Anticancer β-hairpin peptides: membrane-induced folding triggers activity

Several cationic antimicrobial peptides (AMPs) have recently been shown to display anticancer activity via a mechanism that usually entails the disruption of cancer cell membranes. In this work, we designed an 18-residue anticancer peptide, SVS-1, whose mechanism of action is designed to take advantage of the aberrant lipid composition presented on the outer leaflet of cancer cell membranes, which makes the surface of these cells electronegative relative to the surface of noncancerous cells. SVS-1 is designed to remain unfolded and inactive in aqueous solution but to preferentially fold at the surface of cancer cells, adopting an amphiphilic β-hairpin structure capable of membrane disruption. Membrane-induced folding is driven by electrostatic interaction between the peptide and the negatively charged membrane surface of cancer cells. SVS-1 is active against a variety of cancer cell lines such as A549 (lung carcinoma), KB (epidermal carcinoma), MCF-7 (breast carcinoma), and MDA-MB-436 (breast carcinoma). However, the cytotoxicity toward noncancerous cells having typical membrane compositions, such as HUVEC and erythrocytes, is low. CD spectroscopy, appropriately designed peptide controls, cell-based studies, liposome leakage assays, and electron microscopy support the intended mechanism of action, which leads to preferential killing of cancerous cells.     

Antiviral properties of lactoferrin--a natural immunity molecule

Lactoferrin, a multifunctional iron binding glycoprotein, plays an important role in immune regulation and defence mechanisms against bacteria, fungi and viruses. Lactoferrin's iron withholding ability is related to inhibition of microbial growth as well as to modulation of motility, aggregation and biofilm formation of pathogenic bacteria. Independently of iron binding capability, lactoferrin interacts with microbial, viral and cell surfaces thus inhibiting microbial and viral adhesion and entry into host cells. Lactoferrin can be considered not only a primary defense factor against mucosal infections, but also a polyvalent regulator which interacts in viral infectious processes. Its antiviral activity, demonstrated against both enveloped and naked viruses, lies in the early phase of infection, thus preventing entry of virus in the host cell. This activity is exerted by binding to heparan sulphate glycosaminoglycan cell receptors, or viral particles or both. Despite the antiviral effect of lactoferrin, widely demonstrated in vitro studies, few clinical trials have been carried out and the related mechanism of action is still under debate. The nuclear localization of lactoferrin in different epithelial human cells suggests that lactoferrin exerts its antiviral effect not only in the early phase of surface interaction virus-cell, but also intracellularly. The capability of lactoferrin to exert a potent antiviral activity, through its binding to host cells and/or viral particles, and its nuclear localization strengthens the idea that lactoferrin is an important brick in the mucosal wall, effective against viral attacks and it could be usefully applied as novel strategy for treatment of viral infections.     

Interaction of the antimicrobial peptide dicynthaurin with membrane phospholipids at the air-liquid interface

This paper reports the first study on the interaction of the antimicrobial peptide dicynthaurin with 1,2-dipalmitoyl-glycerophosphatidyl-glycerol investigated in monolayers at the air-liquid interface. The influence of the peptide on the two-dimensional phase behavior of the negatively charged lipid was elucidated by means of pressure-area isotherm measurements, fluorescence microscopy, and grazing incidence X-ray diffraction measurements. The pure peptide forms a stable monolayer at the air-liquid interface up to 30 mN/m as shown for both the monomeric and the dimeric cynthaurins. The peptide lipid interaction was monitored in isotherm measurements showing a strong adsorption of the peptide and stabilization at the interface promoted by the lipid monolayer. The X-ray diffraction measurements in agreement with fluorescence microscopy studies showed that the peptide destabilizes the condensed chain lattice, leading to a complete fluidization of the condensed lipid phase on physiological buffer. The adsorption of the peptide to the negatively charged lipid monolayer and the fluidization of the condensed chain lattice suggest a direct link to the peptides' ability to expand the bacterial membrane that would be relevant for the in vivo mode of action.     

HYDROPHOBIZING EFFECT OF CATIONS ON ACIDIC PHOSPHOLIPID MEMBRANES

By means of contact angle measurements with water and aqueous salt solutions, it is shown that plurivalent cations increase the hydrophobicity of negatively charged phospholipid vesicle membranes (consisting of phosphatidic acid, PA, or of phosphatidylserine, PS), but does not influence the hydrophobicity of neutral phospholipid membranes, (e.g., phosphatidylcholine, PC, at up to 200 mM of CaCl₂). The hydrophobizing action of cations on PA and PS membranes is concomitant with the reduction in (negative) zeta potential with increasing cation concentrations. Trivalent cations, La³⁺, showed more effective in hydrophobizing negatively charged phospholipid membranes than divalent and monovalent cations. Except for hydrogen ions, monovalent cations do not show any appreciable hydrophobizing effect on lipid vesicle membranes at concentrations less than 1 M. The hydrophobizing effect on phospholipid membranes can also be used to explain the induction of lateral phase separation into patches of different phospholipids as well as cell fusion.     

