Fabienne Defrise-Quertain

Orientation and structure of the NH2-terminal HIV-1 gp41 peptide in fused and aggregated liposomes.

For several retroviruses, the N-terminal hydrophobic sequence of the viral envelope glycoprotein has been shown to play a crucial role in the interaction between the virus and the host cell membrane. We report here on the interaction of a synthetic 16 residues peptide corresponding to the gp41 NH2-terminal sequence of Human Immunodeficiency Virus with the phospholipid bilayer. Fluorescence energy transfer measurements show that this peptide can induce lipid mixing of large unilamellar vesicles (LUV) of various compositions at pH 7.4 and 37 degrees C. LUV undergo fusion, provided they contained phosphatidylethanolamine (PE) in their lipid composition. To provide insight into the mechanism of the fusion event, the peptide secondary structure and orientation in the lipid bilayer were determined using Fourier Transform Infrared Spectroscopy (FTIR). The peptide adopts mainly a beta-sheet conformation in the absence of lipids. After interaction with LUV the beta-sheet is partly converted into alpha-helix. The orientation of the peptide with respect to the lipid acyl chains depends on the presence of PE in the lipid bilayer. The peptide is inserted into the lipid bilayer with the helix axis oriented parallel to the lipid acyl chains in the fused vesicles, whereas it is adsorbed parallel to the lipid/water interface in the aggregated vesicles. The role of the two kinds of orientation during the fusion event is discussed.

Fusogenic activity of SIV (Simian Immunodeficiency Virus) peptides located in the GP32 NH2 terminal domain

Peptides of 12, 16 and 24 amino acids length corresponding to the NH2 terminal sequence of SIV gp32 were synthesized. Fluorescence energy transfer studies have shown that those peptides can induce lipid mixing of SUV (Small Unilamellar Vesicles) of various compositions at pH 7.4 and 37 degrees C. LUV (Large Unilamellar Vesicles) were shown to undergo fusion, provided they contained PE in their lipid composition. This work is an attempt to determine how the fusogenic activity depends on the structure of the peptide inserted into a lipidic environment. The peptides secondary structure and orientation in the lipid bilayer were determined using Fourier Transform infrared spectroscopy (FTIR). They adopt mainly a beta-sheet conformation in the absence of lipids. After interaction with DOPC SUV, the beta-sheet is partly converted into alpha-helix oriented obliquely with respect to the membrane interface. We bring here evidence that this oblique orientation is a prerequisite to the fusion process.

Spin label partitioning in lipid vesicles a model study for drug encapsulation

In this paper we attempt to outline some features which determine the encapsulation of small molecules into lipid vesicles. Spin labels derived from five carboxylic acids of different lengths were synthesized and incorporated in varying amounts into multilamellar and unilamellar vesicles made up of four different phosphatidylcholines. The influence on the release process of the bilayer rigidity and of the hydrophobicity of the entrapped molecule was systematically studied. The hydrophobicity is of critical importance and was estimated by measuring the partition coefficient (P) between octanol and buffer. In multilamellar vesicles, molecules characterized by extreme P values (log P less than -0.3 and log P greater than 5) can be efficiently entrapped. The rate of leakage is related to the P value according to a bell-shaped curve. Moreover, gel state of the bilayer and long acyl chains of the lipids are properties which favor a good entrapment. Small unilamellar vesicles may be formed in the presence of high concentrations of hydrophilic and lipophilic spin labels. However, the formation of unilamellar vesicles produces a significant reduction of the internal volume and of the entrapped water-soluble spin lables. High fractions of lipid-soluble spin labels can be incorporated in unilamellar vesicles but the vesicle stability is diminished.

Comparison of adriamycin and derivatives uptake into large unilamellar lipid vesicles in response to a membrane potential.

The uptake of adriamycin (ADM) and several derivatives into large unilamellar vesicles (LUV) displaying a transmembrane potential and having a lipid composition close to that of the inner mitochondrial membrane has been measured. Drug association to neutral liposomes, made of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (70:30, w/w) was shown to be potential-dependent: in the absence of potential, accumulation of drug was almost undetectable, whereas between 11 and 50 nmol of drug/mumol phospholipid, depending on the anthracycline used, was associated to LUV exhibiting a membrane potential after 1 h incubation. Association of drugs to LUV with a lipid composition closer to that of the inner mitochondrial (cardiolipin, CL, 20%; PC 50%; PE, 30%, w/w) and displaying a membrane potential is higher than with neutral vesicles (between 40 and 76 nmol of anthracycline/mumol phospholipid after 1 h incubation). Since it is known that ADM and derivatives have a high affinity for CL, a fraction of the associated drug may bind to CL on the outer side of the vesicles. This was confirmed by the fact that, in the absence of potential, between 40 and 56 nmol of anthracycline/mumol phospholipid was still associated to LUV containing CL. In order to discriminate between drug adsorbed at the surface of the LUV and drug accumulated inside the LUV, an anthracycline fluorescence quencher (I-) was used. It was shown on neutral LUV displaying a membrane potential, that between 55 and 81% of the associated drug is actually entrapped inside the vesicles, inaccessible to the quencher. These percentages decreased to between 41 and 68%, respectively, in the presence of LUV containing CL and exhibiting a membrane potential, whereas for LUV of the same composition but displaying no membrane potential almost all the associated drug is adsorbed on the outer face of the LUV, accessible to the quencher, and likely bound to CL. This study brings evidence that antitumour anthracyclines despite important structural homologies do not accumulate to the same extent into vesicles mimicking the lipid composition and the membrane potential of mitoplasts. This ability to reach the matrix compartment of mitochondria could partly explain the differences of cardiotoxicities associated to anthracyclines with closely related molecular structure.

