Biomedical subjects

R Brasseur

Publications and source records attributed to R Brasseur.

At least 73 records · Page 4Linked to original sources

PubMed1997

Composition and structural and functional properties of discoidal and spherical phospholipid-apoE3 complexes.

To model the common structural unit in the system of reverse cholesterol transport, we studied the composition, structure, and physicochemical properties of complexes generated between dipalmitoylphosphatidylcholine (DPPC) or palmitoyllinoleoylphosphatidylcholine (PLPC) and apoE3 in the absence and in the presence of cholesterol (Chol); the data were compared with similar experiments using apoA-I, the major proteins of high-density lipoproteins. The conformation and organization of lipid-binding domains of apoE3 within the complexes were calculated by computer modeling. The transition temperatures of DPPC within discoidal complexes with mean diameters of 116 A (GGE) or 148 A (EM) were higher for complexes versus liposomes both in the absence and in the presence of Chol. Association of apoE3 with DPPC resulted in a more structured state of the apolipoprotein molecule versus the soluble apolipoprotein; this state was characterized by parallel orientation of alpha-helixes of apoE3 and DPPC acyl chains. Substrate efficiency of the apoE3-PLPC-Chol complexes in the lecithin-cholesterol acyltransferase (LCAT) reaction expressed as Vmax/Km was 0.5 mole cholesteryl esters/h per 1 microM. The transformation of discoidal apoE3-DPPC-Chol complexes into spherical particles was induced by LCAT and accumulation of cholesteryl esters was approximately 62% of the total cholesterol. Parallel orientation of phospholipid acyl chains with helical segments disappeared in these particles. Discoidal apoE3-DPPC complexes incorporated unesterified cholesterol released from Chol-loaded J774 macrophages. The data support the concept that association of apoE3 and apoA-I with phospholipids is qualitatively similar due to similar orientation of helical repeats in the C-terminal domains of apoE3 and apoA-I.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1996

Lipid-binding properties of synthetic peptide fragments of human apolipoprotein A-II.

Human apolipoprotein A-II (apo A-II) consists of three potential amphipathic helices of 17 residues each, which contribute to the lipid-binding properties of this apolipoprotein. The conformation and lipid-binding properties of these peptides, either as single-helix or as two-helix peptides, were investigated by turbidity, fluorescence, electron-microscopy and circular-dichroism measurements, and are compared in this article. The lipid affinity of shorter C-terminal segments of apo A-II was compared with those of the single-helix or two-helix peptides, to define the minimal peptide length required for stable complex formation. The properties of the apo-A-II-(13-48)-peptide were further compared with those of the same segment after deletion of the Ser31 and Pro32 residues, because the deleted apo-A-II-(13-30)-(33-48)-peptide, is predicted to form a long uninterrupted helix. The single helices of apo A-II could not form stable complexes with phospholipids, and the helix-turn-helix segment spanning residues 13-48 was not active either. The apo-A-II-(37-77)-peptide and the apo-A-II-(40-73)-peptide could form complexes with lipids, which appear as discoidal particles by negative-staining electron microscopy. The shortest C-terminal domain of apo A-II able to associate with lipids to form stable complexes was the apo-A-II-(40-73)-peptide, which consisted of the C-terminal helix, a beta-turn and part of the preceding helix. The shorter apo-A-II-(49-77)-peptide, and the helical apo-A-II-(13-30)-(33-48)-peptide, could also associate with phospholipids. The complexes formed were, however, less stable, as they dissociated outside the transition temperature range of the phospholipid. These data suggest that the C-terminal pair of helices of apo A-II, which is the most hydrophobic pair, is responsible for the lipid-binding properties of the entire protein. The N-terminal pair of helices of apo A-II at residues 13-48 does not associate tightly with lipids. The degree of internal similarity and the cooperativity between the helical segments of apo A-II is thus less pronounced than in apo A-I or apo A-IV. The N-terminal and C-terminal domains of apo A-II appear to behave as two distinct entities with regard to lipid-protein association.

Amino Acid Sequence↗

PubMed1996

Fusogenic properties of the C-terminal domain of the Alzheimer beta-amyloid peptide.