Statistical Analysis and Modeling of Pelletized Cultivation of Mucor circinelloides for Microbial Lipid Accumulation

Microbial oil accumulation via oleaginous fungi has some potential benefits because filamentous fungi can form pellets during cell growth and these pellets are easier to harvest from the culture broth than individual cells. This research studied the effect of various culture conditions on the pelletized cell growth of Mucor circinelloides and its lipid accumulation. The results showed that cell pelletization was positively correlated to biomass accumulation; however, pellet size was negatively correlated to the oil content of the fungal biomass, possibly due to the mass transfer barriers generated by the pellet structure. How to control the size of the pellet is the key to the success of the pelletized microbial oil accumulation process.     

Reinforcing the membrane-mediated mechanism of action of the anti-tuberculosis candidate drug thioridazine with molecular simulations

Thioridazine is a well-known dopamine-antagonist drug with a wide range of pharmacological properties ranging from neuroleptic to antimicrobial and even anticancer activity. Thioridazine is a critical component of a promising multi-drug therapy against M. tuberculosis. Amongst the various proposed mechanisms of action, the cell membrane-mediated one is peculiarly tempting due to the distinctive feature of phenothiazine drug family to accumulate in selected body tissues. In this study, we employ long-scale molecular dynamics simulations to investigate the interactions of three different concentrations of thioridazine with zwitterionic and negatively charged model lipid membranes. Thioridazine partitions into the interfacial region of membranes and modifies their structural and dynamic properties, however dissimilarly so at the highest membrane-occurring concentration, that appears to be obtainable only for the negatively charged bilayer. We show that the origin of such changes is the drug induced decrease of the interfacial tension, which ultimately leads to the significant membrane expansion. Our findings support the hypothesis that the phenothiazines therapeutic activity may arise from the drug–membrane interactions, and reinforce the wider, emerging view of action of many small, bioactive compounds.     

The chemical ecology of crucifers and their fungal pathogens: boosting plant defenses and inhibiting pathogen invasion

Fungal plant diseases can cause very substantial yield losses in crucifer crops such as rapeseed and canola, or vegetables such as cabbage and broccoli. To devise sustainable methods to prevent and deter crucifer pathogens, the chemical interaction between crucifers and their fungi is under intense investigation. Crucifers produce complex blends of secondary metabolites with diverse ecological roles that include protection against microbial pathogens and other pests. The secondary metabolites involved in crucifer defense, namely phytoalexins and phytoanticipins, and their metabolism by fungal pathogens indicate that some fungi produce different enzymes to detoxify these metabolites and that some fungal detoxifying enzymes are rather specific. Chemical synthesis and screening of phytoalexin analogue libraries using cultures of fungal pathogens, as well as protein extracts, have shown that such detoxification reactions can be inhibited and that some inhibitors are strongly antifungal. Overall results of current work show the feasibility of using selective inhibitors of fungal detoxifying enzymes, i.e., paldoxins, to protect plants by boosting their chemical defenses.     

Kinetics and thermodynamics of chlorpromazine interaction with lipid bilayers: effect of charge and cholesterol

Passive transport across cell membranes is the major route for the permeation of xenobiotics through tight endothelia such as the blood–brain barrier. The rate of passive permeation through lipid bilayers for a given drug is therefore a critical step in the prediction of its pharmacodynamics. We describe a detailed study on the kinetics and thermodynamics for the interaction of chlorpromazine (CPZ), an antipsychotic drug used in the treatment of schizophrenia, with neutral and negatively charged lipid bilayers. Isothermal titration calorimetry was used to study the partition and translocation of CPZ in lipid membranes composed of pure POPC, POPC:POPS (9:1), and POPC:Chol:POPS (6:3:1). The membrane charge due to the presence of POPS as well as the additional charge resulting from the introduction of CPZ in the membrane were taken into account, allowing the calculation of the intrinsic partition coefficients (K(P)) and the enthalpy change (ΔH) associated with the process. The enthalpy change upon partition to all lipid bilayers studied is negative, but a significant entropy contribution was also observed for partition to the neutral membrane. Because of the positive charge of CPZ, the presence of negatively charged lipids in the bilayer increases both the observed amount of CPZ that partitions to the membrane (KP(obs)) and the magnitude of ΔH. However, when the electrostatic effects are discounted, the intrinsic partition coefficient was smaller, indicating that the hydrophobic contribution was less significant for the negatively charged membrane. The presence of cholesterol strongly decreases the affinity of CPZ for the bilayer in terms of both the amount of CPZ that associates with the membrane and the interaction enthalpy. A quantitative characterization of the rate of CPZ translocation through membranes composed of pure POPC and POPC:POPS (9:1) was also performed using an innovative methodology developed in this work based on the kinetics of the heat evolved due to the interaction of CPZ with the membranes.     

