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Toxins May 2022Melittin, the main toxic component in the venom of the European honeybee, interacts with natural and artificial membranes due to its amphiphilic properties. Rather than...
Melittin, the main toxic component in the venom of the European honeybee, interacts with natural and artificial membranes due to its amphiphilic properties. Rather than interacting with a specific receptor, melittin interacts with the lipid components, disrupting the lipid bilayer and inducing ion leakage and osmotic shock. This mechanism of action is shared with pneumolysin and other members of the cholesterol-dependent cytolysin family. In this manuscript, we investigated the inverse correlation for cholesterol dependency of these two toxins. While pneumolysin-induced damage is reduced by pretreatment with the cholesterol-depleting agent methyl-β-cyclodextrin, the toxicity of melittin, after cholesterol depletion, increased. A similar response was also observed after a short incubation with lipophilic simvastatin, which alters membrane lipid organization and structure, clustering lipid rafts. Therefore, changes in toxin sensitivity can be achieved in cells by depleting cholesterol or changing the lipid bilayer organization.
Topics: Animals; Bacterial Proteins; Bees; Cholesterol; Lipid Bilayers; Melitten; Streptolysins
PubMed: 35622592
DOI: 10.3390/toxins14050346 -
International Journal of Molecular... Mar 2022Melanoma is an immunogenic tumor and a serious type of skin cancer. Tumor-associated macrophages (TAMs) express an M2-like phenotype and are involved in all stages of...
Melanoma is an immunogenic tumor and a serious type of skin cancer. Tumor-associated macrophages (TAMs) express an M2-like phenotype and are involved in all stages of melanomagenesis; it is hence a promising target for cancer immunotherapy. We herein investigated whether melittin-dKLA inhibits the growth of melanoma by inducing apoptosis of M2-like macrophages. For the in vitro study, a conditioned medium of macrophages was prepared from M0, M1, or M2-differentiated THP-1 cells with and without melittin-dKLA. The affinity of melittin for M2 macrophages was studied with FITC (fluorescein isothiocyanate)-conjugated melittin. For the in vivo study, murine melanoma cells were inoculated subcutaneously in the right flank of mice, melittin-dKLA was intraperitoneally injected at 200 nmol/kg every three days, and flow cytometry analysis of TAMs was performed. Since melittin binds preferentially to M2-like macrophages, melittin-dKLA induced more caspase 3 expression and cell death in M2 macrophages compared with M0 and M1 macrophages and melanoma cells. Melittin-dKLA significantly inhibited the proliferation and migration of M2 macrophages, resulting in a decrease in melanoma tumor growth in vivo. The CD206 M2-like TAMs were reduced, while the CD86 M1-like TAMs were not affected. Melittin-dKLA is therapeutically effective against melanoma by inducing the apoptosis of M2-like TAMs.
Topics: Animals; Cell Line, Tumor; Immunotherapy; Macrophages; Melanoma; Melitten; Mice; Tumor-Associated Macrophages
PubMed: 35328518
DOI: 10.3390/ijms23063094 -
Biophysical Journal Apr 2022While it is established that the topology of lipid membranes plays an important role in biochemical processes, few direct observations exist regarding how the membranes...
While it is established that the topology of lipid membranes plays an important role in biochemical processes, few direct observations exist regarding how the membranes are actively restructured and its consequences on subsequent reactions. In this work, we investigated how the two major components of bee venom, melittin and phospholipase A (PLA), achieve activation by such membrane remodeling. Their membrane-disrupting functions have been reported to increase when both are present, but the mechanism of this synergism had not been established. Using membrane reconstitution, we found that melittin can form large-scale membrane deformities upon which PLA activity is 25-fold higher. Tracking of single-molecule PLA revealed that its processive behavior on these deformities underlies the enhanced activity. These results show how melittin and PLA work synergistically to enhance the lytic effects of the bee venom. More broadly, they also demonstrate how the membrane topology may be actively altered to modulate cellular membrane-bound reactions.
Topics: Bee Venoms; Melitten; Phospholipases A2
PubMed: 35314142
DOI: 10.1016/j.bpj.2022.03.019 -
Journal of the American Chemical Society Jun 2022Elucidation of the detailed mechanisms by which biological macromolecules undergo major structural conversions, such as folding, complex formation, and self-assembly, is...
