Negatively-charged Lipid Research Articles

Gasdermin D cysteine residues synergistically control its palmitoylation-mediated membrane targeting and assembly.

Gasdermin D (GSDMD) executes the cell death program of pyroptosis by assembling into oligomers that permeabilize the plasma membrane. Here, by single-molecule imaging, we elucidate the yet unclear mechanism of Gasdermin D pore assembly and the role of cysteine residues in GSDMD oligomerization. We show that GSDMD preassembles at the membrane into dimeric and trimeric building blocks that can either be inserted into the membrane, or further assemble into higher-order oligomers prior to insertion into the membrane. The GSDMD residues Cys39, Cys57, and Cys192 are the only relevant cysteines involved in GSDMD oligomerization. S-palmitoylation of Cys192, combined with the presence of negatively-charged lipids, controls GSDMD membrane targeting. Simultaneous Cys39/57/192-to-alanine (Ala) mutations, but not Ala mutations of Cys192 or the Cys39/57 pair individually, completely abolish GSDMD insertion into artificial membranes as well as into the plasma membrane. Finally, either Cys192 or the Cys39/Cys57 pair are sufficient to enable formation of GSDMD dimers/trimers, but they are all required for functional higher-order oligomer formation. Overall, our study unveils a cooperative role of Cys192 palmitoylation-mediated membrane binding and Cys39/57/192-mediated oligomerization in GSDMD pore assembly. This study supports a model in which Gasdermin D oligomerization relies on a two-step mechanism mediated by specific cysteine residues.

Thermodynamics of oligomerization and Helix-to-sheet structural transition of amyloid β-protein on anionic phospholipid vesicles

Understanding oligomerization and aggregation of the amyloid-β protein is important to elucidate the pathological mechanisms of Alzheimer's disease, and lipid membranes play critical roles in this process. In addition to studies reported by other groups, our group has also reported that the negatively-charged lipid bilayers with a high positive curvature induced α-helix-to-β-sheet conformational transitions of amyloid-β-(1–40) upon increase in protein density on the membrane surface and promoted amyloid fibril formation of the protein. Herein, we investigated detailed mechanisms of the conformational transition and oligomer formation of the amyloid-β protein on the membrane surface. Changes in the fractions of the three protein conformers (free monomer, membrane-bound α-helix-rich conformation, and β-sheet-rich conformation) were determined from the fluorescent spectral changes of the tryptophan probe in the protein. The helix-to-sheet structural transition on the surface was described by a thermodynamic model of octamer formation driven by entropic forces including hydrophobic interactions. These findings provide useful information for understanding the self-assembly of amyloidogenic proteins on lipid membrane surfaces.

Hydrophobic moment drives penetration of bacterial membranes by transmembrane peptides

With antimicrobial resistance (AMR) remaining a persistent and growing threat to human health worldwide, membrane-active peptides are gaining traction as an alternative strategy to overcome the issue. Membrane-embedded multi-drug resistant (MDR) efflux pumps are a prime target for membrane-active peptides, as they are a well-established contributor to clinically relevant AMR infections. Here, we describe a series of transmembrane peptides (TMs) to target the oligomerization motif of the AcrB component of the AcrAB-TolC MDR efflux pump from Escherichia coli. These peptides contain an N-terminal acetyl-A-(Sar)3 (sarcosine; N-methylglycine) tag and a C-terminal lysine tag-a design strategy our lab has utilized to improve the solubility and specificity of targeting for TMs previously. While these peptides have proven useful in preventing AcrB-mediated substrate efflux, the mechanisms by which these peptides associate with and penetrate the bacterial membrane remained unknown. In this study, we have shown peptide hydrophobic moment (μH)-the measure of concentrated hydrophobicity on one face of a lipopathic α-helix-drives bacterial membrane permeabilization and depolarization, likely through lateral-phase separation of negatively-charged POPG lipids and the disruption of lipid packing. Our results show peptide μH is an important consideration when designing membrane-active peptides and may be the determining factor in whether a TM will function in a permeabilizing or non-permeabilizing manner when embedded in the bacterial membrane.

Simultaneous membrane and RNA binding by tick-borne encephalitis virus capsid protein.