Adsorption and disruption of lipid bilayers by nanoscale protein aggregates

Nanoparticles taken into biological systems can have biological impacts through their interactions with cell membranes, accompanied by protein adsorption onto the nanoparticle surfaces, forming a so-called protein corona. Our current research aims to demonstrate that nanoscale protein aggregates behave like such nanoparticles with regard to the interaction with lipid membranes. In this study, the adsorption and disruption of the lipid membranes by protein aggregates were investigated using amyloid fibrils and nanoscale thermal aggregates of lysozyme. Both types of protein aggregates had disruptive effects on the negatively charged liposomes, similar to polycationic nanoparticles. Interestingly, adsorption of liposomes on the amyloid fibrils preceding disruption occurred even if the net charge of the liposome was zero, suggesting the importance of hydrophobic interactions in addition to electrostatic interactions. The results of the present study provide new insights into the biological impacts of nanoparticles in vivo.     

Structural and dynamic characterization of the interaction of the putative fusion peptide of the S2 SARS-CoV virus protein with lipid membranes

The SARS coronavirus (SARS-CoV) envelope spike (S) glycoprotein, a Class I viral fusion protein, is responsible for the fusion between the membranes of the virus and the target cell. In the present work, we report a study of the binding and interaction with model membranes of a peptide pertaining to the putative fusion domain of SARS-CoV, SARS FP, as well as the structural changes that take place in both the phospholipid and the peptide molecules upon this interaction. From fluorescence and infrared spectroscopies, the peptide ability to induce membrane leakage, aggregation and fusion, as well as its affinity toward specific phospholipids, was assessed. We demonstrate that SARS FP strongly partitions into phospholipid membranes, more specifically with those containing negatively charged phospholipids, increasing the water penetration depth and displaying membrane-activity modulated by the lipid composition of the membrane. Interestingly, peptide organization is different depending if SARS FP is in water or bound to the membrane. These data suggest that SARS FP could be involved in the merging of the viral and target cell membranes by perturbing the membrane outer leaflet phospholipids and specifically interacting with negatively charged phospholipids located in the inner leaflet.     

Criterion for amino acid composition of defensins and antimicrobial peptides based on geometry of membrane destabilization

Defensins comprise a potent class of membrane disruptive antimicrobial peptides (AMPs) with well-characterized broad spectrum and selective microbicidal effects. By using high-resolution synchrotron small-angle X-ray scattering to investigate interactions between heterogeneous membranes and members of the defensin subfamilies, α-defensins (Crp-4), β-defensins (HBD-2, HBD-3), and θ-defensins (RTD-1, BTD-7), we show how these peptides all permeabilize model bacterial membranes but not model eukaryotic membranes: defensins selectively generate saddle-splay ("negative Gaussian") membrane curvature in model membranes rich in negative curvature lipids such as those with phosphoethanolamine (PE) headgroups. These results are shown to be consistent with vesicle leakage assays. A mechanism of action based on saddle-splay membrane curvature generation is broadly enabling, because it is a necessary condition for processes such as pore formation, blebbing, budding, and vesicularization, all of which destabilize the barrier function of cell membranes. Importantly, saddle-splay membrane curvature generation places constraints on the amino acid composition of membrane disruptive peptides. For example, we show that the requirement for generating saddle-splay curvature implies that a decrease in arginine content in an AMP can be offset by an increase in both lysine and hydrophobic content. This "design rule" is consistent with the amino acid compositions of 1080 known cationic AMPs.     

Methotrexate interaction with a lipid membrane model of DPPC

Dipalmitoylphosphatidylcholine (DPPC) liposomes were employed as membrane models for the investigation of the interaction occurring between methotrexate (MTX) and bilayer lipid matrix. Liposomes were obtained by hydrating a lipid film with 50 mM Tris buffer (pH 7.4). The differential scanning calorimetry (DSC) evaluation of the thermotropic parameters associated with the phase transitions of DPPC liposomes gave useful information about the kind of drug-membrane interaction. The results showed an electrostatic interaction taking place with the negatively charged molecules of MTX and the phosphorylcholine head groups, constituting the outer part of DPPC bilayers. No interaction with the hydrophobic phospholipid bilayer domains was detected, revealing a poor capability of MTX to cross through lipid membranes to reach the interior compartment of a lipid bounded structure. These findings correlate well within vitro biological experiments on MTX cell susceptibility.     