A series of natural peptides and mutants, derived from the Alzheimer beta-amyloid peptide, was synthesized, and the potential of these peptides to induce fusion of unilamellar lipid vesicles was investigated. These peptide domains were identified by computer modeling and correspond to respectively the C-terminal (e.g. residues 29-40 and 29-42) and a central domain (13-28) of the beta-amyloid peptide. The C-terminal peptides are predicted to insert in an oblique way into a lipid membrane through their N-terminal end, while the mutants are either parallel or perpendicular to the lipid bilayer. Peptide-induced vesicle fusion was demonstrated by several techniques, including lipid-mixing and core-mixing assays using pyrene-labeled vesicles. The effect of peptide elongation toward the N-terminal end of the entire beta-amyloid peptide was also investigated. Peptides corresponding to residues 22-42 and 12-42 were tested using the same techniques. Both the 29-40 and 29-42 beta-amyloid peptides were able to induce fusion of unilamellar lipid vesicles and calcein leakage, and the amyloid 29-42 peptide was the most potent fusogenic peptide. Neither the two mutants or the 13-28 beta-amyloid peptide had any fusogenic activity. Circular dichroism measurements showed an increase of the alpha-helical content of the two C-terminal peptides at increasing concentrations of trifluoroethanol, which was accompanied by an increase of the fusogenic potential of the peptides. Our data suggest that the alpha-helical content and the angle of insertion of the peptide into a lipid bilayer are critical for the fusogenic activity of the C-terminal domain of the amyloid peptide. The differences observed between the fusogenic capacity of the amyloid 29-40 and 29-42 peptides might result from differences in the degree of penetration of the peptides into the membrane and the resulting membrane destabilization. The longer peptides, residues 22-42 and 12-42, had decreased, but significant, fusogenic properties associated with perturbation of the membrane permeability. These data suggest that the fusogenic properties of the C-terminal domain of the beta-amyloid peptide might contribute to the cytotoxicity of the peptide by destabilizing the cell membrane.

Alzheimer Disease↗

PubMed1996

Importance of the hydrophobic energy: structural determination of a hypoglycemic drug of the meglitinide family by nuclear magnetic resonance and molecular modeling.

The molecular structure of (2S)-2-benzyl-3-(cis-hexahydro-2-isoindolinylcarbonyl) propionic acid (KAD-1229), a hypoglycemic drug of the meglitinide family, was studied by nuclear magnetic resonance (NMR) and molecular modeling. The results of the NMR experiments indicated that KAD-1229 existed in solution in the form of two stable conformers of equal population, called KADI and KADII in this paper. Three different molecular modelings were then applied: the classical molecular dynamics using the commercial Biosym and Hyperchem softwares and the Prot+ program, which is not based on a dynamical study but on a systematic conformational analysis of the molecule, which includes a term that allows the estimation of the hydrophobic interaction. The modeling results showed the following points. First, in contrast with classical molecular dynamics, which uses restraints from two-dimensional nuclear Overhauser effect (NOE) data, the Prot+ KAD structure provides conformations that support experimental NMR data without any external intervention. In the structures in agreement with NMR data, an important hydrophobic interaction between the phenyl cycle and the perhydroisoindole ring of KAD is observed. This interaction, which seems to play a role in the biological activity of the drug, is lost when no restraints are considered in classical molecular dynamics. Second, the difference between KADI and KADII arises mainly from slight distance geometric differences at the level of the perhydroisoindole and the phenyl rings.

Benzamides↗

PubMed1996

Interaction of the macrolide azithromycin with phospholipids. II. Biophysical and computer-aided conformational studies.

In a comparison paper, we show the azithromycin causes a lysosomal phospholipidosis in cultured cells, binds in vitro to negatively charged bilayers without causing aggregation or fusion, and inhibits lysosomal phospholipase A1. In this paper, we show that azithromycin decreases the mobility of the phospholipids in negatively charged liposomes (using 31P nuclear magnetic resonance) and that it increases the fluidity of the acyl chains close to the hydrophilic/hydrophobic interface, but not deeper into the hydrophobic domain (assessed by measuring the fluorescence polarization of trimethylammonium-diphenylhexatriene and diphenyhexatriene, respectively). Computer-aided conformational analysis of mixed monolayers of azithromycin and phosphatidylinositol shows that the drug can be positioned largely in the hydrophobic domain, but close to the interface, with the macrocycle facing the C1 of the fatty acids (allowing the N9a endocyclic tertiary amine to interact with the phospho-groups), the cladinose located on the hydrophobic side of the lipid/water interface and the desosamine projected into the hydrophobic domain. This position is consistent with the experimental data. Analysis of virtual molecules shows that this unanticipated behavior to the shielding of the ionizable N3' amino-group in the desosamine by methyl-groups, and to the wide dispersion of hydrophobic domains all over the molecule. The interaction of azithromycin with phospholipids may account for some of its unusual pharmacokinetic properties and for its potential to cause lysosomal phospholipidosis.

