Wine tannins and their aggregation/release with lipids and proteins: Review and perspectives for neurodegenerative diseases.

Tannins are amphiphilic molecules, often polymeric, which can be generally described as a core containing hydrophobic aromatic rings surrounded by hydroxyl groups. They have been known for millennia and are part of human culture. They are ubiquitous in nature and are best known in the context of wine and tea tasting and food cultures. However, they are also very useful for human health, as they are powerful antioxidants capable of combating the constant aggressions of everyday life. However, their mode of action is only just beginning to be understood. This review, using physicochemical concepts, attempts to summarize current knowledge and present an integrated view of the complex relationship between tannins, proteins and lipids, in the context of wine drinking while eating. There are many thermodynamic equilibria governing the interactions between tannins, saliva proteins, lipid droplets in food, membranes and the taste receptors embedded in them. Taste sensations can be explained using these multiple equilibria: for example, astringency (dry mouth) can be explained by the strong binding of tannin micelles to the proline-rich proteins of saliva, suppressing their lubricating action on the palate. In the presence of lipid droplets in food, the equilibrium is shifted towards tannin-lipid complexes, a situation that reduces the astringency perceived when consuming a tannic wine with fatty foods, the so-called "camembert effect". Tannins bind preferentially to taste receptors located in mouth membranes, but can also fluidify lipids in the non-keratinized mucous membranes of the mouth, which can impair the functioning of taste receptors there. Cholesterol, present in large quantities in keratinized mucous membranes, stiffens them and thus prevents tannins from disrupting the conduction of information through other taste receptors. As tannins assemble and disassemble depending on whether they are in contact with proteins, lipids or taste receptors, a perspective on their potential use in the context of neurodegenerative diseases where fibrillation is a key phenomenon will also be discussed.

Membrane plasticity induced by myo-inositol derived archaeal lipids: chemical synthesis and biophysical characterization.

Archaeal membrane lipids have specific structures that allow Archaea to withstand extreme conditions of temperature and pressure. In order to understand the molecular parameters that govern such resistance, the synthesis of 1,2-di-O-phytanyl-sn-glycero-3-phosphoinositol (DoPhPI), an archaeal lipid derived from myo-inositol, is reported. Benzyl protected myo-inositol was first prepared and then transformed to phosphodiester derivatives using a phosphoramidite based-coupling reaction with archaeol. Aqueous dispersions of DoPhPI alone or mixed with DoPhPC can be extruded and form small unilamellar vesicles, as detected by DLS. Neutron, SAXS, and solid-state NMR demonstrated that the water dispersions could form a lamellar phase at room temperature that then evolves into cubic and hexagonal phases with increasing temperature. Phytanyl chains were also found to impart remarkable and nearly constant dynamics to the bilayer over wide temperature ranges. All these new properties of archaeal lipids are proposed as providers of plasticity and thus means for the archaeal membrane to resist extreme conditions.

Switchable Lipids: From Conformational Switch to Macroscopic Changes in Lipid Vesicles.

It has been shown that the use of conformationally pH-switchable lipids can drastically enhance the cytosolic drug delivery of lipid vesicles. Understanding the process by which the pH-switchable lipids disturb the lipid assembly of nanoparticles and trigger the cargo release is crucial to optimize the rational design of pH-switchable lipids. Here, we gather morphological observations (FF-SEM, Cryo-TEM, AFM, confocal microscopy), physicochemical characterization (DLS, ELS), as well as phase behavior studies (DSC, 2H NMR, Langmuir isotherm, and MAS NMR) to propose a mechanism of pH-triggered membrane destabilization. We demonstrate that the switchable lipids are homogeneously incorporated with other co-lipids (DSPC, cholesterol, and DSPE-PEG2000) and promote a liquid-ordered phase insensitive to temperature variation. Upon acidification, the protonation of the switchable lipids triggers a conformational switch altering the self-assembly properties of lipid nanoparticles. These modifications do not lead to a phase separation of the lipid membrane; however, they cause fluctuations and local defects, which result in morphological changes of the lipid vesicles. These changes are proposed to affect the permeability of vesicle membrane, triggering the release of the cargo encapsulated in the lipid vesicles (LVs). Our results confirm that pH-triggered release does not require major morphological changes, but can result from small defects affecting the lipid membrane permeability.

Dynamic Sorting of Mobile and Rigid Molecules in Biomembranes by Magic-Angle Spinning 13C NMR.

