Mehmet Nail Nasir

Complementary biophysical tools to investigate lipid specificity in the interaction between bioactive molecules and the plasma membrane: A review.

Plasma membranes are complex entities common to all living cells. The basic principle of their organization appears very simple, but they are actually of high complexity and represent very dynamic structures. The interactions between bioactive molecules and lipids are important for numerous processes, from drug bioavailability to viral fusion. The cell membrane is a carefully balanced environment and any change inflicted upon its structure by a bioactive molecule must be considered in conjunction with the overall effect that this may have on the function and integrity of the membrane. Conceptually, understanding the molecular mechanisms by which bioactive molecules interact with cell membranes is of fundamental importance. Lipid specificity is a key factor for the detailed understanding of the penetration and/or activity of lipid-interacting molecules and of mechanisms of some diseases. Further investigation in that way should improve drug discovery and development of membrane-active molecules in many domains such as health, plant protection or microbiology. In this review, we will present complementary biophysical approaches that can give information about lipid specificity at a molecular point of view. Examples of application will be given for different molecule types, from biomolecules to pharmacological drugs. A special emphasis is given to cyclic lipopeptides since they are interesting molecules in the scope of this review by combining a peptidic moiety and a lipidic tail and by exerting their activity via specific interactions with the plasma membrane.

Interactions of the natural antimicrobial mycosubtilin with phospholipid membrane models

Among the secondary metabolite lipopeptides produced by Bacillus subtilis, mycosubtilin is characterized by its strong antifungal activities. Even though its structure and its cellular target, the cytoplasmic membrane, have been determined, the molecular mechanisms of the biological activity of mycosubtilin have not been completely elucidated. In this work, the interactions between mycosubtilin and cytoplasmic membranes were modelled by using biomimetic systems such as Langmuir monolayers at the air-water interface and lipid multilamellar vesicles. The interactions of mycosubtilin with these biomimetic systems were examined, for the first time, by using specific techniques such as polarization modulation infrared reflection absorption spectroscopy, Brewster angle microscopy and high-resolution magic angle spinning NMR. Our findings indicate that mycosubtilin alone, at the air-water interface, forms a monolayer film and keeps its turn conformation. In the presence of DMPC, mycosubtilin binds to phospholipid monolayers, in a surface pressure-dependent manner. This binding results in the appearance of condensed domains which can be due to the formation of mycosubtilin clusters and/or to the lipopeptide aggregation with some phospholipid molecules and/or the formation of liquid-condensed domains of DMPC. Furthermore, in multilamellar vesicles, the mycosubtilin-DMPC interactions take place at the level of the aliphatic chains of the phospholipid because the phase transition temperature of DMPC decreased in the presence of mycosubtilin.

Specific interactions of mycosubtilin with cholesterol-containing artificial membranes.

Mycosubtilin is a natural antimicrobial lipopeptide produced by Bacillus subtilis strains. It is characterized by its hemolytic and strong antifungal activities. Mycosubtilin interacts with the plasma membranes of sensitive cells. However, the molecular mechanisms of its biological activities have not been completely elucidated. Our purpose was therefore to analyze the interactions of mycosubtilin with biological membranes by using biomimetic membranes such as Langmuir monolayers and multilayers. Structural changes of mycosubtilin, involving its peptide backbone and the side chain of its tyrosyl residue, were observed when the lipopeptide was interacting with cholesterol-containing multilayers. The interactions of mycosubtilin with monolayers constituted by pure lipids and by phosholipid/cholesterol or phospholipid/sphingomyelin/cholesterol were also examined. An original behavior of mycosubtilin toward cholesterol-containing monolayers was found. However, this original behavior was lost when mycosubtilin was interacting with pure cholesterylacetate monolayers. This suggests the involvement of the alcohol group of cholesterol in mycosubtilin-cholesterol interactions within membranes. Moreover, mycosubtilin induced changes in the organization and morphology of cholesterol-containing monolayers, and large condensed domains with different levels of condensation appeared only in the case of DPPC/sphingomyelin/cholesterol monolayer.