Anti-Bacterial Agents↗

PubMed1996

Aminoglycoside antibiotics prevent the formation of non-bilayer structures in negatively-charged membranes. Comparative studies using fusogenic (bis(beta-diethylaminoethylether)hexestrol) and aggregating (spermine) agents.

Aminoglycoside antibiotics cause aggregation but not fusion of negatively-charged liposomes at an extent proportional to their capacity to interact with acidic phospholipids (Van Bambeke et al., 1995, Eur. J. Pharmacol., 289, 321-333). To understand why aggregation is not followed by fusion, we have examined here the influence of two aminoglycosides with markedly different toxic potential (gentamicin > isepamicin) on lipid phase transition in negatively-charged liposomes using 31P-NMR spectroscopy, in comparison with spermine (an aggregating agent) and bis(beta-diethylaminoethylether)hexestrol or DEH (a fusogenic cationic amphiphile). Gentamicin, spermine, and, to a lesser extent, isepamicin inhibit the appearance of the isotropic signal seen upon warming of control liposomes and denoting the presence of mobile structures. This non-bilayer signal appeared most prominently when liposomes were incubated with DEH, a strong fusogenic agent. We conclude that aminoglycosides, like spermine, have the potential to prevent membrane fusion, by inhibiting the development of a critical change in membrane organization, which is associated with fusion. We suggest that this capacity could be a determinant in aminoglycoside toxicity.

Animals↗

PubMed1996

Prediction of signal peptide functional properties: a study of the orientation and angle of insertion of yeast invertase mutants and human apolipoprotein B signal peptide variants.

A number of studies have introduced mutations into the yeast invertase signal peptide, using it as a model system to elucidate features for targeting, translocation and intracellular transport. Using molecular modelling of the invertase signal peptide we have analysed the hydrophobicity potential and the change in dielectric constant of the energy transfer, when the molecule moves from a hydrophobic to a hydrophilic phase at the simulated hydrophobic-hydrophilic interface. This modelling has been carried out on wild type and mutant invertase signal peptides of altered function, previously reported in the literature. While the predicted angle of insertion correlates with the measured extent of invertase secretion, with an optimum angle of 45 degrees, mutations that change the angle of orientation reduce the extent of invertase secretion. We have applied these same molecular modelling principles to the naturally occurring variants of the human apolipo-protein B (apoB) signal peptide, that confer a secretion defective phenotype when fused to yeast invertase and expressed in yeast. Our modelling thus identifies a strong correlation between the predicted angle of insertion of the signal peptide into the membrane and its ability to direct secretion.

Amino Acid Sequence↗

PubMed1996

Subunit 8 of the Saccharomyces cerevisiae cytochrome bc1 complex interacts with succinate-ubiquinone reductase complex.

We have investigated the function of subunit 8 of the cytochrome bc1 complex by generating six site-directed mutants, F46C, R51S, P62V, G64A, R91N, and W69-stop, in the cloned QCR8 gene and expressing the mutated genes in a Saccharomyces cerevisiae strain in which the chromosomal copy of QCR8 is deleted. The W69-stop mutation impairs assembly of the bc1 complex and growth of yeast on nonfermentable carbon sources as does deletion of QCR8 [Maarse, A. C., De Haan, M., Schoppink, P. J., Berden J. A., and Grivell, L. A. (1988) Eur. J. Biochem. 172, 179-184], implying that the C-terminus of subunit 8 is important for assembly and/or the stability of the bc1 complex. The F46C, R51S, P62V, G64A, and R91N mutations do not affect the growth of yeast on nonfermentable carbon sources, not do they lower the activity or alter the inhibitor sensitivity of the bc1 complex. Rather, some of the mutations increase the cytochrome C reductase activity of the bc1 complex by as much as 40%. However, succinate-ubiquinone reductase activity was consistently reduced 40-60% in mitochondrial membranes from these mutants, while NADH-ubiquinone reductase activity was not affected. In addition, the activation of succinate-ubiquinone reductase activity by succinate was diminished by the F46C, R51S, P62V, and G64A mutations. These results indicate that the cytochrome bc1 complex participates in electron transfer from succinate to ubiquinone in situ and also suggest an interaction between succinate-ubiquinone reductase and cytochrome bc1 complex which involves subunit 8 of the bc1 complex.

