RESUMEN
Mycolactone is a lipid-like endotoxin synthesized by an environmental human pathogen, Mycobacterium ulcerans, the causal agent of Buruli ulcer disease. Mycolactone has pleiotropic effects on fundamental cellular processes (cell adhesion, cell death and inflammation). Various cellular targets of mycolactone have been identified and a literature survey revealed that most of these targets are membrane receptors residing in ordered plasma membrane nanodomains, within which their functionalities can be modulated. We investigated the capacity of mycolactone to interact with membranes, to evaluate its effects on membrane lipid organization following its diffusion across the cell membrane. We used Langmuir monolayers as a cell membrane model. Experiments were carried out with a lipid composition chosen to be as similar as possible to that of the plasma membrane. Mycolactone, which has surfactant properties, with an apparent saturation concentration of 1 µM, interacted with the membrane at very low concentrations (60 nM). The interaction of mycolactone with the membrane was mediated by the presence of cholesterol and, like detergents, mycolactone reshaped the membrane. In its monomeric form, this toxin modifies lipid segregation in the monolayer, strongly affecting the formation of ordered microdomains. These findings suggest that mycolactone disturbs lipid organization in the biological membranes it crosses, with potential effects on cell functions and signaling pathways. Microdomain remodeling may therefore underlie molecular events, accounting for the ability of mycolactone to attack multiple targets and providing new insight into a single unifying mechanism underlying the pleiotropic effects of this molecule. This membrane remodeling may act in synergy with the other known effects of mycolactone on its intracellular targets, potentiating these effects.
Asunto(s)
Membrana Dobles de Lípidos , Macrólidos/farmacología , Microdominios de Membrana/efectos de los fármacos , Úlcera de Buruli/microbiología , Adhesión Celular/efectos de los fármacos , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Mycobacterium ulcerans/química , Mycobacterium ulcerans/efectos de los fármacos , Mycobacterium ulcerans/ultraestructura , Tensoactivos/farmacologíaRESUMEN
Sensory properties of red wine tannins are bound to complex interactions between saliva proteins, membranes taste receptors of the oral cavity, and lipids or proteins from the human diet. Whereas astringency has been widely studied in terms of tannin-saliva protein colloidal complexes, little is known about interactions between tannins and lipids and their implications in the taste of wine. This study deals with tannin-lipid interactions, by mimicking both oral cavity membranes by micrometric size liposomes and lipid droplets in food by nanometric isotropic bicelles. Deuterium and phosphorus solid-state NMR demonstrated the membrane hydrophobic core disordering promoted by catechin (C), epicatechin (EC), and epigallocatechin gallate (EGCG), the latter appearing more efficient. C and EGCG destabilize isotropic bicelles and convert them into an inverted hexagonal phase. Tannins are shown to be located at the membrane interface and stabilize the lamellar phases. These newly found properties point out the importance of lipids in the complex interactions that happen in the mouth during organoleptic feeling when ingesting tannins.
Asunto(s)
Liposomas/química , Taninos/química , Gusto/fisiología , Vino/análisis , Catequina/análogos & derivados , Catequina/química , Humanos , Espectroscopía de Resonancia MagnéticaRESUMEN
Many in vitro studies have pointed out the interaction between amyloids and membranes, and their potential involvement in amyloid toxicity. In a previous study, we generated a yeast toxic mutant (M8) of the harmless model amyloid protein HET-s((218-289)). In this study, we compared the self-assembling process of the nontoxic wild-type (WT) and toxic (M8) protein at the air-water interface and in interaction with various phospholipid monolayers (DOPE, DOPC, DOPI, DOPS and DOPG). We first demonstrate using ellipsometry measurements and polarization-modulated infrared reflection absorption spectroscopy (PMIRRAS) that the air-water interface promotes and modifies the assembly of WT since an amyloid-like film was instantaneously formed at the interface with an antiparallel ß-sheet structuration instead of the parallel ß-sheet commonly observed for amyloid fibers generated in solution. The toxic mutant (M8) behaves in a similar manner at the air-water interface or in bulk, with a fast self-assembling and an antiparallel ß-sheet organization. The transmission electron microscopy (TEM) images established the fibrillous morphology of the protein films formed at the air-water interface. Second, we demonstrate for the first time that the main driving force between this particular fungus amyloid and membrane interaction is based on electrostatic interactions with negatively charged phospholipids (DOPG, DOPI, DOPS). Interestingly, the toxic mutant (M8) clearly induces perturbations of the negatively charged phospholipid monolayers, leading to a massive surface aggregation, whereas the nontoxic (WT) exhibits a slight effect on the membrane models. This study allows concluding that the toxicity of the M8 mutant could be due to its high propensity to interact with membranes.