Tick-borne encephalitis virus is an enveloped, pathogenic, RNA virus in the family Flaviviridae, genus Flavivirus. Viral particles are formed when the nucleocapsid, consisting of an RNA genome and multiple copies of the capsid protein, buds through the endoplasmic reticulum membrane and acquires the viral envelope and the associated proteins. The coordination of the nucleocapsid components to the sites of assembly and budding are poorly understood. Here, we investigate the interactions of the wild-type and truncated capsid proteins with membranes with biophysical methods and model membrane systems. We show that capsid protein initially binds membranes via electrostatic interactions with negatively-charged lipids, which is followed by membrane insertion. Additionally, we show that membrane-bound capsid protein can recruit viral genomic RNA. We confirm the biological relevance of the biophysical findings by using mass spectrometry to show that purified virions contain negatively-charged lipids. Our results suggest that nucleocapsid assembly is coordinated by negatively-charged membrane patches on the endoplasmic reticulum and that the capsid protein mediates direct contacts between the nucleocapsid and the membrane.

Enzyme mediated production of asymmetrically charged liposomes.

In asymmetric liposomes, the inner and outer leaflets of the lipid bilayer have different compositions. As such, these liposomes are appealing platforms for modeling and studying membrane asymmetry in biological membranes. In addition, asymmetric liposomes can serve as innovative drug delivery vehicles in which, the inner leaflet can be designed for maximal cargo encapsulation while the outer leaflet can be optimized for minimal toxicity. This study aims to explore the use of a membrane-active enzyme, phospholipase D (PLD), to produce liposomes with asymmetrically-charged membranes. PLD is an interfacial enzyme that catalyzes the hydrolysis of zwitterionic phosphatidylcholine (PC) lipids to produce choline and negatively-charged phosphatidic acid (PA) lipids. For this work, we introduced PLD to pre-formed PC liposomes and monitored the zeta potential of these liposomes at different time points. Zeta potential of these liposomes showed a time-dependent drop, presumably due to PLD activity and production of negatively-charged PA lipids in the membrane. Using a calibration curve, the zeta potential values were converted to the amount of PA formed in the outer leaflet of liposomes. Notably, the use of heat-deactivated PLD enzyme did not have an impact on liposomal zeta values. In addition, the results of a choline assay that was used to quantify the produced choline and thus PA in the system were comparable to those from zeta measurements. Furthermore, performing these studies with different PLD concentrations showed that the rate of PA production varied with the concentration of enzyme. Lastly, experiments of dye leakage from liposomes suggested that this enzymatic activity did not have a significant impact on liposomal membrane integrity. Together, these findings indicate that PLD enzymatic activity can be utilized to generate liposomes with asymmetrically charged leaflets, providing exciting opportunities for membrane biophysical studies as well as drug delivery applications.

Cetylpyridinium chloride (CPC) reduces zebrafish mortality from influenza infection: Super-resolution microscopy reveals CPC interference with multiple protein interactions with phosphatidylinositol 4,5-bisphosphate in immune function

The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.1 μM, 1 h) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5–10 μM) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP2); here, we show that nanoscale co-localization of HA with the PIP2-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP2 is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP2-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.

Conformation-specific perturbation of membrane dynamics by structurally distinct oligomers of Alzheimer's amyloid-β peptide.

The accumulation of toxic soluble oligomers of the amyloid-β peptide (Aβ) is a key step in the pathogenesis of Alzheimer's disease. There are mainly two conformationally distinct oligomers, namely, prefibrillar and fibrillar oligomers, that are recognized by conformation-specific antibodies, anti-amyloid oligomer antibody (A11) and anti-amyloid fibrillar antibody (OC), respectively. Previous studies have shown that the interaction of Aβ oligomers with the lipid membrane is one of the key mechanisms of toxicity produced by Aβ oligomers. However, the mechanism by which structurally distinct Aβ oligomers interact with the lipid membrane remains elusive. In this work, we dissect the molecular mechanism underlying the interaction of structurally distinct Aβ42 oligomers with the lipid membrane derived from the brain total lipid extract. Using picosecond time-resolved fluorescence spectroscopy, we show that the A11-positive Aβ42 oligomers undergo a membrane-induced conformational change that promotes the deeper immersion of these oligomers into the lipid hydrocarbon region and results in an increase in the membrane micro-viscosity. In sharp contrast, OC-positive Aβ42 oligomers interact with the lipid membrane via electrostatic interactions between the negatively-charged lipid headgroup and positively-charged residues of Aβ42 without perturbing the membrane dynamics. We show that the two structurally distinct Aβ42 oligomers demonstrating different interaction mechanisms with the lipid membrane eventually lead to the formation of typical amyloid fibrils. Our findings provide the mechanistic underpinning of the perturbation of lipid membranes by two conformationally distinct Aβ42 oligomers and can be of prime importance in designing anti-Alzheimer's therapeutic agents targeting Aβ-membrane interactions.