Amino Acid Sequence↗

PubMed1995

Conformational analysis of non-sulfonylurea hypoglycemic agents of the meglitinide family.

Non-sulfonylurea hypoglycemic agents of the meglitinide family such as S3075, repaglinide, KAD-1229, and A-4166, were found to display a comparable U-shaped conformation by molecular modelling, with hydrophobic cycles placed at the extremity of each branch and a peptidic bond placed at the bottom of the U. A comparable conformation was observed with the hypoglycemic sulfonylureas glibenclamide and glimepiride. A different conformation with a greater distance between the hydrophobic cycles at the extremity of each branch was found, however, with the biologically inactive enantiomers of A-4166 and repaglinide and the poorly efficient insulinotropic agent meglitinide. The identification of a common conformation of these hypoglycemic agents may help in the design of highly active compounds and provide an imprint of their postulated target receptor on the pancreatic B-cell plasma membrane.

Benzamides↗

PubMed1995

Synthesis and antimicrobial and toxicological studies of amino acid and peptide derivatives of kanamycin A and netilmicin.

Amino acid and peptide derivatives of aminoglycosides have been obtained by substitution of the 1-N or 6'-N amino functions of kanamycin A and netilmicin via the temporary complexation of vicinal and nonvicinal amino and hydroxy functions by copper ion [1-N kanamycin A derivatives: L-Ala (6a), D-Ala (6b), Gly (6c), L-Asp (6d), L-Ala-L-Ala (6e). 6'-N kanamycin A derivatives: L-Ala (3a), D-Ala (3b), Gly (3c), L-Ala-L-Ala (3e), L-Leu (3f). 6'-N netilmicin derivatives: L-Ala (9a), D-Ala (9b), Gly (9c), L-Asp (9d), L-Ala-L-Ala (9e)]. Characterization was made by FAB-MS, IR, 1H-NMR, and 13C-NMR. All derivatives were essentially inactive. The nephrotoxic potential of the derivatives obtained in sufficient quantities (3b,e and 9a-e) was assessed by measuring their inhibitory potential toward the activity of lysosomal phospholipase A1 acting on phosphatidylcholine embedded in negatively-charged membranes. One compound, 6'-N-L-Ala-netilmicin (9a), showed a 2-fold decrease of inhibitory potency compared to its parent drug. A conformational analysis revealed that it adopts two equally probable conformations and orientations when interacting with phosphatidylinositol. The first in which the drug lies parallel to the hydrophobic-hydrophilic interface, is similar to that of netilmicin. The second, in which the drug inserts itself in the bilayer across the hydrophilic/hydrophobic interface, is similar to that described for streptomycin, an almost non-nephrotoxic aminoglycoside.

Amino Acids↗

PubMed1995

Piracetam-induced changes to membrane physical properties. A combined approach by 31P nuclear magnetic resonance and conformational analysis.

Piracetam, Nootropil (2-oxo-1-pyrrolidine acetamide), is a drug promoting erythrocyte deformability. To establish the mode of action of this compound, we have investigated its influence on the organization of model phospholipid membranes. 31P NMR data show that the drug induces a structural modification in liposomes made of phosphatidylcholine and phosphatidylethanolamine. Our conformational analysis results have allowed the interpretation of the effect of piracetam on these model membranes: the specific interaction between the drug molecules and the phosphate headgroups induces a new organization of the lipids favouring formation of mobile drug-phospholipid complexes that exhibit an isotropic-type signal in the 31P NMR spectra.