Unraveling dendrimer translocation across cell membrane mimics

Poly(amidoamine) (PAMAM) dendrimers are promising candidates in several applications within the medical field. However, it is still to date not fully understood whether they are able to passively translocate across lipid bilayers. Recently, we used fluorescence microscopy to show that PAMAM dendrimers induced changes in the permeability of lipid membranes but the dendrimers themselves could not translocate to be released into the vesicle lumen. Because of the lack of resolution, these experiments could not assess whether the dendrimers were able to translocate but remained attached to the membrane. Using quartz crystal microbalance with dissipation monitoring and neutron reflectivity, a structural investigation was performed to determine how dendrimers interact with zwitterionic and negatively charged lipid bilayers. We hereby show that dendrimers adsorb on top of lipid bilayers without significant dendrimer translocation, regardless of the lipid membrane surface charge. Thus, most likely dendrimers are actively transported through cell membranes by protein-mediated endocytosis in agreement with previous cell studies. Finally, the higher activity of PAMAM dendrimers for phosphoglycerol-containing membranes is in line with their high antimicrobial activity against Gram-negative bacteria.     

F-肌动蛋白与负电荷磷脂膜的相互作用研究

细胞膜的内膜含有大量的负电荷磷脂,研究F-肌动蛋白与负电荷磷脂的相互作用将有助于更深入地了解细胞骨架与细胞膜的体内相互作用机制.在金片和金电极上分别构建了负电荷磷脂的杂化双层磷脂膜,通过表面等离子体共振方法(SPR)和电化学阻抗技术研究了F-肌动蛋白与负电荷磷脂膜的相互作用.结果表明,F-肌动蛋白可以在没有中间联系蛋白的情况下,直接与负电荷磷脂膜发生相互作用.钙离子可以有效地促进它们的相互作用,表明钙离子在其中发挥了重要作用.高浓度的KCl显著抑制它们的相互作用,表明这种相互作用主要受静电作用影响.实验结果进一步证明在F-肌动蛋白与负电荷磷脂膜相互作用时,除了可以通过其它蛋白发生间接相互作用外,还可以与磷脂膜发生直接的相互作用.     

Antimicrobial peptides in burns and wounds

Burn-induced immunosuppression not only increases susceptibility to infection, but also predisposes burn patients to related adverse sequelae, including systemic inflammatory response syndrome and sepsis. Although burn-related immunosuppression is not fully understood, it is characterized by decreased T- and B-lymphocyte function and by impaired functions of circulating leukocytes and complement. Alterations in defensins, a family of cationic, naturally occurring antimicrobial peptides, may underlie these immune deficiency patterns. Defensins are considered important components of the innate immune system, as they inhibit bacterial, fungal, and viral colonization. They also chemoattract immature dendritic cells and T lymphocytes, recruit neutrophils, macrophages, and monocytes, modulate complement and adjuvant activity, and promote inflammation and wound healing. Infectious states are associated with upregulation of circulating defensins, which suggests an underlying antimicrobial role. In addition, data from our laboratory demonstrated diminished levels of certain defensins in burned tissue. The inference is that decreased defensin levels in burn injury may facilitate infection and subsequent sepsis. It may also alter functions of T- and B-lymphocytes, neutrophils, macrophages, and complement, thereby contributing to the pathophysiology of burn-related systemic inflammatory responses. This article is a comprehensive review on the role of antimicrobial peptides in burns and wounds.     

Interactions of membrane-active peptides with thick, neutral, nonzwitterionic bilayers

Alamethicin is a well-studied channel-forming peptide that has a prototypical amphipathic helix structure. It permeabilizes both microbial and mammalian cell membranes, causing loss of membrane polarization and leakage of endogenous contents. Antimicrobial peptide-lipid systems have been studied quite extensively and have led to significant advancements in membrane biophysics. These studies have been performed on lipid bilayers that are generally charged or zwitterionic and restricted to a thickness range of 3-5 nm. Bilayers of amphiphilic diblock copolymers are a relatively new class of membranes that can have significantly different physicochemical properties compared with those of lipid membranes. In particular, they can be made uncharged, nonzwitterionic, and much thicker than their lipid counterparts. In an effort to extend studies of membrane-protein interactions to these synthetic membranes, we have characterized the interactions of alamethicin and several other membrane-active peptides with diblock copolymer bilayers. We find that although alamethicin is too small to span the bilayer, the peptide interacts with, and ruptures, thick polymer membranes.     