Asunto(s)
Amiloide/toxicidad , Membranas Artificiales , Fosfolípidos/metabolismo , Aire , Amiloide/metabolismo , Humanos , Microscopía Electrónica de Transmisión , Análisis Espectral , AguaRESUMEN
Wine is very often consumed with a meal. However, although it is well known to tasters that the taste of wine changes in the presence of food, the influence of dietary lipids on wine astringency and bitterness caused by grape tannins is not well established from a molecular point of view. In this context, we investigated wine tannin-lipid interactions by combining biophysical techniques to sensory analysis. Nuclear magnetic resonance and optical and electron microscopy showed an interaction between catechin, a majority component of grape tannins, and lipid droplets from a phospholipid-stabilized oil-in-water emulsion, characterized by (a) an increase in the droplet size in the presence of catechin, (b) slowing of their size growth over time, and (c) an increase in lipid dynamics in the droplet interfacial layer. Those results were strengthened by sensory analysis, which demonstrated that dietary oils decrease the perception of astringency of grape tannin solutions. Our results highlight that dietary lipids are crucial molecular agents impacting our sensory perception during wine consumption.
Asunto(s)
Vitis , Vino , Percepción , Taninos/análisis , Gusto , Vino/análisisRESUMEN
Amyloids are thought to be involved in various types of neurodegenerative disorders. Several kinds of intermediates, differing in morphology, size, and toxicity, have been identified in the multistep amyloidogenesis process. However, the mechanisms explaining amyloid toxicity remain unclear. We previously generated a toxic mutant of the nontoxic HET-s((218-289)) amyloid in yeast. Here we report that toxic and nontoxic amyloids differ not only in their structures but also in their assembling process. We used multiple and complementary methods to investigate the intermediates formed by these two amyloids. With the methods used, no intermediates were observed for the nontoxic amyloid; however, under the same experimental conditions, the toxic mutant displayed visible oligomeric and fibrillar intermediates.
Asunto(s)
Amiloide/química , Amiloide/toxicidad , Proteínas Fúngicas/química , Proteínas Fúngicas/toxicidad , Proteínas Mutantes/toxicidad , Priones/química , Priones/toxicidad , Amiloide/ultraestructura , Rojo Congo/metabolismo , Concentración de Iones de Hidrógeno/efectos de los fármacos , Luz , Modelos Biológicos , Proteínas Mutantes/química , Estructura Cuaternaria de Proteína , Dispersión de Radiación , Solubilidad/efectos de los fármacos , Soluciones , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The amyloid aggregation pathway is a multistep process, and many in vitro studies have highlighted the role of particular intermediates in the cellular toxicity of various amyloid diseases. In a previous study, we generated a yeast toxic mutant (M8) of the harmless model amyloid protein Het-s(218-289). In this study, we compared the aggregation characteristics of the wild-type (WT) and the toxic mutant at the molecular level. Both proteins formed fibrillar amyloid aggregates but with different dye-binding properties and X-ray diffraction patterns. The toxic amyloid formed very unusual short (80 nm) unbranched fibers visible on transmission electron microscopy. Fourier transform infrared spectroscopy demonstrated that M8 beta-sheets were essentially organized into a mixed parallel and antiparallel structure, whereas the WT protein displayed a predominantly parallel organization. Cellular toxicity may therefore be related to assembly of the toxic amyloid in a new aggregation pathway.
Asunto(s)
Amiloide/química , Mutación , Multimerización de Proteína , Amiloide/genética , Amiloide/toxicidad , Amiloidosis , Pliegue de Proteína , Estructura Secundaria de Proteína , Espectroscopía Infrarroja por Transformada de Fourier , Difracción de Rayos X , LevadurasRESUMEN
Wine tasting results in interactions of tannin-ethanol solutions with proteins and lipids of the oral cavity. Among the various feelings perceived during tasting, astringency and bitterness most probably result in binding events with saliva proteins, lipids and receptors. In this work, we monitored the conjugated effect of the grape polyphenol catechin and ethanol on lipid membranes mimicking the different degrees of keratinization of oral cavity surfaces by varying the amount of cholesterol present in membranes. Both catechin and ethanol fluidify the membranes as evidenced by solid-state 2H NMR of perdeuterated lipids. The effect is however depending on the cholesterol proportion and may be very important and cumulative in the absence of cholesterol or presence of 18 mol % cholesterol. For 40 mol % cholesterol, mimicking highly keratinized membranes, both ethanol and catechin can no longer affect membrane dynamics. These observations can be accounted for by phase diagrams of lipid-cholesterol mixtures and the role played by membrane defects for insertion of tannins and ethanol when several phases coexist. These findings suggest that the behavior of oral membranes in contact with wine should be different depending of their cholesterol content. Astringency and bitterness could be then affected; the former because of a potential competition between the tannin-lipid and the tannin-saliva protein interaction, and the latter because of a possible fluidity modification of membranes containing taste receptors. The lipids that have been up to now weakly considered in oenology may be become a new actor in the issue of wine tasting.