Liposomes↗

PubMed1995

Regulation of the beta-lactamase BlaL of Streptomyces cacaoi: the product of the blaB regulatory gene is an internal membrane-bound protein.

The beta-lactamase-encoding gene blaL, cloned from Streptomyces cacaoi in Streptomyces lividans, is inducible by beta-lactam compounds. This regulation has been shown to depend on the products of two open reading frames, ORF1 (blaA) and ORF2 (blaB) [Lenzini, Magdalena, Fraipont, Joris, Matagne and Dusart (1992) Mol. Gen. Genet. 235, 41-48]. BlaA belongs to the LysR family of transcription activators, whereas BlaB shares some features with the penicillin-recognizing proteins. BlaB has now been overexpressed in Escherichia coli, purified and used for antibody preparation. Immunoblotting of cell-fractionated materials from S. cacaoi showed that BlaB is attached to the internal face of the cytoplasmic membrane. It could not be released by high salt concentrations or EDTA, but only by protease treatment. Under the assay conditions, BlaB did not act as a penicillin-binding protein, a beta-lactamase, a D-amino-peptidase or a target in a phosphorylation step.

Cell Membrane↗

PubMed1995

Helix-helix interactions in reconstituted high-density lipoproteins.

In this work we calculated the ionic interactions between adjacent amphipathic helices of apo A-I and apo A-IV. The calculation of the electrostatic potential around the helices helps identify the charged residues susceptible to form salt bridges between adjacent helices. An estimation of the stability of the different pairs of helices is derived from the calculation of the energy of interaction between contiguous helices at a water/lipid interface after energy minimization. The most stable energetic conformation corresponds to the 17-residue helices oriented anti-parallel and separated by a stretch of 5 residues in an extended beta-strand conformation, as calculated through the 'stereo alphabet' calculation procedure. In a pair of helices, the hydrophobic faces are directed towards the lipid core of the discoidal phospholipid-apolipoprotein complex and the hydrophobic lipid-protein interactions are major determinants for the stability of the complex. Interactions between polar residues located on the opposite face of the helix and water molecules can also contribute to the overall energy of the system. Finally, salt bridge formation between residues of opposite charge along the edge of the helical segments contribute to the cooperativity of the phospholipid-apolipoprotein complex formation. The mode of assembly of the amphipathic helical repeats of the apolipoproteins around the edge of a discoidal complex is therefore determined both by the hydrophobic character of the residues and by the charge complementarity along the edge of the helices which increases the structural stability and determines the relative orientation of the helices.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Structure, chromosomal location, and expression pattern of three mouse genes homologous to the human MAGE genes.

The human MAGE1 gene directs the expression of an antigen recognized on a melanoma by autologous cytolytic T lymphocytes. MAGE1 belongs to a family of genes that are expressed in a number of tumors of various histological types but not in normal tissues except testis. The MAGE genes are arranged in two groups that are located within two different regions of the human X chromosome (Xq26-qter and Xp21.3). By hybridizing mouse genomic libraries with a MAGE1 probe, we identified three homologous genes. Two of these mouse genes, Smage1 and Smage2, are more than 99% identical to each other and encode the same protein of 330 aa. The 5' noncoding region of Smage2 provides the potential for regulating the expression of the gene through several different promoters located in front of alternative first exons. The third gene, Smage3, has the structure of a processed transcript. It codes for a protein with only 11 aa substitutions with respect to the Smage1/2 product. Somatic cell hybrids and interspecific backcross analysis showed that Smage3 is autosomal and that Smage1 and Smage2 are located between the Dmd and the Ar loci on the mouse X chromosome. Since this region is syntenic to the human Xp21.1-p22.1 region, we conclude that Smage1 and Smage2 are homologous to the MAGE-Xp rather than to the MAGE-Xq genes. Smage1/2 transcripts were detected in several tumor and embryonal cell lines but not in normal mouse tissues with the exception of testis. Expression of Smage3 was found in embryos from Day 11 to Day 15.

Amino Acid Sequence↗

PubMed1995

Aminoglycoside antibiotics induce aggregation but not fusion of negatively-charged liposomes.