Aromatic-rich C-terminal region of LCI is a potent antimicrobial peptide in itself

LCI is a 47-residue antimicrobial peptide produced by Bacillus subtilis. The peptide displays potent activity against plant pathogens, Xanthomonas and Pseudomonas. The peptide takes a compact 3-dimensional structure characterized by a four-stranded β-sheet. The peptide is unusually rich in aromatic residues; 10 of the 47 residues are aromatic and 8 of them lie in the C-terminal region, LCI22-47. Here we report the antimicrobial activity of this C-terminal region against Gram-positive and Gram-negative bacteria. The C-terminal-amidated peptide displays potent activity against E. coli, methicillin and gentamicin-resistant S. aureus, and Xanthomonas oryzae pv. oryzae with lethal concentrations ≤4 μM. Membrane-binding assays indicate preferential binding to the negatively-charged lipids. The peptide permeabilizes the outer-membrane of E. coli indicating membrane-permeabilization as one of the mechanisms of killing. Interestingly, however, no inner-membrane permeabilization was observed, indicating that the membrane-permeabilization may not be the sole mechanism of action.

Characterization of Tachyplesin Peptides and Their Cyclized Analogues to Improve Antimicrobial and Anticancer Properties.

Tachyplesin I, II and III are host defense peptides from horseshoe crab species with antimicrobial and anticancer activities. They have an amphipathic β-hairpin structure, are highly positively-charged and differ by only one or two amino acid residues. In this study, we compared the structure and activity of the three tachyplesin peptides alongside their backbone cyclized analogues. We assessed the peptide structures using nuclear magnetic resonance (NMR) spectroscopy, then compared the activity against bacteria (both in the planktonic and biofilm forms) and a panel of cancerous cells. The importance of peptide-lipid interactions was examined using surface plasmon resonance and fluorescence spectroscopy methodologies. Our studies showed that tachyplesin peptides and their cyclic analogues were most potent against Gram-negative bacteria and melanoma cell lines, and showed a preference for binding to negatively-charged lipid membranes. Backbone cyclization did not improve potency, but improved peptide stability in human serum and reduced toxicity toward human red blood cells. Peptide-lipid binding affinity, orientation within the membrane, and ability to disrupt lipid bilayers differed between the cyclized peptide and the parent counterpart. We show that tachyplesin peptides and cyclized analogues have similarly potent antimicrobial and anticancer properties, but that backbone cyclization improves their stability and therapeutic potential.

Preparation of Asymmetric Charged Large Unilamellar Vesicles Containing Both Cationic and Anionic Lipids

Liposomes have important applications for drug delivery. Whether asymmetric large unilamellar vesicles (asymmetric LUVs) with different charges on different leaflets can be efficiently prepared and have practical applications have advantages for efficient drug encapsulation and efficiency has not been explored. To examine this we have developed methods to prepare asymmetric LUV with opposite charges on the inner and outer leaflet. We investigated liposomes mixtures containing one of three different kinds of negative charged lipids, POPG, POPS and POPA, one of two different kinds of positive charged lipids, POePC and DOTAP, and a zwitterionic lipid, POPC. Using the cyclodextrin (CD) assisted exchange methods developed by our group, we have prepared asymmetric LUVs with about 25 % of POPS or POPA in the outer or inner leaflet (monolayer) and about 25 % of POePC or DOTAP on the opposite leaflet, with about 75 % of lipids in each leaflets being POPC. Exchange with POPG was somewhat less efficient. To investigate asymmetry of the LUVs a TMA-DPH binding assay was developed. Future studies will investigate drug entrapment ability and the efficiency of drug delivery using these liposomes.

Thymol in cellular membrane models formed by negative charged lipids causes aggregation at the air-water interface

The physical-chemical effects of drugs/lipidic interfaces interactions are still little known, justifying investigations using model systems. Here, we investigated a drug with potential biological activity against microbial and tumorigenic cells, thymol, using phosphatidylserine as cellular membrane models. Surface pressure-area isotherms showed that selected amounts of thymol expand the monolayers and decreased its elasticity. Vibrational spectroscopy and Brewster angle microscopy pointed that thymol adsorbs on the lipid polar heads, affecting the aliphatic chain gauche conformations, causing aggregation at the interface. This indicated distinctive molecular accommodations of thymol along the phospholipidic structures, which is associated to its biological effect in natural membranes.

