Over the past decades an extensive effort has been made to provide a more comprehensive understanding of Wnt signaling, yet many regulatory and structural aspects remain elusive. Among these, the ability of Dishevelled (DVL) protein to relocalize at the plasma membrane is a crucial step in the activation of all Wnt pathways. The membrane binding of DVL was suggested to be mediated by the preferential interaction of its C-terminal DEP domain with phosphatidic acid (PA). However, due to the scarcity and fast turnover of PA, we investigated the role on the membrane association of other more abundant phospholipids. The combined results from computational simulations and experimental measurements with various model phospholipid membranes, demonstrate that the membrane binding of DEP/DVL constructs is governed by the concerted action of generic electrostatics and finely-tuned intermolecular interactions with individual lipid species. In particular, while we confirmed the strong preference for PA lipid, we also observed a weak but non-negligible affinity for phosphatidylserine, the most abundant anionic phospholipid in the plasma membrane, and phosphatidylinositol 4,5-bisphosphate. The obtained molecular insight into DEP-membrane interaction helps to elucidate the relation between changes in the local membrane composition and the spatiotemporal localization of DVL and, possibly, other DEP-containing proteins.
Wild-type cells of Candida albicans, the most common human fungal pathogen, are able to grow at very low micromolar concentrations of potassium in the external milieu. One of the reasons behind that behaviour is the existence of three different types of K+ transporters in their plasma membrane: Trk1, Acu1 and Hak1. This work shows that the transporters are very differently regulated at the transcriptional level upon exposure to saline stress, pH alterations or K+ starvation. We propose that different transporters take the lead in the diverse environmental conditions, Trk1 being the "house-keeping" one, and Acu1/Hak1 dominating upon K+ limiting conditions. Heterologous expression of the genes coding for the three transporters in a Saccharomyces cerevisiae strain lacking its endogenous potassium transporters showed that all of them mediated cation transport but with very different efficiencies. Moreover, expression of the transporters in S. cerevisiae also affected other physiological characteristics such as sodium and lithium tolerance, membrane potential or intracellular pH, being, in general, CaTrk1 the most effective in keeping these parameters close to the usual wild-type physiological levels.
- MeSH
- buněčná membrána genetika metabolismus MeSH
- Candida albicans genetika metabolismus MeSH
- draslík metabolismus MeSH
- fungální proteiny genetika metabolismus MeSH
- lidé MeSH
- proteiny přenášející kationty genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DivIVA is a crucial membrane-binding protein that helps to localize other proteins to negatively curved membranes at cellular poles and division septa in Gram-positive bacteria. The N-terminal domain of DivIVA is responsible for membrane binding. However, to which lipids the domain binds or how it recognizes the membrane negative curvature remains elusive. Using computer simulations, we demonstrate that the N-terminal domain of Streptomyces coelicolor DivIVA adsorbs to membranes with affinity and orientation dependent on the lipid composition. The domain interacts non-specifically with lipid phosphates via its arginine-rich tip and the strongest interaction is with cardiolipin. Moreover, we observed a specific attraction between a negatively charged side patch of the domain and ethanolamine lipids, which addition caused the change of the domain orientation from perpendicular to parallel alignment to the membrane plane. Similar but less electrostatically dependent behavior was observed for the N-terminal domain of Bacillus subtilis. The domain propensity for lipids which prefer negatively curved membranes could be a mechanism for the cellular localization of DivIVA protein.
- MeSH
- Bacillus subtilis genetika MeSH
- bakteriální proteiny genetika MeSH
- lipidy genetika MeSH
- membránové proteiny genetika MeSH
- proteinové domény genetika MeSH
- proteiny buněčného cyklu genetika MeSH
- Streptomyces coelicolor genetika MeSH
- vazba proteinů genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The cell membrane is mainly composed of lipid bilayers with inserted proteins and carbohydrates. Lipid bilayers made of purified or synthetic lipids are widely used for estimating the effect of target compounds on cell membranes. However, the composition of such biomimetic membranes is much simpler than the composition of biological membranes. Interactions between compounds and simple composition biomimetic membranes might not demonstrate the effect of target compounds as precisely as membranes with compositions close to real organisms. Therefore, the aim of our study is to construct biomimetic membrane closely mimicking the state of natural membranes. Liposomes were prepared from lipids extracted from L-α-phosphatidylcholine, Escherichia coli, yeast (Saccharomyces cerevisiae) and bovine liver cells through agitation and sonication. They were immobilized onto silicon dioxide (SiO2) sensor surfaces using N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer with calcium chloride. The biomimetic membranes were successfully immobilized onto the SiO2 sensor surface and detected by nanoplasmonic sensing. The immobilized membranes were exposed to choline carboxylates. The membrane disruption effect was, as expected, more pronounced with increasing carbohydrate chain length of the carboxylates. The results correlated with the toxicity values determined using Vibrio fischeri bacteria. The yeast extracted lipid membranes had the strongest response to introduction of choline laurate while the bovine liver lipid extracted liposomes were the most sensitive towards the shorter choline carboxylates. This implies that the composition of the cell membrane plays a crucial role upon interaction with choline carboxylates, and underlines the necessity of testing membrane systems of different origin to obtain an overall image of such interactions.