Understanding the membrane dynamics of complex systems is essential to follow their function. As molecules in membranes can be in a rigid or mobile state depending on external (temperature, pressure) or internal (pH, domains, etc.) conditions, we propose an in-depth examination of NMR methods to filter highly mobile molecular parts from others that are in more restricted environments. We have thus developed a quantitative magic-angle spinning (MAS) 13C NMR approach coupled with cross-polarization (CP) and/or Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) on rigid and fluid unlabeled model membranes. We demonstrate that INEPT can detect only very mobile lipid headgroups in gel (solid-ordered) phases; the remaining rigid parts are only detected with CP. A direct correlation is established between the normalized line intensity as obtained by CP and the C-H (C-D) order parameters measured by wide-line 2H NMR or extracted from molecular dynamics: ICP/IDPeq ≈ 5|SCH|, indicating that when the order is greater than 0.2-0.3 (maximum value of 0.5 for chain CH2), only rigid parts can be filtered and detected using CP techniques. In very fluid (liquid-disordered) membranes, where there are many more active motions, both INEPT and CP detect resonances, with, however, a clear propensity of each technique to detect mobile and restricted molecular parts, respectively. Interestingly, the 13C NMR chemical shift of lipid hydrocarbon chains can be used to monitor order-disorder phase transitions and calculate the fraction of chain defects (rotamers) and the part of the transition enthalpy due to bond rotations (6-7 kJ·mol-1 for dimyristolphosphatidylcholine, DMPC). Cholesterol-containing membranes (liquid-ordered phases) can be dynamically contrasted as the rigid-body sterol is mainly detected by the CP technique, with a contact time of 1 ms, and the phospholipid by INEPT. Our work opens up a straightforward, robust, and cost-effective route for the determination of membrane dynamics by taking advantage of well-resolved conventional 13C NMR experiments without the need of isotopic labeling.

Solid-state NMR molecular snapshots of Aspergillus fumigatus cell wall architecture during a conidial morphotype transition.

While establishing an invasive infection, the dormant conidia of Aspergillus fumigatus transit through swollen and germinating stages, to form hyphae. During this morphotype transition, the conidial cell wall undergoes dynamic remodeling, which poses challenges to the host immune system and antifungal drugs. However, such cell wall reorganization during conidial germination has not been studied so far. Here, we explored the molecular rearrangement of Aspergillus fumigatus cell wall polysaccharides during different stages of germination. We took advantage of magic-angle spinning NMR to investigate the cell wall polysaccharides, without employing any destructive method for sample preparation. The breaking of dormancy was associated with a significant change in the molar ratio between the major polysaccharides ß-1,3-glucan and α-1,3-glucan, while chitin remained equally abundant. The use of various polarization transfers allowed the detection of rigid and mobile polysaccharides; the appearance of mobile galactosaminogalactan was a molecular hallmark of germinating conidia. We also report for the first time highly abundant triglyceride lipids in the mobile matrix of conidial cell walls. Water to polysaccharides polarization transfers revealed an increased surface exposure of glucans during germination, while chitin remained embedded deeper in the cell wall, suggesting a molecular compensation mechanism to keep the cell wall rigidity. We complement the NMR analysis with confocal and atomic force microscopies to explore the role of melanin and RodA hydrophobin on the dormant conidial surface. Exemplified here using Aspergillus fumigatus as a model, our approach provides a powerful tool to decipher the molecular remodeling of fungal cell walls during their morphotype switching.

Bio-membranes: Picosecond to second dynamics and plasticity as deciphered by solid state NMR.

Since the first membrane models in the 1970s, the concept of biological membranes has evolved considerably. The membrane is now seen as a very complex mixture whose dynamic behavior is even more complex. Solid-state NMR is well suited for such studies as it can probe the movements of the membrane from picoseconds to seconds. Two NMR observables can be used: motionally averaged spectra and relaxation times. They bring information on order parameters, phase transitions, correlation times, activation energies and membrane elasticity. Spectra are used to determine the nature of the membrane phase. The order parameters can be measured directly from spectra that are dominated by quadrupolar, dipolar and chemical shielding magnetic interactions and allow describing the lipid membrane as being very rigid at the glycerol and chain level and very fluid at its center and surface. Correlation times and activation energies can be measured for intramolecular motions (pico to nanoseconds), molecular motions (nano to 100 ns) and collective modes of membrane deformation (microseconds). Sterols modulate membrane phases, order parameters, correlation times and membrane elasticity. In general terms, sterols tend to act to reduce the impact of environmental changes on molecular order and dynamics. They can be described as regulators of membrane dynamics by keeping them in a state of dynamics that changes very little when the temperature or other factors change. The presence of such large-scale membrane dynamics is proposed as a means of adapting to evolutionary constraints.