Structural characteristics and membrane interactions of tandem \u03b1-synuclein oligomers

Pre-fibrillar oligomers of α-synuclein are thought to be pathogenic molecules leading to neurotoxicity associated with Parkinson’s disease and other neurodegenerative disorders. However, small oligomers are difficult to isolate for study. To gain better insight into the properties of small α-synuclein oligomers, we investigated engineered oligomers of specific size (dimers, tetramers, and octamers) linked head-to-tail in tandem, comparing the behavior of the oligomers to monomeric α-synuclein. All oligomeric constructs remained largely disordered in solution, as determined from dynamic light scattering and size-exclusion chromatography. Electron microscopy revealed that each construct could aggregate to form fibrils similar to those formed by monomeric α-synuclein. The interactions with large unilamellar vesicles (LUVs) composed of negatively-charged lipids differed depending on size, with smaller oligomers forming more extensive helical structure as determined by CD spectroscopy. Monitoring the influx of a fluorescence bleaching agent into vesicles showed that larger oligomers were somewhat more effective at degrading vesicular integrity and inducing membrane permeabilization.

Identification of Cardiolipin Binding Sites on Membrane Proteins using an Accelerated Computational Membrane Model

Cardiolipin (CDL), a negatively-charged lipid found primarily in bacterial and mitochondrial membranes, is believed to play a major role in membrane morphology, some hereditary heart diseases, as well as acting as a proton reservoir in the inner mitochondrial membrane. CDL has been shown to regulate many integral membrane proteins involved in bioenergetics, often binding with nanomolar affinity. In this study we incorporate CDL into the Highly Mobile Membrane Mimetic (HMMM), a membrane model consisting of lipid head groups partitioned by an organic solvent mimicking hydrophobic acyl tails, which allows for fast lipid diffusion, nanosecond spontaneous membrane insertion of proteins, and providing atomistic sampling of CDL-protein interactions, which gives rise to the identification of potential CDL binding residues. As an application, we chose CLC chloride transporter (CLC-ec1), an integral membrane protein involved in maintaining proton gradients, which initial experimental results show that CDL increases CLC-ec1 activity, likely due to CDLs ability to act as a proton reservoir. The increased lipid diffusion and sampling of CDL containing HMMM membranes aided in determining potential binding sites for ClC Chloride transporter. Incorporating CDL into the HMMM model involved applying harmonic restraints to each carbonyl carbon for each tail to maintain membrane thickness, as well as applying RMSD based collective variable restraints on ClC-ec1. Using ten one-hundred nanosecond HMMM simulations of CLC-ec1 embedded into a 80:20 POPE:CDL membrane, interactions between CDL and protein residues were measured. The overall microsecond simulation time, together with the enhanced dynamics offered by the HMMM membrane, provided a large sample size, which led to narrowing down the potential CDL binding sites to two sites, each stabilized by two basic side chains, thus expediting and guiding ClC-ec1 mutagenesis studies.

Monte Carlo simulations of the distributions of intra- and extra-vesicular ions and membrane associated charges in hybrid liposomes composed of negatively charged tetraether and zwitterionic diester phospholipids

Here, we model a negatively charged lipid vesicle, composed of a mixture of bipolar tetraether and diester (or diether) phospholipid molecules, by a spherical shell that has zero ion permeability. We take into consideration all the charge-charge interactions between intra-vesicular ions, extra-vesicular ions, and membrane lipid associated charges. Monte Carlo simulations result in homogeneous and double-exponential ion distribution, respectively, in the intra- and extra-vesicular space. The extra-vesicular ion concentration close to the membrane surface is proportional to the total amount of the membrane charges (Nm) and is independent of the partitioning of the membrane charges between the outer (Nom) and inner membrane (Nim) surface. This result shows that one should not disregard the effect of the charges on the inner membrane surface when calculating the ion distributions around a charged vesicle. If the partitioning of the membrane charges is not restricted (i.e., lipid flip-flop is allowed), then at different Nm, the Nom/Nim ratio remains constant and the value of Nom/Nim, as a consequence of the interaction between every charges of the model, is close to, but significantly higher than, the ratio of the outer to the inner surface area of the membrane. These results indicate that the amount and the orientation of the negatively-charged tetraether lipids in the membrane are important determinants of membrane properties in tetraether/zwitterionic diester phospholipid liposomes. Finally we compared the results of our discrete charge model and continuous models based on the solutions of the Poisson-Boltzmann equation and pointed out qualitative similarities and sometimes major quantitative differences between these two types of models.