- MeSH
- biomimetické materiály chemie MeSH
- buněčná membrána chemie MeSH
- cholin analogy a deriváty MeSH
- liposomy chemie MeSH
- membránové lipidy chemie MeSH
- Saccharomyces cerevisiae MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Surfactin, a cyclic lipoheptapeptide produced by Bacillus subtilis, is a surface-active antimicrobial that targets the barrier function of lipid membranes. It inserts itself into the membrane, where it forms conductive pores. Depending on its concentration, it eventually disintegrates the membrane in a detergent-like manner. The molecular details of this activity are not yet sufficiently understood, nor are the mechanisms that the surfactin producer employs to resist its own toxic product. We have previously shown that B. subtilis modifies its membrane lipid composition upon the onset of surfactin production, mainly increasing the cardiolipin content. Here we show that the increased cardiolipin content leads to a decreased surfactin-induced leakage of liposomes reconstituted from lipids isolated from the surfactin producer. This stabilizing effect of cardiolipin is concentration-dependent. Using a propidium iodide-based cell permeabilization assay, we further confirmed that the cytoplasmic membrane of the mutant B. subtilis strain lacking cardiolipin was substantially more susceptible to the action of surfactin, even though the amount of bound surfactin was the same as in the wild-type strain. We propose that membrane remodelling; due to the increase in cardiolipin content, contributes to the surfactin tolerance of B. subtilis.
Two distinct conformers of the adenylate cyclase toxin (CyaA) appear to accomplish its two parallel activities within target cell membrane. The translocating conformer would deliver the N-terminal adenylyl cyclase (AC) enzyme domain across plasma membrane into cytosol of cells, while the pore precursor conformer would assemble into oligomeric cation-selective pores and permeabilize cellular membrane. Both toxin activities then involve a membrane-interacting 'AC-to-Hly-linking segment' (residues 400 to 500). Here, we report the NMR structure of the corresponding CyaA411-490 polypeptide in dodecylphosphocholine micelles and show that it consists of two α-helices linked by an unrestrained loop. The N-terminal α-helix (Gly418 to His439) remained solvent accessible, while the C-terminal α-helix (His457 to Phe485) was fully enclosed within detergent micelles. CyaA411-490 weakly bound Ca2+ ions (apparent KD 2.6 mM) and permeabilized negatively charged lipid vesicles. At high concentrations (10 μM) the CyaA411-490 polypeptide formed stable conductance units in artificial lipid bilayers with applied voltage, suggesting its possible transmembrane orientation in the membrane-inserted toxin. Mutagenesis revealed that two clusters of negatively charged residues within the 'AC-to-Hly-linking segment' (Glu419 to Glu432 and Asp445 to Glu448) regulate the balance between the AC domain translocating and pore-forming capacities of CyaA in function of calcium concentration.
- MeSH
- adenylátcyklasový toxin chemie metabolismus MeSH
- AMP cyklický metabolismus MeSH
- biologický transport genetika MeSH
- Bordetella pertussis chemie metabolismus MeSH
- buněčná membrána chemie metabolismus MeSH
- hemolýza genetika MeSH
- konformace proteinů, alfa-helix genetika MeSH
- lidé MeSH
- lipidové dvojvrstvy chemie metabolismus MeSH
- permeabilita buněčné membrány genetika MeSH
- vápník metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Tear Film Lipid Layer (TFLL) covering the surface of the aqueous film at human cornea forms a first barrier between the eye and environment. Its alterations are related to dry eye disease. TFLL is formed by a complex mixture of lipids, with an excess of nonpolar components and a minor fraction of polar molecules. Its thickness is up to 160 nm, hence a multilayer-like structure of TFLL is assumed. However, details of TFLL organization are mostly unavailable in vivo due to the dynamic nature of the human tear film. To overcome this issue, we employ a minimalistic in vitro lipid model of TFLL. We study its biophysical characteristics by using a combination of the Langmuir trough with fluorescence microscopy. The model consists of two-component polar-nonpolar lipid films with a varying component ratio spread on the aqueous subphase at physiologically relevant temperature. We demonstrate that the model lipid mixture undergoes substantial structural reorganization as a function of lateral pressure and polar to nonpolar lipid ratio. In particular, the film is one-molecule-thick and homogenous under low lateral pressure. Upon compression, it transforms into a multilayer structure with inhomogeneities in the form of polar-nonpolar lipid assemblies. Based on this model, we hypothesize that TFLL in vivo has a duplex polar-nonpolar structure and it contains numerous mixed lipid aggregates formed because of film restructuring. These findings, despite the simplified character of the model, seem relevant for TFLL physiology as well as for understanding pathological conditions related to the lipids of the tear film.
