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1.
Interaction of Halictine-Related Antimicrobial Peptides with Membrane Models.
Pazderková, M, Maloň, P, Zíma, V, Hofbauerová, K, Kopecký, V, Kočišová, E, Pazderka, T, Čeřovský, V, Bednárová, L
International journal of molecular sciences. 2019;(3)
Abstract
We have investigated structural changes of peptides related to antimicrobial peptide Halictine-1 (HAL-1) induced by interaction with various membrane-mimicking models with the aim to identify a mechanism of the peptide mode of action and to find a correlation between changes of primary/secondary structure and biological activity. Modifications in the HAL-1 amino acid sequence at particular positions, causing an increase of amphipathicity (Arg/Lys exchange), restricted mobility (insertion of Pro) and consequent changes in antimicrobial and hemolytic activity, led to different behavior towards model membranes. Secondary structure changes induced by peptide-membrane interaction were studied by circular dichroism, infrared spectroscopy, and fluorescence spectroscopy. The experimental results were complemented by molecular dynamics calculations. An α-helical structure has been found to be necessary but not completely sufficient for the HAL-1 peptides antimicrobial action. The role of alternative conformations (such as β-sheet, PPII or 310-helix) also seems to be important. A mechanism of the peptide mode of action probably involves formation of peptide assemblies (possibly membrane pores), which disrupt bacterial membrane and, consequently, allow membrane penetration.
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2.
The C-Terminal VPRTES Tail of LL-37 Influences the Mode of Attachment to a Lipid Bilayer and Antimicrobial Activity.
de Miguel Catalina, A, Forbrig, E, Kozuch, J, Nehls, C, Paulowski, L, Gutsmann, T, Hildebrandt, P, Mroginski, MA
Biochemistry. 2019;(19):2447-2462
Abstract
Cathelicidins are a family of host defense antimicrobial peptides in mammalian species. Among them, LL-37 is the only peptide of this family found in humans. Although LL-37 has been intensively investigated in the past, the mode of exerting its bactericidal activity through the specific interactions with bacterial membranes remains elusive. In this work, we combined microbiological and computational approaches with a tool box of experimental biophysical techniques, including conventional and surface-enhanced infrared absorption spectroscopy as well as fluorescence spectroscopy to characterize the structural and dynamic properties of LL-37 and shorter variants adsorbed on POPC/POPG (9:1) lipid bilayers as mimics of bacterial membranes. First, microbiological assays demonstrate that, while LL-32 and, in a lesser degree, LL-37 show hemolysis and antimicrobial activity, LL-20 remains practically inactive. Second, by comparing experimental and computational data of LL-37 with LL-20, we explained the bactericidal activity of the active peptide core as a consequence of an increased flexibility of the peptide structure, leading to reactive dangling charged side chains. Third, permeabilization assays showed a concentration-dependent membrane disruption activity of LL-37 and LL-32: at high peptide concentrations, LL-32 shows higher activity than LL-37, while, at low peptide concentrations, both peptides show similar activities. Responsible for this behavior is the C-terminal VPRTES tail (Ct-VPRTES tail), which, according to atomistic simulations, is able to promote the insertion of the peptide in the membrane and plays an essential role in controlling ordered peptide oligomerization on the surface of the membrane.
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3.
Steady state analysis of influx and transbilayer distribution of ergosterol in the yeast plasma membrane.
Wüstner, D
Theoretical biology & medical modelling. 2019;(1):13
Abstract
BACKGROUND The transbilayer sterol distribution between both plasma membrane (PM) leaflets has long been debated. Recent studies in mammalian cells and in yeast show that the majority of sterol resides in the inner PM leaflet. Since sterol flip-flop in model membranes is rapid and energy-independent, a mechanistic understanding for net enrichment of sterol in one leaflet is lacking. Import of ergosterol in yeast can take place via the ABC transporters Aus1/Pdr11 under anaerobic growth conditions, eventually followed by rapid non-vesicular sterol transport to the endoplasmic reticulum (ER). Little is known about how these transport steps are dynamically coordinated. METHODS Here, a kinetic steady state model is presented which considers sterol import via Aus1/Pdr11, sterol flip-flop across the PM, bi-molecular complex formation and intracellular sterol release followed by eventual transport to and esterification of sterol in the ER. The steady state flux is calculated, and a thermodynamic analysis of feasibility is presented. RESULTS It is shown that the steady state sterol flux across the PM can be entirely controlled by irreversible sterol import via Aus1/Pdr11. The transbilayer sterol flux at steady state is a non-linear function of the chemical potential difference of sterol between both leaflets. Non-vesicular release of sterol on the cytoplasmic side of the PM lowers the attainable sterol enrichment in the inner leaflet. Including complex formation of sterol with phospholipids or proteins can explain several puzzling experimental observations; 1) rapid sterol flip-flop across the PM despite net sterol enrichment in one leaflet, 2) a pronounced steady state sterol gradient between PM and ER despite fast non-vesicular sterol exchange between both compartments and 3) a non-linear dependence of ER sterol on ergosterol abundance in the PM. CONCLUSIONS A steady state model is presented that can account for the observed sterol asymmetry in the yeast PM, the strong sterol gradient between PM and ER and threshold-like expansion of ER sterol for increasing sterol influx into the PM. The model also provides new insight into selective uptake of cholesterol and its homeostasis in mammalian cells, and it provides testable predictions for future experiments.