Charge boosting effect of cholesterol on cationic liposomes

Cationic liposomes that have been used as drug or gene delivery vehicles can be obtained by mixing cationic surfactants in phospholipid liposomes. Cytotoxicity of cationic molecules is one of the problems of the cationic nanocarriers. Therefore we aimed to formulate highly positively-charged cationic liposomes with low content of cationic surfactant, which have both higher cellular uptake ability and lower cytotoxicity. We have investigated the zeta potential behavior in water/soy bean lecithin/cationic surfactant/cholesterol systems by the changing composition as well as the molecular structure of the cationic surfactant. Mixing of monoalkyl (C12, C16 and C18) and dialkyl (C12 and C18) quaternary ammonium chlorides were increased the zeta potential of liposomes from negative to positive values (more than +50mV). Mixing the cholesterol into the cationic liposomes increased the maximum zeta potential further and also induced the large shift of the negative-positive transition point with respect to the cationic surfactant mixing fraction. It means much less cationic surfactant is needed to obtain highly positively-charged liposomes. The significant cholesterol effects on the zeta potential of liposomes were explained based on the lipid distribution inhomogeneity caused by the increased fluidity of liquid ordered phase membranes and the large critical packing parameter of the negatively-charged lipid.

Lecithin‐acrylamido‐2‐methylpropane sulfonate based crosslinked phospholipid nanoparticles as drug carrier

ABSTRACTIn this study, a novel paclitaxel (PTX) loaded and a crosslinked solid phospholipid nanoparticles (SLN‐PTX) with negative surface charge was prepared by UV polymerization for drug delivery. Capping of positive charge of zwitterionic lecithin with negative charge of sodium 2‐acrylamido‐2‐methyl‐1‐propanesulfonate (AMPS‐Na) through cation exchange interaction produced a lecithin‐AMPS (L‐AMPS) complex. The amphiphilic and negative charged lipid complex was emulsified in the presence of emulsifier, paclitaxel, initiator, and methacrylated poly ε‐caprolacton‐diol (PCL‐MAC) as a spacer. The colloidal system was subjected to UV‐irradiation to obtain crosslinked nanoparticles. Completion of the UV‐polymerization was monitored with differential scanning calorimetry (DSC), which indicated the disappearance of exothermic peaks of vinyl groups. The nanoparticle system, having an average size of 200 nm, exhibited high drug encapsulation (96%) with negatively charged surface (zeta potential had an average of −70 mV). PTX release profiles of the crosslinked and uncrosslinked SLN‐PTXs were studied and their pharmacological properties were compared. The crosslinked nanoparticles exhibited more controlled release behavior with longer release time compared to the uncrosslinked ones. In vitro cytotoxicity test was conducted on MCF‐7 human breast adenocarcinoma cell line, which indicated that the crosslinked SLN‐PTXs have a potential therapeutic effect for breast cancer treatments. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 44105.

Peptide translocation through the plasma membrane of human cells: Can oxidative stress be exploited to gain better intracellular access?

ABSTRACTCell-penetrating peptides (CPPs) enter cells primarily by escaping from endosomal compartments or by directly translocating across the plasma membrane. Due to their capability of permeating into the cytosolic space of the cell, CPPs are utilized for the delivery of cell-impermeable molecules. However, the fundamental mechanisms and parameters associated with the penetration of CPPs and their cargos through the lipid bilayer have not been fully determined. This in turn has hampered their usage in biotechnological or therapeutic applications. We have recently reported that the cell penetration activity of poly-arginine CPPs (PACPPs) is dependent on the oxidation status of the plasma membrane of cells. Our data support a model where the positively-charged PACPP binds negatively-charged lipids exposed on the cell surface as a result of oxidative damage. The PACPP then crosses the membrane via formation of inverted micelles with these anionic lipids. This model provides a plausible explanation for the high variability in the cell delivery efficiency of a PACPP often observed in different settings. Notably, taking into account the current literature describing the effects of lipid oxidation, our data point to a highly complex and underappreciated interplay between PACPPs and oxidized membrane species. Overall, a better understanding of oxidation-dependent cell penetration might provide a fundamental basis for development of optimal cell permeable peptides (including cyclic peptides, stapled peptides, peptoids, etc…) and of robust delivery protocols.