- MeSH
- fluorescenční mikroskopie MeSH
- lidé MeSH
- lipidy chemie MeSH
- povrchové vlastnosti MeSH
- rohovka chemie metabolismus MeSH
- slzy chemie MeSH
- voda chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (>250 nm) than those containing non-raft proteins (<20 nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid Lo phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different - the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts.
- MeSH
- anizotropie MeSH
- buněčná membrána chemie MeSH
- cholesterol chemie MeSH
- detergenty chemie MeSH
- gelová chromatografie MeSH
- Jurkat buňky MeSH
- lidé MeSH
- lipidové dvojvrstvy chemie MeSH
- lipidy chemie MeSH
- maleáty chemie MeSH
- mastné kyseliny chemie MeSH
- membránové mikrodomény chemie MeSH
- membránové proteiny chemie MeSH
- membrány umělé MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- polymery chemie MeSH
- radiační rozptyl MeSH
- rozpustnost MeSH
- styren chemie MeSH
- světlo MeSH
- T-lymfocyty cytologie MeSH
- ultracentrifugace MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Free radical scavengers like α-phenyl-N-tert-butylnitrone (PBN) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) have been widely used as protective agents in various biomimetic and biological models. A series of three amphiphilic Trolox and PBN derivatives have been designed by adding to those molecules a perfluorinated chain as well as a sugar group in order to render them amphiphilic. In this work, we have studied the interactions between these derivatives and lipid membranes to understand how they influence their ability to prevent membrane lipid oxidation. We showed the derivatives better inhibited the AAPH-induced oxidation of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLiPC) small unilamellar vesicles (SUVs) than the parent compounds. One of the derivatives, bearing both PBN and Trolox moieties on the same fluorinated carrier, exhibited a synergistic antioxidant effect by delaying the oxidation process. We next investigated the ability of the derivatives to interact with DLiPC membranes in order to better understand the differences observed regarding the antioxidant properties. Surface tension and fluorescence spectroscopy experiments revealed the derivatives exhibited the ability to form monolayers at the air/water interface and spontaneously penetrated lipid membranes, underlying pronounced hydrophobic properties in comparison to the parent compounds. We observed a correlation between the hydrophobic properties, the depth of penetration and the antioxidant properties and showed that the location of these derivatives in the membrane is a key parameter to rationalize their antioxidant efficiency. Molecular dynamics (MD) simulations supported the understanding of the mechanism of action, highlighting various key physical-chemical descriptors.
- MeSH
- antioxidancia farmakologie MeSH
- chromany (dihydrobenzopyrany) chemie MeSH
- fluor chemie MeSH
- membránové lipidy chemie MeSH
- membrány umělé MeSH
- oxidace-redukce MeSH
- oxidy dusíku chemie MeSH
- peroxidace lipidů MeSH
- synergismus léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In Saccharomyces cerevisiae, K+-uptake under K+-limiting conditions is largely mediated by the cation translocation systems Trk1 and Trk2 belonging to the family of SKT proteins. They are related to two-transmembrane-domain (inward rectifying K-) channels but unlike the symmetrical tetrameric structure of K-channels, a single Trk contains four pore-forming domains (A-D) encoded on one polypeptide chain. Between domains A and B Trks contain large cytosolic regions dubbed "long hydrophilic loop" (LHL). LHLs are not homologous/similar to any other identified protein (domain) and also show little similarity between Trk1 and Trk2. Here we demonstrate that Trk1 is functional without LHL. However, in growth experiments NaCl sensitivity of Trk1[ΔLHL] expressing cells is increased under K+-limiting conditions compared to full-length Trk1. Non-invasive ion flux measurements showed that K+-influx through Trk1 and Trk1[ΔLHL] is decreased in the presence of surplus Na+ due to permeability of the proteins for both cations and competition between them. Trk1[ΔLHL] is less affected than full-length Trk1 because it is more selective for K+ over Na+. Furthermore, K+ re-uptake after starvation is delayed and decreased in Trk1[ΔLHL]. Thus, a role of LHL is regulating cation fluxes through Trk1 by (i) allowing also Na+ to pass if monovalent cations (mainly K+) are limiting and (ii) by accelerating/enhancing a switch from low to high affinity ion translocation. We propose that LHL could modulate Trk1 transport properties via direct influence on a transmembrane helix (M2A) which can switch between bent and straight conformation, thereby directly modifying the radius of the pore and selectivity filter.
- MeSH
- dimerizace MeSH
- draslík metabolismus MeSH
- hydrofobní a hydrofilní interakce MeSH
- iontový transport MeSH
- kationty metabolismus MeSH
- proteiny přenášející kationty chemie metabolismus MeSH
- Saccharomyces cerevisiae - proteiny chemie metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
