissue.
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Project description:As a naturally occurring cytolytic peptide, melittin (Mel) has strong cytolytic activity and is a potent therapeutic peptide for cancer therapy. However, the serious hemolytic activity of Mel largely impedes its clinical applications. In this work, based on the strong interactions between proteins/peptides and polyphenols, we develop a tannic acid-Fe3+ metal-phenolic network (MPN)-based strategy that can convert Mel from foe to friend via shielding its positive charges and reducing its hemolytic activity. Besides, an immune adjuvant resiquimod (R848) is also introduced for immunostimulation, affording the final Mel- and R848-coloaded nanodrug. The Mel-caused membrane disruption can induce immunogenic cell death for immunostimulation, R848 can act as an immune adjuvant to further facilitate the immunostimulatory effect, and the tannic acid-Fe3+ MPN-mediated Fenton reaction can produce reactive oxygen species for cancer treatment. Further experiments reveal that the nanodrug can effectively cause immunogenic cell death of tumor cells and arouse robust intratumoral and systemic antitumor immunostimulation. In the bilateral tumor-bearing mouse models, the nanodrug considerably destroys the primary tumor and also boosts the abscopal effect to ablate the distant tumor. Collectively, the MPN-facilitated "foe-to-friend" strategy may promote the practical applications of Mel and foster the development of cancer immunotherapeutics.
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Project description:The cosolvent effect on the equilibrium of peptide aggregation is reviewed from the energetic perspective. It is shown that the excess chemical potential is stationary against the variation of the distribution function for the configuration of a flexible solute species and that the derivative of the excess chemical potential with respect to the cosolvent concentration is determined by the corresponding derivative of the solvation free energy averaged over the solute configurations. The effect of a cosolvent at low concentrations on a chemical equilibrium can then be addressed in terms of the difference in the solvation free energy between pure-water solvent and the mixed solvent with the cosolvent, and illustrative analyses with all-atom model are presented for the aggregation of an 11-residue peptide by employing the energy-representation method to compute the solvation free energy. The solvation becomes more favorable with addition of the urea or DMSO cosolvent, and the extent of stabilization is smaller for larger aggregate. This implies that these cosolvents inhibit the formation of an aggregate, and the roles of such interaction components as the electrostatic, van der Waals, and excluded-volume are discussed.
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Project description:Red blood cells (RBCs) are the most abundant cells in the human blood that have been extensively studied under morphology, ultrastructure, biochemical and molecular functions. Therefore, RBCs are excellent cell models in the study of biologically active compounds like drugs and toxins on the structure and function of the cell membrane. The aim of the present study was to explore erythrocyte ghost's proteome to identify changes occurring under the influence of three bee venom peptides-melittin, tertiapin, and apamin. We conducted preliminary experiments on the erythrocyte ghosts incubated with these peptides at their non-hemolytic concentrations. Such preparations were analyzed using liquid chromatography coupled with tandem mass spectrometry. It was found that when higher concentrations of melittin and apamin were used, fewer proteins were identified. Moreover, the results clearly indicated that apamin demonstrates the greatest influence on the RBCs ghosts proteome. Interestingly, the data also suggest that tertiapin exerted a stabilizing effect on the erythrocyte membrane. The experiments carried out show the great potential of proteomic research in the projects focused on the toxin's properties as membrane active agents. However, to determine the specificity of the effect of selected bee venom peptides on the erythrocyte ghosts, further proteomic research should be focused on the quantitative analysis.
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