Interactions of the antifungal mycosubtilin with ergosterol-containing interfacial monolayers

Mycosubtilin, an antimicrobial lipopeptide produced by Bacillus subtilis, is characterized by strong antifungal activities. The molecular mechanisms of its biological activities on the membranes of the sensitive yeasts or fungi have not yet been clearly elucidated. Our purpose was to mimic the mycosubtilin interactions with these membranes using various Langmuir monolayers. Since the major sterol of yeasts or fungi is ergosterol, the interactions of mycosubtilin with monolayers constituted by ergosterol, DPPC/ergosterol or DPPC/sphingomyelin/ergosterol were examined at different initial surface pressures (Πi). Plotting the mycosubtilin-induced surface pressure increases versus Πi allowed to determine that the exclusion pressures of mycosubtilin from these different monolayers is higher than the surface prevailing within the biological membranes. However, this behavior was lost when mycosubtilin was interacting with ergosteryl acetate-containing monolayers. This suggests the involvement of the sterol alcohol group in the mycosubtilin interactions within membranes. Furthermore, the behavior of mycosubtilin with stigmasterol, similar to that observed with ergosterol, differs from that previously observed with cholesterol, suggesting a role of the alkyl side chain of the sterols. The adsorption of mycosubtilin to ergosterol monolayers induced changes in the lipopeptide orientation at the air-water interface as revealed by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). Moreover, imaging the air-water interface by Brewster angle microscopy (BAM) indicates that mycosubtilin induced changes in the organization and morphology of monolayers containing pure ergosterol with the appearance of small condensed dots, suggesting again that the target of mycosubtilin might be the ergosterol present in the membranes of the sensitive yeasts or fungi.

Conformational and interfacial analyses of K3A18K3 and alamethicin in model membranes.

The involvement of membrane-bound peptides and the influence of protein conformations in several neurodegenerative diseases lead us to analyze the interactions of model peptides with artificial membranes. Two model peptides were selected. The first one, an alanine-rich peptide, K3A18K3, was shown to be in alpha-helix structures in TFE, a membrane environment-mimicking solvent, while it was mostly beta-sheeted in aqueous buffer as revealed by infrared spectroscopy. The other, alamethicin, a natural peptide, was in a stable alpha-helix structure. To determine the role of the peptide conformation on the nature of its interactions with lipids, we compared the structure and topology of the conformational-labile peptide K3A18K3 and of the alpha-helix rigid alamethicin in both aqueous and phospholipid environments (Langmuir monolayers and multilamellar vesicles). K3A18K3 at the air-water interface showed a pressure-dependent orientation of its beta-sheets, while the alpha-helix axis of alamethicin was always parallel to the interface, as probed by polarization modulation infrared reflection absorption spectroscopy. The beta-sheeted K3A18K3 peptide was uniformly distributed into DPPC condensed domains, while the helical-alamethicin insertion distorted the DPPC condensed domains, as evidenced by Brewster angle microscopy imaging of the air/interface. The beta-sheeted K3A18K3 interacted with DMPC multilamellar vesicles via hydrophilic interactions with polar heads and the helical-alamethicin via hydrophobic interactions with alkyl chains, as shown by infrared spectroscopy and solid state NMR. Our findings are consistent with the prevailing assumption that the conformation of the peptide predetermines the mode of interaction with lipids. More precisely, helical peptides tend to be inserted via hydrophobic interactions within the hydrophobic region of membranes, while beta-sheeted peptides are predisposed to interact with polar groups and stay at the surface of lipid layers.

Annexins as organizers of cholesterol- and sphingomyelin-enriched membrane microdomains in Niemann-Pick type C disease.

Growing evidence suggests that membrane microdomains enriched in cholesterol and sphingomyelin are sites for numerous cellular processes, including signaling, vesicular transport, interaction with pathogens, and viral infection, etc. Recently some members of the annexin family of conserved calcium and membrane-binding proteins have been recognized as cholesterol-interacting molecules and suggested to play a role in the formation, stabilization, and dynamics of membrane microdomains to affect membrane lateral organization and to attract other proteins and signaling molecules onto their territory. Furthermore, annexins were implicated in the interactions between cytosolic and membrane molecules, in the turnover and storage of cholesterol and in various signaling pathways. In this review, we focus on the mechanisms of interaction of annexins with lipid microdomains and the role of annexins in membrane microdomains dynamics including possible participation of the domain-associated forms of annexins in the etiology of human lysosomal storage disease called Niemann-Pick type C disease, related to the abnormal storage of cholesterol in the lysosome-like intracellular compartment. The involvement of annexins and cholesterol/sphingomyelin-enriched membrane microdomains in other pathologies including cardiac dysfunctions, neurodegenerative diseases, obesity, diabetes mellitus, and cancer is likely, but is not supported by substantial experimental observations, and therefore awaits further clarification.

