Alessio Ausili

Edelfosine Is Incorporated into Rafts and Alters Their Organization

The effect of edelfosine (1- O-octadecyl-2- O-methyl-rac-glycero-3-phosphocholine or ET-18-OCH3) on model membranes containing 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine/sphingomyelin/cholesterol (POPC/SM/cholesterol) was studied by several physical techniques. The sample POPC/SM (1:1 molar ratio) showed a broad phase transition as seen by DSC, X-ray diffraction, and 2H NMR. The addition of edelfosine to this sample produced isotropic structures at temperatures above the phase transition, as seen by 2H NMR and by 31P NMR. When cholesterol was added to give a POPC/SM/cholesterol (at a molar ratio 1:1:1), no transition was observed by DSC nor X-ray diffraction, and 2H NMR indicated the presence of a liquid ordered phase. The addition of 10 mol % edelfosine increased the thickness of the membrane as seen by X-ray diffraction and led to bigger differences in the values of the molecular order of the membrane detected at high and low temperatures, as detected through the M 1 first spectral moment from 2H NMR. These differences were even greater when 20 mol % edelfosine was added, and a transition was now clearly visible by DSC. In addition, a gel phase was clearly indicated by X-ray diffraction at low temperatures. The same technique pointed to greater membrane thickness in this mixture and to the appearance of a second membrane structure, indicating the formation of two separated phases in the presence of edelfosine. All of these data strongly suggest that edelfosine associating with cholesterol alter the phase status present in a POPC/SM/cholesterol (1:1:1 molar ratio) mixture, which is reputed to be a model of a raft structure. However, cell experiments showed that edelfosine colocalizes in vivo with rafts and that it may reach concentrations higher than 20 mol % of total lipid, indicating that the concentrations used in the biophysical experiments were within what can be expected in a cell membrane. The conclusion is that molecular ways of action of edelfosine in cells may involve the modification of the structure of rafts.

Curcumin Disorders 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine Membranes and Favors the Formation of Nonlamellar Structures by 1,2-Dielaidoyl-sn-glycero-3-phosphoethanolamine

Curcumin is a polyphenol present in turmeric, a spice widely used in Asian traditional medicine and cooking. It has many and diverse biological effects and is incorporated in cell membranes. This paper describes the mode in which curcumin modulates the physical properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dielaidyl-sn-glycero-3-phosphoetnanolamine (DEPE) multilamellar membranes. Curcumin disordered DPPC membranes at temperatures below T(c) as seen by DSC, FT-IR, (2)H NMR, WAXD, and SAXD. The decrease induced by curcumin in T(c) suggested that it is oriented in the bilayer with its main axis parallel to the acyl chains. Above T(c), too, curcumin introduced disorder as seen by infrared spectroscopy which showed that curcumin also alters the conformation of the polar group of DPPC, increasing the percentage of unhydrated C=O groups, but does not form hydrogen bonds with either the C=O group or the phosphate group of DPPC. Small angle X-ray diffraction showed a notable increase in the repeating spacings as a result of the presence of curcumin, suggesting the formation of a rippled phase. Increasing concentrations of curcumin progressively modified the onset and completion of the phase transition and also DeltaH up to a 6:1 DPPC/curcumin molar ratio. A further increase of curcumin concentration did not produce effects on the transition parameters, suggesting that there is a limit for the solubility of curcumin in DPPC. Additionally, when DEPE was used to test the effect of curcumin on the phospholipid polymorphism, it was found that the temperature at which the H(II) phase is formed decreased, indicating that curcumin favors negative curvature of the membrane, which may be important for explaining its effect on membrane dynamics and on membrane proteins or on proteins which may be activated through membrane insertion.

The interaction of the Bax C-terminal domain with negatively charged lipids modifies the secondary structure and changes its way of insertion into membranes

Fourier transform infrared spectroscopy (FTIR) was used to study the secondary structure of peptides which imitate the amino acid sequences of the C-terminal domain of the pro-apoptotic protein Bax (Bax-C) when incorporated into different lipid vesicles with or without negatively charged phospholipids. The infrared spectroscopy results showed that while the beta-sheet components are predominant in the membrane-free Bax-C secondary structure as well as in the presence of phosphatidylcholine vesicles, the peptide changes its secondary structure in the presence of negatively charged membranes, including phospholipids such as phosphatidylglycerol or phosphatidylinositol, depending on both the lipid composition and their molar ratio. The negative charges in the model membrane surface caused a marked change from beta-sheet to alpha-helix structure. Moreover, using attenuated total reflection infrared spectroscopy (ATR-FTIR), we investigated the orientation of Bax-C alpha-helical structures with respect to the normal to the internal reflection element. The orientation of Bax-C in membranes was also affected by negatively charged lipids, the presence of phosphatidylglycerol reduced the angle it forms with the normal to the germanium plate from 45 degrees in phosphatidylcholine to 27 degrees in phosphatidylglycerol vesicles. These results highlight the importance of lipid-protein interaction for the correct folding of membrane proteins and they suggest that the C-terminal domain of Bax will only span membranes with a net negative charge in their surface.