Interaction of Cecropin B with Zwitterionic and Negatively Charged Lipid Bilayers Immobilized at Gold Electrode Surface

Membranolytic properties of cationic antimicrobial peptide cecropin B were investigated using electrochemical techniques, atomic force microscopy and quartz crystal microbalance with dissipation monitoring. Two types of artificial lipid bilayers supported on gold electrode were used as model systems composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol (Chol) at 7:3 molar ratio and L-α-phosphatidylethanolamine (E. coli) (PE), L-α-phosphatidylglycerol sodium salt (E. coli) (PG) at 8:2 molar ratio. Thus the lipid content was intended to represent either mammalian or bacterial membrane respectively. Model bilayers were exposed to cecropin B at 1μM concentration and the changes in bilayer structure, permeability and morphology were monitored as a function of time. We have found that cecropin B does not show any pronounced effect on POPC/Chol bilayer, while PE/PG system was strongly affected in the presence of the peptide. This observation suggests that cecropin B shows some selectivity with respect to lipid composition of the membrane. In case of PE/PG membrane, we have observed that peptide action involves electrostatically driven adsorption of the cecropin B at the top of the bilayer with simultaneous fluidization and swelling of the membrane. The latter may facilitate the rearrangement and insertion of the molecules into the core of the lipid bilayer, which leads to further rupture and degradation of the film through formation of mixed peptide-lipid aggregates.

Structure-Activity Relationship Studies Reveal that the Spider Toxin Protx-II has Unusual Membrane-Binding Properties and Inhibits NAV1.7 Channel at the Membrane Surface

ProTx-II, a peptide toxin isolated from spider venom, has large potential as a pain therapeutic. This disulfide-rich peptide inhibits the membrane voltage-gated sodium channel Nav 1.7, a recognized therapeutic target to treat pain, through binding to the voltage-sensor domains of the channel inserted in the membrane. The exact peptide-channel binding site and the importance of the membrane in its inhibition activity remain unknown. In the current study we examined the structure and the membrane-binding properties of ProTx-II, and of a series of analogues, using NMR spectroscopy, surface plasmon resonance and fluorescence spectroscopy methodologies. Our studies reveal that ProTx-II has an amphipathic surface and high affinity for lipid bilayers with preference for negatively-charged lipids. Interestingly, despite of each of the four Trp residues being essential for its membrane-binding properties, the peptide has a shallow location in the bilayer. Furthermore, contrary to other membrane-active peptides, replacement of Lys with Arg residues decreases the membrane-binding properties of ProTx-II. Our studies also suggest that sphingomyelinase D, an enzyme present in spider venoms that converts the neutral sphingomyelin into a negatively-charged lipid, increases the ability of ProTx-II to bind to membranes that mimic raft-like domains where the ion channels are located. Overall these results suggest: 1) ProTx-II interacts with Nav 1.7 voltage sensor domain at the membrane surface; 2) sphingomyelinase D in spider venoms increases the concentration of positively-charged toxins in the channel vicinity by converting the neutral raft-domains into negatively-charged surfaces.

Dispersion of atmospheric fine particulate matters in simulated lung fluid and their effects on model cell membranes

Atmospheric fine particulate matter (PM2.5) was collected to investigate its dispersion in simulated lung fluid (SLF) and its interaction with model cell membranes. Organic acids, NH4+, SO42− and NO3− were detected in PM2.5 soluble fraction, and heavy metals were detected from the total mass. The insoluble fraction contained kaolinite, CaCO3, aliphatic carbons, aromatic rings, carboxyl and hydroxyl groups reflected by the infrared spectra. Proteins dispersed PM2.5 in SLF, resulted in smaller hydrodynamic diameter (dH) and slower sedimentation rate. Conversely, phospholipids increased dH value and accelerated sedimentation rate. Giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs) were used as model cell membranes. PM2.5 adhered on and disrupted the membrane containing positively-charged lipids but not the membrane containing neutrally- and negatively-charged lipids, which was monitored by microscopy and a quartz crystal microbalance with dissipation (QCM-D). The cationic sites on membrane were necessary for PM2.5 adhesion, but membrane should be disrupted by the combined action of electrostatic forces and hydrogen bonds between PM2.5 oxygen containing groups and the lipid phosphate groups. Our results specified the roles of proteins and phospholipids in PM2.5 dispersion and transport, highly suggested that the health hazard of PM2.5 was related to the biomolecules in the lung fluid and the particle surface groups.