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4.
Co-Translational Protein Folding in Lipid Membranes.
Harris, NJ, Booth, PJ
Trends in biochemical sciences. 2019;(8):729-730
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5.
Nanostructured cochleates: a multi-layered platform for cellular transportation of therapeutics.
Shende, P, Khair, R, Gaud, RS
Drug development and industrial pharmacy. 2019;(6):869-881
Abstract
Among lipid-based nanocarriers, multi-layered cochleates emerge as a novel delivery system because of prevention of oxidation of hydrophobic and hydrophilic drugs, enhancement in permeability, and reduction in dose of drugs. It also improves oral bioavailability and increases the safety of a drug by targeting at a specific site with less side effects. Nanostructured cochleates are used as a carrier for the delivery of water-insoluble or hydrophobic drugs of anticancer, antiviral and anti-inflammatory action. This review article focuses on different methods for preparation of cochleates, mechanism of formation of cochleates, mechanism of action like cochleate undergoes macrophagic endocytosis and release the drug into the systemic circulation by acting on membrane proteins, phospholipids, and receptors. Advanced methods such as calcium-substituted and β-cyclodextrin-based cochleates, novel techniques include microfluidic and modified trapping method. Cochleates showed enhancement in oral bioavailability of amphotericin B, delivery of factor VII, oral mucosal vaccine adjuvant-delivery system, and delivery of volatile oil. In near future, cochleate will be one of the interesting delivery systems to overcome the stability and encapsulation efficiency issues associated with liposomes. The current limiting factors for commercial preparation of cochleates involve high cost of manufacturing, lack of standardization, and specialized equipments.
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6.
The effects of oxidised phospholipids and cholesterol on the biophysical properties of POPC bilayers.
Schumann-Gillett, A, O'Mara, ML
Biochimica et biophysica acta. Biomembranes. 2019;(1):210-219
Abstract
Oxidation of unsaturated membrane phospholipids by oxidative stress is associated with inflammation, infection, numerous diseases and neurodegenerative disorders. Lipid oxidation is observed in experimental samples when the parent lipid is exposed to oxidative stressors. The effect of phospholipid oxidation on the properties of biological membranes are still being explored, while low concentrations (0.1-2.0 mol%) of oxidised phospholipids are associated with disease states [1]. Previous computational studies have focused on the effect of high concentrations (~50 mol%) of oxidised phospholipids on binary lipid bilayers. This work systematically characterises the effect of lower concentrations (~10 mol%) of two oxidised lipid species, PoxnoPC (1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine) or PazePC (1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine), on POPC/cholesterol and pure POPC bilayers. During μs atomistic simulations in pure POPC bilayers, PoxnoPC and PazePC reoriented their oxidised sn-2 acyl chains towards the solution, and PazePC adopted an extended conformation. The addition of 20 mol% cholesterol not only modulated the fluidity of the bilayers; it also modulated the flexibility of the PoxnoPC oxidised sn-2 tail, reducing bilayer disorder. In contrast, the addition of cholesterol had little effect on bilayers containing PazePC. Our studies show that the effect of oxidised lipids on the biophysical properties of a multicomponent bilayer cannot be intuitively extrapolated from a binary lipid system.
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7.
A New Computational Method for Membrane Compressibility: Bilayer Mechanical Thickness Revisited.
Doktorova, M, LeVine, MV, Khelashvili, G, Weinstein, H
Biophysical journal. 2019;(3):487-502
Abstract
Because lipid bilayers can bend and stretch in ways similar to thin elastic sheets, physical models of bilayer deformation have utilized mechanical constants such as the moduli for bending rigidity (κC) and area compressibility (KA). However, the use of these models to quantify the energetics of membrane deformation associated with protein-membrane interactions, and the membrane response to stress is often hampered by the shortage of experimental data suitable for the estimation of the mechanical constants of various lipid mixtures. Although computational tools such as molecular dynamics simulations can provide alternative means to estimate KA values, current approaches suffer significant technical limitations. Here, we present a novel, to our knowledge, computational framework that allows for a direct estimation of KA values for individual bilayer leaflets. The theory is based on the concept of elasticity and derives KA from real-space analysis of local thickness fluctuations sampled in molecular dynamics simulations. We explore and validate the model on a large set of single and multicomponent bilayers of different lipid compositions and sizes, simulated at different temperatures. The calculated bilayer compressibility moduli agree with values estimated previously from experiments and those obtained from a standard computational method based on a series of constrained tension simulations. We further validate our framework in a comparison with an existing polymer brush model and confirm the polymer brush model's predicted linear relationship with proportionality coefficient of 24, using elastic parameters calculated from the simulation trajectories. The robustness of the results that emerge from the method allows us to revisit the origins of the bilayer mechanical (compressible) thickness and in particular its dependence on acyl-chain unsaturation and the presence of cholesterol.