PH effects on drug interactions with lipid bilayers by liposome electrokinetic chromatography

Liposome electrokinetic chromatography (LEKC) provides convenient and rapid methods for studying drug interactions with lipid bilayers using liposomes as a pseudostationary phase. LEKC was used to determine the effects of pH on the partitioning of basic drugs into liposomes composed of zwitterionic phosphatidylcholine (PC), anionic phosphatidylglycerol (PG), and cholesterol, which mimic the composition of natural cell membranes. An increase in pH results in a smaller degree of ionization of the basic drugs and consequently leads to a lower degree of interaction with the negatively charged membranes. From the LEKC retention data, the fractions of drugs distributed in the bulk aqueous and the liposome phase were determined at various pH values. Finally, lipid mediated shifts in the ionization constants of drugs were examined.     

Chemical Composition,Antipathogenic and Cytotoxic Activity of the Essential Oil Extracted from Amorpha fruticosa Fruits

The purpose of this paper was to characterize and investigate the antimicrobial potential of Amorpha fruticosa fruits essential oil (EO). The EO was extracted by hydrodistillation, analyzed by GC-MS, and then evaluated for its interaction with microbial and mammalian cells. The antimicrobial activity was assessed against bacterial and fungal strains, in a planktonic and adherent growth state, using qualitative and quantitative assays. The main components identified in A. fruticosa fruits EO were δ-cadinene, γ-muurolene, and α-muurolene. The Gram-positive strains proved to be more susceptible than Gram-negative bacteria and fungal strains. The EO exhibited good antibiofilm activity, inhibiting the microbial adherence to the inert (96-well plates and Foley catheter section) and cellular substrata. The flow cytometry analysis revealed as one of the possible mechanisms of antimicrobial action the alteration of cell membrane hydrophobicity. The cytotoxicity on the L929 cell line occurred at concentrations higher than 0.3 mg/mL. Taken together, our results demonstrate that A. fruticosa fruits EO contains active compounds with selective inhibitory effect on different microbial strains in planktonic and biofilm growth state, explained at least partially by the interference with microbial membranes due to their hydrophobic character.     

Competitive adsorption of Ca and local anesthetics on lipid membrane surfaces

Adsorption fo tertriary amine local anesthetics and Ca²⁺ onto lipid membranes having various negative surface charge densities was studied by measuring lipid vesicle electrophoretic mobility.

As the surface charge density of the membrane was reduced, the adsorption of the local anesthetics dominated that of the divalent cation. For a relatively high negatively charged membrane, the adsorption of both local anesthetic and Ca²⁺ became comparable and competitive.

It is deduced that the major factor for the adsorption of local anesthetic onto lipid membranes is due to simple physical partitioning between aqueous and membrane phases, and not due to ionic type of binding as seen for divalent cations with membranes. However, the adsorption of anesthetics is influenced by the surface potential of membranes which is in turn related to the surface concentration of local anesthetics near the membrane.

The amounts of competitive adsorption of divalent cations and local anesthetics are analyzed with respect to their bulk concentrations and various surface charge densities of the membranes. With the results of the above studies, a possible interpretation for the interaction site as well as the mode of adsorption of local anesthetics onto axon membranes is made in relation to divalent cation concentrations in the bulk phases.     

An electrodynamics-based model for ion diffusion in microbial polysaccharides

An electrodynamics-based model was formulated for simulation of ion diffusion in microbial polysaccharides. The fixed charges and electrostatic double layers that may associate with microbial polysaccharides and their effects on ion diffusion were explicitly built into the model. The model extends a common multicomponent ion diffusion formulation that is based on irreversible thermodynamics under a zero ionic charge flux condition, which is only applicable to the regions without fixed charges and electrostatic double layers. An efficient numerical procedure was presented to solve the differential equations in the model. The model well described key features of experimental observations of ion diffusion in negatively charged microbial polysaccharides including accelerated diffusive transport of cations, exclusion of anions, and increased rate of cation transport with increasing negative charge density. The simulated diffusive fluxes of cations and anions were consistent with a cation exchange diffusion concept in negatively charged polysaccharides at the interface of plant roots and soils; and the developed model allows to mathematically study such diffusion phenomena. An illustrative example was also provided to simulate dynamic behavior of ionic current during ion diffusion within a charged bacterial cell wall polysaccharide and the effects of the ionic current on the compression or expansion of the bacterial electrostatic double layer at the interface of the cell wall and bulk solution.