Wine tannins, saliva proteins and membrane lipids.

Polyphenols have been part of human culture for about 6000 years. However, their mode of action in relation to wine tasting while eating is only beginning to be understood. This review, using analytical techniques and physicochemical concepts, attempts to summarize current knowledge and present an integrated view of the complex relationship between tannins, salivary proteins, lipids in food and in oral membranes. The action of tannins on taste sensations and astringency depends on their colloidal state. Although taste sensations are most likely due to interactions with taste receptors, astringency results from strong binding to proline-rich salivary proteins that otherwise lubricate the palate. Tannins disorder non-keratinized mucosa in mouth, possibly perturbing taste receptor function. The 10-15% ethanol present in wines potentiates this action. Cholesterol present in large quantities in keratinized mucosa prevents any disordering action on these oral membranes. Polyphenols bind strongly to the lipid droplets of fatty foods, a situation that reduces the astringency perceived when drinking a tannic wine, the so-called "camembert effect". Based on binding constants mainly measured by NMR, a comprehensive thermodynamic model of the interrelation between polyphenols, salivary proteins, lipids and taste receptors is presented.

Bicelles and nanodiscs for biophysical chemistry.

Membrane nanoobjects are very important tools to study biomembrane properties. Two types are described herein: Bicelles and Nanodiscs. Bicelles are obtained by thorough water mixing of long chain and short chain lipids and may take the form of membranous discs of 10-50 nm. Temperature-composition-hydration diagrams have been established for Phosphatidylcholines and show limited domains of existence. Bicelles can be doped with charged lipids, surfactants or with cholesterol and offer a wide variety of membranous platforms for structural biology. Internal dynamics as measured by solid-state NMR is very similar to that of liposomes in their fluid phase. Because of the magnetic susceptibility anisotropy of the lipid chains, discs may be aligned along or perpendicular to the magnetic field. They may serve as weak orienting media to provide distance information in determining the 3D structure of soluble proteins. In different conditions they show strong orienting properties which may be used to study the 3D structure, topology and dynamics of membrane proteins. Lipid Bicelles with biphenyl chains or doped with lanthanides show long lasting remnant orientation after removing the magnetic field due to smectic-like properties. An alternative to pure lipid Bicelles is provided by nanodiscs where the half torus composed by short chain lipids is replaced by proteins. This renders the nano-objects less fragile as they can be used to stabilize membrane protein assemblies to be studied by electron microscopy. Internal dynamics is again similar to liposomes except that the phase transition is abolished, possibly due to lateral constrain imposed by the toroidal proteins limiting the disc size. Advantages and drawbacks of both nanoplatforms are discussed.

Tandem NMR and Mass Spectrometry Analysis of Human Nuclear Membrane Lipids.

The human nuclear membrane is composed of a double bilayer, the inner membrane being linked to the protein lamina network and the outer nuclear membrane continuous with the endoplasmic reticulum. Nuclear membranes can form large invaginations inside the nucleus; their specific roles still remain unknown. Although much of the protein identification has been determined, their lipid composition remains largely undetermined. In order to understand the mechanical and dynamic properties of nuclear membranes we investigated their lipid composition by two quantitative methods, namely, 31P and 1H multidimensional NMR and mass spectrometry, using internal standards. We also developed a nondetergent nuclei extraction protocol allowing to produce milligram quantities of nuclear membrane lipids. We found that the nuclear membrane lipid extract is composed of a complex mixture of phospholipids with different phosphatidylcholine species present in large amounts. Negatively charged lipids, with elevated amounts of phosphatidylinositol (PI), were also present. Mass spectrometry confirmed the phospholipid composition and provided further information on acyl-chain length and unsaturation. Lipid chain lengths ranged between 30 and 38 carbon atoms (two chains summed up) with a high proportion of 34 carbon atom length for most species. PI lipids have high amounts of chain lengths with 36-38 carbons. Independent of the chain length unsaturations were highly elevated with one to two double bonds per lipid species.

The unprecedented membrane deformation of the human nuclear envelope, in a magnetic field, indicates formation of nuclear membrane invaginations.