Redox State of Coenzyme Q10 Determines Its Membrane Localization

The interaction between oxidized (ubiquinone-10) and reduced (ubiquinol-10) coenzyme Q 10 with dimyristoylphosphatidylcholine has been examined by differential scanning microcalorimetry, X-ray diffraction, infrared spectroscopy, and (2)H NMR. Microcalorimetry experiments showed that ubiquinol-10 perturbed considerably more the phase transition of the phospholipids than ubiquinone-10, both forms giving rise to a shoulder of the main transition peak at lower temperatures. Small angle X-ray diffraction showed an increase in d-spacing suggesting a thicker membrane in the presence of both ubiquinone-10 and ubiquinol-10, below the phase transition and a remarkable broadening of the peaks indicating a loss of the repetitive pattern of the lipid multilamellar vesicles. Infrared spectroscopy showed an increase in wavenumbers of the maximum of the CH 2 stretching vibration at temperatures below the phase transition, in the presence of ubiquinol-10, indicating an increase in the proportion of gauche isomers in the gel phase, whereas this effect was smaller for ubiquinone-10. A very small effect was observed at temperatures above the phase transition. (2)H NMR spectroscopy of perdeuterated DMPC showed only modest changes in the spectra of the phospholipids occasioned by the presence of coenzyme Q 10. These small changes were reflected, in the presence of ubiquinol-10, by a decrease in resolution indicating that the interaction between coenzyme Q and phospholipids changed the motion of the lipids. The change was also visible in the first spectral moment (M1), which is related with membrane order, which was slightly decreased at temperatures below the phase transition especially with ubiquinol-10. A slight decrease in M 1 values was also observed above the phase transition but only for ubiquinol-10. These results can be interpreted to indicate that most ubiquinone-10 molecules are localized in the center of the bilayer, but a considerable proportion of ubiquinol-10 molecules may span the bilayer interacting more extensively with the phospholipid acyl chains.

Membrane docking of the C2 domain from protein kinase C alpha as seen by polarized ATR-IR. The role of PIP2.

We have used attenuated total internal reflection infrared spectroscopy (ATR-IR) spectroscopy to study the association of the C2 domain from protein kinase Cα (PKCα) with different phospholipid membranes, so as to characterise the mode of membrane docking and its modulation by the second-messenger lipid PIP₂. In parallel, we have also examined the membrane interaction of the C2 domain from cytosolic phospholipase A₂. PIP₂ did not induce significant changes in secondary structure of the membrane-bound PKCα-C2 domain, nor did binding of the PKCα-C2 domain change the dichroic ratios of the lipid chains, whereas the C2 domain from phospholipase A₂ did perturb the lipid chain orientation. Measurements of the dichroic ratios for the amide I and amide II protein bands were combined so as to distinguish the tilt of the β-sheets from that of the β-strands within the sheet. When associated with POPC/POPS membranes, the β-sandwich of the PKCα-C2 domain is inclined at an angle α=35° to the membrane normal, i.e., is oriented more nearly perpendicular than parallel to the membrane. In the process of membrane docking, the tilt angle increases to α=44° in the presence of PIP₂, indicating that the β-sandwich comes closer to the membrane surface, so confirming the importance of this lipid in determining docking of the C2 domain and consequent activation of PKCα.

The membrane binding kinetics of full-length PKCα is determined by membrane lipid composition

Protein kinase Cα (PKCα) is activated by its translocation to the membrane. Activity assays show the importance of PIP(2) in determining the specific activity of this enzyme. A FRET stopped flow fluorescence study was carried out to monitor the rapid kinetics of protein binding to model membranes containing POPC/POPS/DOG and eventually PIP(2). The results best fitted a binding mechanism in which protein bound to the membrane following a two-phase mechanism with a first bimolecular reaction followed by a slow unimolecular reaction. In the absence of PIP(2), the rapid protein binding rate was especially dependent on POPS concentration. Formation of the slow high affinity complex during the second phase seems to involve specific interactions with POPS and DOG since it is only sensitive to changes within relatively low concentration ranges of these lipids. Both the association and dissociation rate constants fell in the presence of PIP(2). We propose a model in which PKCα binds to the membranes via a two-step mechanism consisting of the rapid membrane initial recruitment of PKCα driven by interactions with POPS and/or PIP(2) although interactions with DOG are involved too. PKCα searches on the lipid bilayer in two dimensions to establish interactions with its specific ligands.