Asunto(s)
Catequina/química , Colesterol/química , Etanol/química , Taninos/química , Vitis/química , Biomimética , Catequina/metabolismo , Colesterol/metabolismo , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Etanol/metabolismo , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Mucosa Bucal/química , Mucosa Bucal/metabolismo , Espectroscopía de Protones por Resonancia Magnética/métodos , Taninos/metabolismo , Gusto , Termodinámica , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismo , Vino/análisisRESUMEN
Dietary polyphenols widespread in vegetables and beverages like red wine and tea have been reported to possess antioxidant properties that could have positive effects on human health. In this study, we propose a new in situ and non-invasive method based on proton liquid-state nuclear magnetic resonance (NMR) to determine the antioxidant efficiency of red wine tannins on a twice-unsaturated phospholipid, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLiPC), embedded in a membrane model. Four tannins were studied: (+)-catechin (C), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG). The lipid degradation kinetics was determined by measuring the loss of the bis-allylic protons during oxidation induced by a radical initiator, 2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH). The antioxidant efficiency, i.e. the ability of tannins to slow down the lipid oxidation rate, was shown to be higher for galloylated tannins, ECG and EGCG. Furthermore, the mixture of four tannins was more efficient than the most effective tannin, EGCG, demonstrating a synergistic effect. To better understand the antioxidant action mechanism of polyphenols on lipid membranes, the tannin location was investigated by NMR and molecular dynamics. A correlation between antioxidant action of tannins and their location at the membrane interface (inserted at the glycerol backbone level) could thus be established.
Asunto(s)
Catequina/análogos & derivados , Catequina/química , Lípidos de la Membrana/química , Vino , Antioxidantes/química , Antioxidantes/farmacología , Catequina/farmacología , Clorobencenos/química , Clorobencenos/metabolismo , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Relación Dosis-Respuesta a Droga , Cinética , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Espectroscopía de Resonancia Magnética , Lípidos de la Membrana/metabolismo , Simulación de Dinámica Molecular , Estructura Molecular , Oxidación-Reducción/efectos de los fármacos , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Factores de TiempoRESUMEN
The folding and interactions of amyloid proteins are at the heart of the debate as to how these proteins may or may not become toxic to their host. Although little is known about this issue, the structure seems to be clearly involved with effects on molecular events. To understand how an amyloid may be toxic, we previously generated a yeast toxic amyloid (mutant 8) from the nontoxic HET-s((218-289)) prion domain of Podospora anserina. Here, we performed a comprehensive structure-toxicity study by mutating individually each of the 10 mutations found in mutant 8. The study of the library of new mutants generated allowed us to establish a clear link between Fourier transform infrared antiparallel signature and amyloid toxicity. All of the mutants that form parallel ß-sheets are not toxic. Double mutations may be sufficient to shift a parallel structure to antiparallel amyloids, which are toxic to yeast. Our findings also suggest that the toxicity of antiparallel structured mutants may be linked to interaction with membranes.
Asunto(s)
Amiloide/química , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Amiloide/genética , Amiloide/metabolismo , Podospora/metabolismo , Priones/química , Priones/genética , Priones/metabolismo , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
Puroindolines (PINs), basic and cysteine-rich proteins of wheat endosperm, are composed of two proteins, puroindoline-a (PIN-a) and puroindoline-b (PIN-b). Using a monolayer assay at the air/liquid interface, both PIN-a and PIN-b were studied in pure components and mixed with wheat galactolipids, 1,2-di-O-acyl-3-O-(beta-d-galactopyranosyl)- sn-glycerol (MGDG) and 2-di-O-acyl-3-O-(beta-d-galactopyranosyl-1,6-beta-d-galactopyranosyl)-sn-glycerol (DGDG). Following the adsorption of PINs at the air/liquid interface thanks to surface pressure increases, we concluded that PIN-a displays a more amphipathic character than PIN-b. Compression isotherms combined with ellipsometric measurements showed that the area per molecule is smaller and the protein film is more condensed for PIN-a than for PIN-b. According to the polarization modulation-infrared reflection-absorption spectroscopy data, both proteins display a highly alpha-helical structure and the alpha-helices are oriented rather parallel to the interface. By measuring the overpressure due to PIN adsorption into MGDG and DGDG monolayers, we observed that PIN-a interacts more strongly into lipid films than PIN-b. The observation by atomic force microscopy of mixed protein/lipid films showed that the nature of the lipid plays a significant role in the organization of PINs, particularly for PIN-a. The presence of galactolipids at the interface stabilizes the alpha-helical structure of PINs, but significant changes were observed concerning the orientation of the alpha-helices. They adopt a perfect parallel orientation to the interface in the MGDG monolayer, whereas the bundle of alpha-helices orients normal to the interface in the DGDG film.