Human nuclear membrane (hNM) invaginations are thought to be crucial in fusion, fission and remodeling of cells and present in many human diseases. There is however little knowledge, if any, about their lipid composition and dynamics. We therefore isolated nuclear envelope lipids from human kidney cells, analyzed their composition and determined the membrane dynamics after resuspension in buffer. The hNM lipid extract was composed of a complex mixture of phospholipids, with high amounts of phosphatidylcholines, phosphatidylinositols (PI) and cholesterol. hNM dynamics was determined by solid-state NMR and revealed that the lamellar gel-to-fluid phase transition occurs below 0 °C, reflecting the presence of elevated amounts of unsaturated fatty acid chains. Fluidity was higher than the plasma membrane, illustrating the dual action of Cholesterol (ordering) and PI lipids (disordering). The most striking result was the large magnetic field-induced membrane deformation allowing to determine the membrane bending elasticity, a property related to hydrodynamics of cells and organelles. Human Nuclear Lipid Membranes were at least two orders of magnitude more elastic than the classical plasma membrane suggesting a physical explanation for the formation of nuclear membrane invaginations.

MRI assessment of multiple dipolar relaxation time (T1D) components in biological tissues interpreted with a generalized inhomogeneous magnetization transfer (ihMT) model.

T1D, the relaxation time of dipolar order, is sensitive to slow motional processes. Thus T1D is a probe for membrane dynamics and organization that could be used to characterize myelin, the lipid-rich membrane of axonal fibers. A mono-component T1D model associated with a modified ihMT sequence was previously proposed for in vivo evaluation of T1D with MRI. However, experiments have suggested that myelinated tissues exhibit multiple T1D components probably due to a heterogeneous molecular mobility. A bi-component T1D model is proposed and implemented. ihMT images of ex-vivo, fixed rat spinal cord were acquired with multiple frequency alternation rate. Fits to data yielded two T1Ds of about 500 µs and 10 ms. The proposed model seems to further explore the complexity of myelin organization compared to the previously reported mono-component T1D model.

Heat-Triggered Crystallization of Liquid Crystalline Macrocycles Allowing for Conductance Switching through Hysteretic Thermal Phase Transitions.

A polymesomorphic thermal phase-transition of a macrocyclic amphiphile consisting of aromatic groups and oligoethylene glycol (OEG) chains is reported. The macrocyclic amphiphile exists in a highly-ordered liquid crystal (LC) phase at room temperature. Upon heating, this macrocycle shows phase-transition from columnar-lamellar to nematic LC phases followed by crystallization before melting. Spectroscopic studies suggest that the thermally induced crystallization is triggered by a conformational change at the OEG chains. Interestingly, while the macrocycle returns to the columnar-lamellar phase after cooling from the isotropic liquid, it retains the crystallinity after cooling from the thermally-induced crystal. Thanks to this bistability, conductance switching was successfully demonstrated. A different macrocyclic amphiphile also shows an analogous phase-transition behavior, suggesting that this molecular design is universal for developing switchable and memorizable materials, by means of hysteretic phase-transition processes.

The potent effect of mycolactone on lipid membranes.

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.

Lipid Internal Dynamics Probed in Nanodiscs.

Nanodiscs offer a very promising tool to incorporate membrane proteins into native-like lipid bilayers and an alternative to liposomes to maintain protein functions and protein-lipid interactions in a soluble nanoscale object. The activity of the incorporated membrane protein appears to be correlated to its dynamics in the lipid bilayer and by protein-lipid interactions. These two parameters depend on the lipid internal dynamics surrounded by the lipid-encircling discoidal scaffold protein that might differ from more unrestricted lipid bilayers observed in vesicles or cellular extracts. A solid-state NMR spectroscopy investigation of lipid internal dynamics and thermotropism in nanodiscs is reported. The gel-to-fluid phase transition is almost abolished for nanodiscs, which maintain lipid fluid properties for a large temperature range. The addition of cholesterol allows fine-tuning of the internal bilayer dynamics by increasing chain ordering. Increased site-specific order parameters along the acyl chain reflect a higher internal ordering in nanodiscs compared with liposomes at room temperature; this is induced by the scaffold protein, which restricts lipid diffusion in the nanodisc area.

Singular Interaction between an Antimetastatic Agent and the Lipid Bilayer: The Ohmline Case.