Elucidation of the detailed mechanisms by which biological macromolecules undergo major structural conversions, such as folding, complex formation, and self-assembly, is a central concern of biophysical chemistry that will benefit from new experimental methods. We describe a simple technique for initiating a structural conversion process by a rapid decrease in the temperature of a solution, i.e., a rapid inverse temperature jump. By pumping solutions through copper capillary tubes that are thermally anchored to heated and cooled blocks, solution temperatures can be switched from 95 to 30 °C (or lower) in about 0.8 ms. For time-resolved solid-state nuclear magnetic resonance (ssNMR), solutions can then be frozen rapidly by spraying into cold isopentane after a variable structural evolution time τ. As an initial demonstration, we use this "inverse T-jump" technique to characterize the kinetics and mechanism by which the 26-residue peptide melittin converts from its primarily disordered, monomeric state at 95 °C to its α-helical, tetrameric state at 30 °C. One- and two-dimensional ssNMR spectra of frozen solutions with various values of τ, recorded at 25 K with signal enhancements from dynamic nuclear polarization, show that both helical secondary structure and intermolecular contacts develop on the same time scale of about 6 ms. The dependences on τ of both intraresidue crosspeak patterns and inter-residue crosspeak volumes in two-dimensional spectra can be fit with a unidirectional dimerization model, consistent with dimerization being the rate-limiting step for melittin tetramer formation.
Topics: Kinetics; Magnetic Resonance Spectroscopy; Melitten; Protein Conformation, alpha-Helical; Temperature
PubMed: 35617672
DOI: 10.1021/jacs.2c02704 -
Biophysical Journal Nov 2022The antimicrobial peptide, melittin, is a potential next-generation antibiotic because melittin can spontaneously form pores in bacterial cell membranes and cause...
The antimicrobial peptide, melittin, is a potential next-generation antibiotic because melittin can spontaneously form pores in bacterial cell membranes and cause cytoplasm leakage. However, the organizations of melittin peptides in cell membranes remain elusive, which impedes the understanding of the poration mechanism. In this work, we use coarse-grained and all-atom molecular dynamics (MD) simulations to investigate the organizations of melittin peptides during and after spontaneous penetration into DPPC/POPG lipid bilayers. We find that the peptides in lipid bilayers adopt either a transmembrane conformation or a U-shaped conformation, which are referred to as T- and U-peptides, respectively. Several U-peptides and/or T-peptides aggregate to form stable pores. We analyze a T-pore consisting of four T-peptides and a U-pore consisting of three U-peptides and one T-peptide. In both pores, peptides are organized in a manner such that polar residues face inward and hydrophobic residues face outward, which stabilizes the pores and produces water channels. Compared with the U-pore, the T-pore has lower energy, larger pore diameter, and higher permeability. However, the T-pore occurs less frequently than the U-pore in our simulations, probably because the formation of the T-pore is kinetically slower than the U-pore. The stability and permeability of both pores are confirmed by 300 ns all-atom MD simulations. The peptide organizations obtained in this work should deepen the understanding of the stability, poration mechanism, and permeability of melittin, and facilitate the optimization of melittin to enhance the antibacterial ability.
Topics: Melitten; Lipid Bilayers; Molecular Dynamics Simulation; Peptides; Cell Membrane
PubMed: 36199252
DOI: 10.1016/j.bpj.2022.10.002 -
Archiv Der Pharmazie Apr 2024Melittin (MLT), a peptide containing 26 amino acids, is a key constituent of bee venom. It comprises ∼40%-60% of the venom's dry weight and is the main pricing index... (Review)
Review
Melittin (MLT), a peptide containing 26 amino acids, is a key constituent of bee venom. It comprises ∼40%-60% of the venom's dry weight and is the main pricing index for bee venom, being the causative factor of pain. The unique properties of MLT extracted from bee venom have made it a very valuable active ingredient in the pharmaceutical industry as this cationic and amphipathic peptide has propitious effects on human health in diverse biological processes. It has the ability to strongly impact the membranes of cells and display hemolytic activity with anticancer characteristics. However, the clinical application of MLT has been limited by its severe hemolytic activity, which poses a challenge for therapeutic use. By employing more efficient mechanisms, such as modifying the MLT sequence, genetic engineering, and nano-delivery systems, it is anticipated that the limitations posed by MLT can be overcome, thereby enabling its wider application in therapeutic contexts. This review has outlined recent advancements in MLT's nano-delivery systems and genetically engineered cells expressing MLT and provided an overview of where the MLTMLT's platforms are and where they will go in the future with the challenges ahead. The focus is on exploring how these approaches can overcome the limitations associated with MLT's hemolytic activity and improve its selectivity and efficacy in targeting cancer cells. These advancements hold promise for the creation of innovative and enhanced therapeutic approaches based on MLT for the treatment of cancer.
Topics: Humans; Melitten; Structure-Activity Relationship; Bee Venoms; Neoplasms; Peptides
PubMed: 38251938
DOI: 10.1002/ardp.202300569 -
Advanced Healthcare Materials May 2019Antimicrobial peptides (AMPs) promise a fundamental solution to the devastating threat of drug-resistant bacteria. However, drawbacks of AMPs (e.g., poor cell membrane...