Vesicle formation by double long-chain amidines

The formation and characterization of synthetic surfactant vesicles prepared from aqueous suspensions of a new class of amphiphiles with an amidine function as the polar head group are reported.

Phospholipase inactivation induced by an amino-piperazine derivative: a study at the lipid-water interface.

1‐Amino‐4‐octylpiperazine, AP 22, an antiviral agent causes lipid accumulation in nervous tissue cultures. A physicochemical membrane model was used to demonstrate the formation of a lipid‐AP 22 complex hindering phospholipase A2 action. A well defined amphiphilic balance seems essential to explain the mode of action of the drug. The hydrophilic group prevents enzyme‐substrate complex formation whereas the hydrophobic group allows the penetration in the lipid layer and determines the stability of the drug‐lipid complex. This stability of the drug‐lipid association has a direct influence on phospholipase A2 activity but does not affect phospholipase C activity. No inactivation of phospholipase A2 due to a drug‐enzyme interaction could be detected.

Hydrolysis of phosphatidylcholine liposomes by lysosomal phospholipase A is maximal at the phase transition temperature of the lipid substrate

We have measured the rate of hydrolysis of liposomes made of DL-α-dipalmitoylphosphatidylcholine (DPPC) and L-α-dimyristoylphosphatidylcholine by a soluble fraction of highly purified lysosomes isolated from rat liver. Phospholipids are hydrolyzed into lysophospho-lipids and fatty acids at a rate which is maximal near the temperature characteristic of the gel to liquid crystalline phase transition of the lipid bilayer. This strong influence of the physical properties of the substrate on the enzyme activity suggests a structural analogy between the lysosomal phospholipases of the A type (EC 3.1.1.32 and EC 3.1.1.4) and the pancreatic phospholipase A2.

Properties of mixed monolayers of lecithin and spin probe: I. Study of the interactions at the air—water interface

Abstract The perturbations induced in a lipid layer by the presence of a spin probe molecule (4-palmitamido-2,2,6,6-tetramethylpiperidine-l-oxyl, TAP) have been investigated in a model membrane. Mixed monolayers of probe and dipalmitoyl- dl -α-phosphatidylcholine (DPPC) were formed at the air-water interface and studied using classic surface techniques. In mixed films, surface pressure measurements showed that the spin probe induces a modification of the monolayer lateral compressibility giving rise to a more expanded structure. Two other methods, i.e., surface viscosity and enzymic hydrolysis, offered the possibility of detecting the propagation of the probe perturbation by observing modifications of the DPPC properties. The surface viscosity approach is based on a very specific property of the probe: The monolayer, even in a closepacked state, shows no detectable surface viscosity. In these conditions surface viscosity measurements carried out with mixed monolayers will depend only on the second component of the system, i.e., the lecithin. Any deviation from the predicted viscosity value indicates a change in the DPPC packing. Viscosity results indicate that the probe induces a more packed organization of the DPPC molecules. The changes in the enzymic hydrolysis kinetics of the mixed monolayers confirm these results. The phospholipases activity is strongly dependent on the lipid packing. Indeed, the probe molecule cannot be hydrolyzed and the inhibition observed must be interpreted in terms of a specific modification in the lipid organization induced by the probe.

Fusion and lipid exchange in vesicles containing lipophilic spin labels.

Lipophilic non-electrolyte spin labels greatly accelerate the fusion of unilamellar vesicles of dipalmitoylphosphatidylcholine when the system is maintained below the lipid phase transition. Differential scanning calorimetry and centrifugation measurements show that the transformed vesicles are large and probably unilamellar. Differential scanning calorimetry and fluorescence depolarization measurements were also carried out on mixtures of labeled dipalmitoylphosphatidylcholine vesicles and of vesicles composed of pure dimyristoylphosphatidylcholine. A mixing of the membrane components is observed when the vesicles are incubated above the transition temperature of the two constituent lipids. However, the process does not involve a real fusion of the entire vesicles. An exchange of lipid and label monomers between the two lipid phases seems to occur. These observations are discussed in view of the molecular organization of the spin label within the dipalmitoylphosphatidylcholine matrix below and above the lipid transition temperature.