SK3 channels are abnormaly expressed in metastatic cells, and Ohmline (OHM), an ether lipid, has been shown to reduce the activity of SK3 channels and the migration capacity of cancer cells. OHM incorporation into the plasma membrane is proposed to dissociate the protein complex formed between SK3 and Orai1, a potassium and a calcium channel, respectively, and would lead to a modification in the lipid environment of both the proteins. Here, we report the synthesis of deuterated OHM that affords the determination, through solid-state NMR, of its entire partitioning into membranes mimicking the SK3 environment. Use of deuterated lipids affords the demonstration of an OHM-induced membrane disordering, which is dose-dependent and increases with increasing amounts of cholesterol (CHOL). Molecular dynamics simulations comfort the disordering action and show that OHM interacts with the carbonyl and phosphate groups of stearoylphosphatidylcholine and sphingomyelin and to a minor extent with CHOL. OHM is thus proposed to remove the CHOL OH moieties away from their main binding sites, forcing a new rearrangement with other lipid groups. Such an interaction takes its origin at the lipid-water interface, but it propagates toward the entire lipid molecules and leads to a cooperative destabilization of the lipid acyl chains, that is, membrane disordering. The consequences of this reorganization of the lipid phases are discussed in the context of the OHM-induced inhibition of SK3 channels.

Sequestration of Proteins by Fatty Acid Coacervates for Their Encapsulation within Vesicles.

Encapsulating biological materials in lipid vesicles is of interest for mimicking cells; however, except in some particular cases, such processes do not occur spontaneously. Herein, we developed a simple and robust method for encapsulating proteins in fatty acid vesicles in high yields. Fatty acid based, membrane-free coacervates spontaneously sequester proteins and can reversibly form membranous vesicles upon varying the pH value, the precrowding feature in coacervates allowing for protein encapsulation within vesicles. We then produced enzyme-enriched vesicles and show that enzymatic reactions can occur in these micrometric capsules. This work could be of interest in the field of synthetic biology for building microreactors.

Grape tannin catechin and ethanol fluidify oral membrane mimics containing moderate amounts of cholesterol: Implications on wine tasting?

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.

Triggering bilayer to inverted-hexagonal nanostructure formation by thiol-ene click chemistry on cationic lipids: consequences on gene transfection.

The ramification of cationic amphiphiles on their unsaturated lipid chains is readily achieved by using the thiol-ene click reaction triggering the formation of an inverted hexagonal phase (HII). The new ramified cationic lipids exhibit different bio-activities (transfection, toxicity) including higher transfection efficacies on 16HBE 14o-cell lines.

Clouding in fatty acid dispersions for charge-dependent dye extraction.

The clouding phenomenon in non-ionic surfactant systems is a common feature that remains rare for ionic detergents. Here, we show that fatty acid (negatively charged) systems cloud upon cooling hot dispersions depending on the concentration or when adding excess guanidine hydrochloride. The clouding of these solutions yields the formation of enriched fatty acid droplets in which they exhibit a polymorphism that depends on the temperature: upon cooling, elongated wormlike micelles transit to rigid stacked bilayers inside droplets. Above this transition temperature, droplets coalesce yielding a phase separation between a fatty acid-rich phase and water, allowing extraction of dyes depending on their charge and lipophilicity. Positively charged and zwitterionic dyes were sequestered within the droplets (and then in the fatty acid-rich upper phase) whereas the negatively charged ones were found in both phases. Our results show an additional case of negatively charged surfactant which exhibit clouding phenomenon and suggest that these systems could be used for extracting solutes depending on their charge and lipophilicity.

Monitoring the Interactions of a Ternary Complex Using NMR Spectroscopy: The Case of Sugars, Polyphenols, and Proteins.

Gaining insight into intermolecular interactions between multiple species is possible at an atomic level by looking at different parameters using different NMR techniques. In the specific case of the astringency sensation, in which at least three molecular species are involved, different NMR techniques combined with dynamic light scattering and molecular modeling contribute to decipher the role of each component in the interaction mode and to assess the thermodynamic parameters governing this complex interaction. The binding process between a saliva peptide, a polyphenol, and polysaccharides was monitored by following 1H chemical shift variations, changes in NMR peak areas, and size of the formed complex. These NMR experiments deliver a complete picture of the association pathway, assessed by dynamic light scattering and molecular dynamics simulations: all of the data collected converge toward a comprehensive mode of interaction in which sugars indirectly play a role in astringency by sequestering part of the polyphenols, reducing their effective concentration to bind saliva proteins.