The interaction of the Bax C-terminal domain with membranes is influenced by the presence of negatively charged phospholipids

The C-terminal domain of the pro-apoptotic protein Bax (Bax-C) is supposed to act as a membrane anchor motif when Bax is activated leading to programmed cell death. A synthetic peptide which imitates this domain has been used to study the mechanism of peptide-phospholipid interaction. We have used static and MAS-NMR techniques to show that the interaction of Bax-C with membranes is modulated by the presence of a negatively charged phospholipid like phosphatidylglycerol. Bax-C slightly shifted upfield the (31)P resonances coming from phosphatidylglycerol and phosphatidylcholine. However the width of the resonance peaks was considerably higher when phosphatidylglycerol was present. Bax-C substantially decreased the T(1) relaxation times of phosphatidylglycerol and those of phosphatidylcholine when mixtured with phosphatidylglycerol, but T(1) values were not decreased when phosphatidylcholine was the only phospholipid present in the membrane. (13)C-MAS-NMR showed that T(1) values were decreased when Bax-C was incorporated into the lipid vesicles and this reduction affected similarly to carbons located in different regions of the membrane when the only phospholipid present was phosphatidylcholine. However, when phosphatidylglycerol was also present, the decrease in T(1) affected considerably more to some carbons in the polar region. These results indicate that Bax-C interacts differently with the polar part of the membrane depending on whether phosphatidylglycerol is present or not, suggesting that an electrostatic interaction of Bax-C with the membrane determines the location of this domain. Fluorescence spectroscopy showed that the Trp residues of Bax-C were placed in a microenvironment more hydrophobic and less accessible to quenching by acrylamide when phosphatidylglycerol was present.

Alcohol dehydrogenase from the hyperthermophilic archaeon Pyrobaculum aerophilum: Stability at high temperature

The structural and stability properties of a novel zinc-dependent alcohol dehydrogenase from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII) were investigated by Fourier transformed infrared spectroscopy (FTIR). This enzyme is a thermostable homo-tetramer belonging to the GroES-fold motif proteins characterized by an irregular β-barrel conformation. Our results revealed a protein with a secondary structure rich in β-sheet (32% of the total secondary elements) and it showed a three-step thermal unfolding pathway. The complete enzyme denaturation was preceded by the formation of a relaxed tertiary/quaternary structure and previously by an excited native-like conformation. Two-dimensional correlation spectroscopy analysis (2D-COS) and differential scanning calorimetry (DSC) experiments supported these data and allowed us to determine the protein melting temperature at 96.9 °C as well as to suggest the sequence of the events that occurred during the protein denaturation process.

Capsaicin Fluidifies the Membrane and Localizes Itself near the Lipid-Water Interface.

Capsaicin is the chemical responsible for making some peppers spicy hot, but additionally it is used as a pharmaceutical to alleviate different pain conditions. Capsaicin binds to the vanilloid receptor TRPV1, which plays a role in coordinating chemical and physical painful stimuli. A number of reports have also shown that capsaicin inserts in membranes and its capacity to modify them may be part of its molecular mode of action, affecting the activity of other membrane proteins. We have used differential scanning calorimetry, X-ray diffraction, (31)P NMR, and (2)H NMR spectroscopy to show that capsaicin increases the fluidity and disorder of 1,2-palmitoyl-sn-glycero-3-phosphocholine membrane models. By using (1)H NOESY MAS NMR based on proton-proton cross-peaks between capsaicin and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine resonances, we determined the location profile of this molecule in a fluid membrane concluding that it occupies the upper part of the phospholipid monolayer, between the lipid-water interface and the double bond of the acyl chain in position sn-2. This location explains the disorganization of the membrane of both the lipid-water interface and the hydrophobic palisade.

The anticancer agent edelfosine exhibits a high affinity for cholesterol and disorganizes liquid ordered membrane structures

Edelfosine is an anticancer drug with an asymmetric structure because, being a derivative of glycerol, it possesses two hydrophobic substituents of very different lengths. We showed that edelfosine destabilizes liquid-ordered membranes formed by either 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine, sphingomyelin (SM), and cholesterol (1:1:1 molar ratio) or SM and cholesterol (2:1 molar ratio). This was observed by differential scanning calorimetry in which phase transition arises from either of these membrane systems after the addition of edelfosine. The alteration in the liquid-ordered domains was characterized by using a small-angle X-ray diffraction that revealed the formation of gel phases as a consequence of the addition of edelfosine at low temperatures and by a wide-angle X-ray diffraction that confirmed changes in the membranes, indicating the formation of these gel phases. The increase in phase transition derived by the edelfosine addition was further confirmed by Fourier-transform infrared spectroscopy. The effect of edelfosine was compared with that of structurally analogue lipids: platelet-activating factor and 1-palmitoyl-2-acetyl- sn-glycero-3-phosphocholine, which also have the capacity of destabilizing liquid-ordered domains, although they are less potent than edelfosine for this activity, and lysophosphatidylcholine, which lacks this capacity. It was concluded that edelfosine may be associated with cholesterol favorably competing with sphingomyelin, and that this sets sphingomyelin free to undergo a phase transition. Finally, the experimental observations can be described by molecular dynamics calculations in terms of intermolecular interaction energies in phospholipid-cholesterol membranes. Higher interaction energies between asymmetric phospholipids and cholesterol than between sphingomyelin and cholesterol were obtained. These results are interesting because they biophysically characterize one of the main molecular mechanisms to trigger apoptosis of the cancer cells.