The binding of aminoglycoside antibiotics to acidic phospholipids of membranes is an essential step in the development of both their renal and auditory toxicities, which could be associated with critical modifications of the membrane properties. This work examines the capacity of aminoglycosides to induce membrane aggregation and fusion. Three techniques were used in parallel: (i) measurement of the dequenching rate of a lipid-soluble fluorescent probe (octadecylrhodamine B) incorporated at self-quenched concentration in membranes; (ii) measurement of the increase in the energy transfer between two fluorescent derivatives of phospholipids; and (iii) electron microscopy of negatively-stained replicas. The results were compared with those obtained with spermine (an aggregating polycation) and melittin (a fusogenic peptide). The three approaches indicate that aminoglycosides induce liposomes aggregation, but not fusion. Aggregation is related to the capacity of each drug studied to bind phosphatidylinositol, as evaluated by its energy of interaction with this acidic phospholipid, and to its toxic potential. Membrane aggregation occurring in vivo could therefore contribute to, or be a determinant of this toxicity, which could rationally be screened for new derivatives by the methods applied here.

Aminoglycosides↗

PubMed1995

Secondary structure of monoamine oxidase by FTIR spectroscopy.

The secondary structure of human monoamine oxidase A and bovine monoamine oxidase B has been investigated by Fourier Transform Attenuated Total Reflection Spectroscopy (FTIR ATR). The experimental results are compared for both isoenzymes and the data are incorporated in a statistical attribution of secondary structure of the enzyme describing the distinct folding and molecular specificity of the two types of monoamine oxidase.

Amino Acid Sequence↗

PubMed1995

Proposition of a three-dimensional representation of the constitutive protein of the hepatitis B surface antigen particles.

Hepatitis B surface antigen particles are composed of the major viral envelope protein, the S protein, embedded into a lipid shell. The description of the folding of this protein within the particle membrane could provide helpful information for replacing surface-exposed protein domains by foreign sequences without destabilizing the particle structure. Since the crystallization of the protein in its lipid environment remains inaccessible in the near future, alternative approaches had to be envisaged. We combine here the available experimental structural and topological data with a conformational procedure to identify membrane-associated domains of the HBsAg protein and to propose a three-dimensional description of their assembly within the particle membrane. The proposed protein structure is composed of four membrane-spanning helices and an amphipatic helix located on the inner surface membrane. The transmembrane helices are assembled into a highly hydrophobic complex in which no access to the water environment is allowed. The approach could be extended to other membrane-associated proteins.

Amino Acid Sequence↗

PubMed1995

Simulating the folding of small proteins by use of the local minimum energy and the free solvation energy yields native-like structures.

Assuming that the protein primary sequence contains all information required to fold a protein into its native tertiary structure, we propose a new computational approach to protein folding by distributing the total energy of the macromolecular system along the torsional axes. We further derive a new semiempirical equation to calculate the total energy of a macromolecular system including its free energy of solvation. The energy of solvation makes an important contribution to the stability of biological structures. The segregation of hydrophilic and hydrophobic domains is essential for the formation of micelles, lipid bilayers, and biological membranes, and it is also important for protein folding. The free energy of solvation consists of two components: one derived from interactions between the atoms of the protein, and the second resulting from interactions between the protein and the solvent. The latter component is expressed as a function of the fractional area of protein atoms accessible to the solvent. The protein-folding procedure described in this article consists of two successive steps: a theoretical transition from an ideal alpha helix to an ideal beta sheet is first imposed on the protein conformation, in order to calculate an initial secondary structure. The most stable secondary structure is built from a combination of the lowest energy structures calculated for each amino acid during this transition. An angular molecular dynamics step is then applied to this secondary structure. In this computational step, the total energy of the system consisting of the sum of the torsional energy, the van der Waals energy, the electrostatic energy, and the solvation energy is minimized. This process yields 3-D structures of minimal total energy that are considered to be the most probable native-like structures for the protein. This method therefore requires no prior hypothesis about either the secondary or the tertiary structure of the protein and restricts the input of data to its sequence. The validity of the results is tested by comparing the crystalline and computed structures of four proteins, i.e., the avian and bovine pancreatic polypeptide (36 residues each), uteroglobin (70 residues), and the calcium-binding protein (75 residues); the C alpha-C alpha maps show significant homologies and the position of secondary structure domains; that of the alpha helices is particularly close.

Animals↗