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8.
Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers.
Iwamoto, M, Oiki, S
Journal of visualized experiments : JoVE. 2019;(143)
Abstract
Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions under various membrane lipid compositions. Among them, the droplet interface bilayer has gained popularity; however, the large membrane size hinders the recording of low electrical background noise. We have established a contact bubble bilayer (CBB) method that combines the benefits of planar lipid bilayer and patch-clamp methods, such as the ability to vary the lipid composition and to manipulate the bilayer mechanics, respectively. Using the setup for conventional patch-clamp experiments, CBB-based experiments can be readily performed. In brief, an electrolyte solution in a glass pipette is blown into an organic solvent phase (hexadecane), and the pipette pressure is maintained to obtain a stable bubble size. The bubble is spontaneously lined with a lipid monolayer (pure lipids or mixed lipids), which is provided from liposomes in the bubbles. Next, the two monolayer-lined bubbles (~50 µm in diameter) at the tip of the glass pipettes are docked for bilayer formation. Introduction of channel-reconstituted liposomes into the bubble leads to the incorporation of channels in the bilayer, allowing for single-channel current recording with a signal-to-noise ratio comparable to that of patch-clamp recordings. CBBs with an asymmetric lipid composition are readily formed. The CBB is renewed repeatedly by blowing out the previous bubbles and forming new ones. Various chemical and physical perturbations (e.g., membrane perfusion and bilayer tension) can be imposed on the CBBs. Herein, we present the basic procedure for CBB formation.
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9.
Effects of mono- and di-valent metal cations on the morphology of lipid vesicles.
Hong, J, Yang, H, Pang, D, Wei, L, Deng, C
Chemistry and physics of lipids. 2018;:19-28
Abstract
Lipid vesicles are an attractive model membrane experimental platform that is widely used in a biological context. The stability of vesicles can affect their performance and depends on various experimental conditions. How bio-related ions affect vesicle morphology is poorly understood in some cases. Herein, we investigated changes in vesicle morphology influenced by cation in the static and flowing environments. The effects of different mono- and di-valent metal cations on the morphology of lipid vesicles were systematically studied using the various techniques. The results showed that divalent cations caused significant aggregation or fusion of lipid vesicles, but monovalent cations had little effect on the vesicle morphology. Cation binding increased the net surface potential of vesicles, leading to changes in the zeta potential. The same qualitative kinetics were observed for cations that had the same valence at the same ionic strength. However, different types of cations gave different quantitative effects. The order of the ability to destroy the vesicle morphology was Cu2+ > Mg2+ > Ca2+ > Na+ > K+. These results are of practical value in the use of lipid vesicles as a bionic model, and help to shed light on the role of ions at membrane surfaces and interfaces.
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10.
A simple supported tubulated bilayer system for evaluating protein-mediated membrane remodeling.
Schenk, NA, Dahl, PJ, Hanna, MG, Audhya, A, Tall, GG, Knight, JD, Anantharam, A
Chemistry and physics of lipids. 2018;:18-28
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Abstract
Fusion and fission of cellular membranes involve dramatic, protein-mediated changes in membrane curvature. Many of the experimental methods useful for investigating curvature sensing or generation require specialized equipment. We have developed a system based on supported lipid bilayers (SLBs) in which lipid tubules are simple to produce and several types of membrane remodeling events can be readily imaged using widely available instrumentation (e.g., tubule fission and/or membrane budding). Briefly, high ionic strength during lipid bilayer deposition results in incorporation of excess lipids in the SLB. After sequentially washing with water and physiological ionic strength buffer solutions, lipid tubules form spontaneously. We find that tubule formation results from solution-dependent spreading of the SLB; washing from water into physiological ionic strength buffer solution leads to expansion of the bilayer and formation of tubules. Conversely, washing from physiological buffer into water results in contraction of the membrane and loss of tubules. We demonstrate the utility of these supported tubulated bilayers, termed "STuBs," with an investigation of Sar1B, a small Ras family G-protein known to influence membrane curvature. The addition of Sar1B to STuBs results in dramatic changes in tubule topology and eventual tubule fission. Overall, STuBs are a simple experimental system, useful for monitoring protein-mediated effects on membrane topology in real time, under physiologically relevant conditions.