Bolaamphiphiles Derived from Alkenyl L-Rhamnosides and Alkenyl D-Xylosides: Importance of the Hydrophilic Head

The two step synthesis of a new bolaamphiphile derived from alkenyl L-rhamnosides was described. The general synthetic strategy of bolaamphiphiles derived from L-rhamnose was based on a previous work describing the synthesis of bolaamphiphiles derived from D-xylose. The conformational properties of this new compound were investigated by FTIR spectroscopy in an aqueous film in order to obtain a reference for further studies about the membrane-interacting properties. Moreover, the surface activity of this new bolaamphiphile was analyzed by Langmuir balance technology and was compared with that of the analogous bolaamphiphile derived from alkenyl D-xylosides. The findings indicate that the rhamnoside-based bolaform has an increased surface activity and a better ability to form aggregates than xyloside-based one.

Structural basis for plant plasma membrane protein dynamics and organization into functional nanodomains

Plasma Membrane is the primary structure for adjusting to ever changing conditions. PM sub-compartmentalization in domains is thought to orchestrate signaling. Yet, mechanisms governing membrane organization are mostly uncharacterized. The plant-specific REMORINs are proteins regulating hormonal crosstalk and host invasion. REMs are the best-characterized nanodomain markers via an uncharacterized moiety called REMORIN C-terminal Anchor. By coupling biophysical methods, super-resolution microscopy and physiology, we decipher an original mechanism regulating the dynamic and organization of nanodomains. We showed that targeting of REMORIN is independent of the COP-II-dependent secretory pathway and mediated by PI4P and sterol. REM-CA is an unconventional lipid-binding motif that confers nanodomain organization. Analyses of REM-CA mutants by single particle tracking demonstrate that mobility and supramolecular organization are critical for immunity. This study provides a unique mechanistic insight into how the tight control of spatial segregation is critical in the definition of PM domain necessary to support biological function. DOI: http://dx.doi.org/10.7554/eLife.26404.001

Analysis of calcium-induced effects on the conformation of fengycin

Fengycin is a natural lipopeptide with antifungal and eliciting properties and able to inhibit the activity of phospholipase A2. A combination of CD, FT-IR, NMR and fluorescence spectroscopic techniques was applied to elucidate its conformation in a membrane-mimicking environment and to investigate the effect of calcium ions on it. We mainly observed that fengycin adopts a turn conformation. Our results showed that calcium ions are bound by the two charged glutamates. The calcium binding has an influence on the fengycin conformation and more particularly, on the environment of the tyrosine residues. The modulation of the fengycin conformation by the environmental conditions may influence its biological properties.

Interactions of sugar-based bolaamphiphiles with biomimetic systems of plasma membranes

Glycolipids constitute a class of molecules with various biological activities. Among them, sugar-based bolaamphiphiles characterized by their biocompatibility, biodegradability and lower toxicity, became interesting for the development of efficient and low cost lipid-based drug delivery systems. Their activity seems to be closely related to their interactions with the lipid components of the plasma membrane of target cells. Despite many works devoted to the chemical synthesis and characterization of sugar-based bolaamphiphiles, their interactions with plasma membrane have not been completely elucidated. In this work, two sugar-based bolaamphiphiles differing only at the level of their sugar residues were chemically synthetized. Their interactions with membranes have been investigated using model membranes containing or not sterol and with in silico approaches. Our findings indicate that the nature of sugar residues has no significant influence for their membrane interacting properties, while the presence of sterol attenuates the interactions of both bolaamphiphiles with the membrane systems. The understanding of this distinct behavior of bolaamphiphiles towards sterol-containing membrane systems could be useful for their applications as drug delivery systems.