Antimicrobial peptides (AMPs) promise a fundamental solution to the devastating threat of drug-resistant bacteria. However, drawbacks of AMPs (e.g., poor cell membrane penetration efficiency) seriously block their clinical use. In this work, rational design of a hybrid complex of melittin (as a representative AMP) and graphene or graphene oxide (Gra or GO) nanosheets for enhanced antibacterial ability is achieved, via combining in-silico prediction and in-tube test. In comparison to pristine melittin, the specifically designed AMP-Gra (/GO) complex exhibits remarkable efficiency in transmembrane perforation with an over tenfold decrease in the threshold working concentration of peptide; moreover, it has an up to 20-fold enhancement in antibacterial activity against both Gram-negative and Gram-positive bacteria. Such improvement is ascribed to the synergetic insertion of nanosheets and melittin due to similarity in antibacterial mechanism between them and is further regulated by the structural factors of the complex, including the intersheet spacing and surface functionalization of the Gra/GO sheets, etc. These results provide practical guidelines to engineer AMPs with nanotechnology for improved antimicrobial performances, especially based on targeted functionalization of the Gra/GO nanosheets.
Topics: Anti-Bacterial Agents; Cell Membrane; Gram-Negative Bacteria; Gram-Positive Bacteria; Graphite; Melitten; Microbial Sensitivity Tests
PubMed: 30866165
DOI: 10.1002/adhm.201801521 -
Journal of Toxicology and Environmental... Feb 2024Africanized bees have spread across the Americas since 1956 and consequently resulted in human and animal deaths attributed to massive attacks related to exposure from... (Review)
Review
Africanized bees have spread across the Americas since 1956 and consequently resulted in human and animal deaths attributed to massive attacks related to exposure from Argentina to the USA. In Brazil, more than 100,000 accidents were registered in the last 5 years with a total of 303 deaths. To treat such massive attacks, Brazilian researchers developed the first specific antivenom against Africanized honey bee sting exposure. This unique product, the first of its kind in the world, has been safely tested in 20 patients during a Phase 2 clinical trial. To develop the antivenom, a standardized process was undertaken to extract primary venom antigens from the Africanized bees for immunization of serum-producing horses. This process involved extracting, purifying, fractionating, characterizing, and identifying the venom (apitoxin) employing mass spectrometry to generate standardized antigen for hyperimmunization of horses using the major toxins (melittin and its isoforms and phospholipase A2). The current guide describes standardization of the entire production chain of venom antigens in compliance with good manufacturing practices (GMP) required by regulatory agencies. Emphasis is placed upon the welfare of bees and horses during this process, as well as the development of a new biopharmaceutical to ultimately save lives.
Topics: Bees; Humans; Animals; Antivenins; Insect Bites and Stings; Bee Venoms; Melitten; Phospholipases A2; Antigens
PubMed: 38247328
DOI: 10.1080/10937404.2023.2300786 -
Colloids and Surfaces. B, Biointerfaces May 2022Biomembranes are involved in diverse cellular activities. How membranes and proteins interact in the activities might hinge on the former's physical characteristics,...
Biomembranes are involved in diverse cellular activities. How membranes and proteins interact in the activities might hinge on the former's physical characteristics, which in turn are influenced by packing of lipid molecules. Yet, the validity of this understanding and its mechanism are unclear. By varying chain saturation of membranes, we explored correlations between lipid packing and peptide-mediated membrane disruption for the antimicrobial peptide, melittin, and amyloidogenic peptide, β-amyloid (1-42). Remarkably, reducing molecular packing flexibility enhanced the membrane disruption, possibly due to a shift from membrane perforation to micellization. A theoretical analysis suggested the energetic basis of this shift. This mechanistically shows that a peptide's mechanism might be dictated not only by its intrinsic properties but also by physical characteristics of membranes.
Topics: Lipid Bilayers; Lipids; Melitten; Peptides
PubMed: 35151994
DOI: 10.1016/j.colsurfb.2022.112384 -
Current Topics in Membranes 2016Due to the recent advances in computer hardware and software, we can now use molecular dynamics and Monte Carlo computer simulation techniques to study systems with... (Review)
Review
Due to the recent advances in computer hardware and software, we can now use molecular dynamics and Monte Carlo computer simulation techniques to study systems with large conformational spaces. It is demonstrated here that computer simulations allow us to get a glimpse at the structural and dynamical properties of membranes and also at the interaction of membranes with other molecules. Specifically two examples are considered: (1) structural properties of lipid rafts in model membranes and (2) interaction of model membranes with an antimicrobial peptide, melittin.
Topics: Cell Membrane; Melitten; Membrane Microdomains; Models, Biological
PubMed: 26781828
DOI: 10.1016/bs.ctm.2015.10.002