Biomedical subjects

R Brasseur

Publications and source records attributed to R Brasseur.

At least 91 records · Page 5Linked to original sources

PubMed1995

Insulinotropic action of (2S)-2-benzyl-3-(cis-hexahydro-2-isoindolinylcarbonyl) propionate. II. Ionophoretic and conformational aspects.

1. The non-sulphonylurea insulinotropic agent sodium (2S)-2-benzyl-3-(cis-hexahydro-2-isoindolinylcarbonyl) propionate (KAD-1229) was found to display calcium ionophoretic activity in an artificial membrane model. 2. Conformation analysis indicated that a complex between calcium and KAD-1229, with a 1:2 stoichiometry, indeed displays favourable attributes for ionophoretic activity across a hydrophobic environment. 3. It is speculated that the ionophoretic property of KAD-1229 might participate to the remodelling of cationic fluxes evoked by this insulinotropic agent in pancreatic islet cells.

Animals↗

PubMed1995

BAGE: a new gene encoding an antigen recognized on human melanomas by cytolytic T lymphocytes.

Several tumor antigens are recognized by autologous cytolytic T lymphocytes (CTL) on human melanoma MZ2-MEL. Some of them are encoded by genes MAGE-1 and MAGE-3, which are not expressed in normal tissues except in testis. Here, we report the identification of a new gene that codes for another of these antigens. This gene, named BAGE, codes for a putative protein of 43 aa and seems to belong to a family of several genes. The antigen recognized by the autologous CTL consists of BAGE-encoded peptide AARAVFLAL bound to an HLA-Cw 1601 molecule. Gene BAGE is expressed in 22% of melanomas, 30% of infiltrating bladder carcinomas, 10% of mammary carcinomas, 8% of head and neck squamous cell carcinomas, and 6% of non-small cell lung carcinomas. Like the MAGE genes, it is silent in normal tissues with the exception of testis. Because of its tumor-specific expression, the BAGE-encoded antigen may prove useful for cancer immunotherapy.

Amino Acid Sequence↗

PubMed1995

Molecular parameters involved in aminoglycoside nephrotoxicity.

Aminoglycoside antibiotics are hydrophilic molecules consisting of an animated cyclitol associated with amino sugar. They bind in vivo as well as in vitro to negatively charged membranes. Their use as chemotherapeutic agents is unfortunately accompanied by oto- and nephrotoxic reactions, and the purpose of this review is to examine the role of the molecular interactions between aminoglycosides and membranes in the development of nephrotoxicity. 31P Nuclear magnetic resonance (NMR) and fluorescence depolarization have been used to characterize the effect of aminoglycosides on phosphate heads and fatty acyl chains of phospholipids. 15N NMR has been used to obtain interesting information on regioselective interactions of amino groups of antibiotics with phospholipids. The binding of aminoglycosides with negatively charged membranes is associated with impairment of phospholipid catabolism, change in membrane permeability, and membrane aggregation. Biochemical analysis and 1H NMR spectroscopy have brought information on the molecular mechanism involved in the impairment of phospholipid catabolism. Nephrotoxic aminoglycosides could induce sequestration of phosphatidylinositol and therefore reduce the amount of negative charge available for optimal lysosomal phospholipase activity toward phosphatidylcholine included in liposomes that also contain cholesterol and sphingomyelin. Conformational analysis shows that aminoglycosides, which have a high potency to inhibit lysosomal phospholipase activity, adopt an orientation parallel to the lipid/water interface. This orientation of the aminoglycoside molecule at the interface is also critical to explain the marked increase of membrane permeability induced by less nephrotoxic aminoglycosides such as isepamicin and amikacin. This effect is indeed only observed with aminoglycosides oriented perpendicular to this interface, probably related to the creation of a local condition of disorder. The impairment of phospholipid catabolism, which is considered to be an early and significant step in the development of aminoglycoside toxicity, is therefore not related to the change in membrane permeability. However, the role of this latter phenomenon and of membrane aggregation for aminoglycoside nephrotoxicity could be further investigated.

Aminoglycosides↗

PubMed1995

Prediction of the antigenic sites of the cystic fibrosis transmembrane conductance regulator protein by molecular modelling.

Antibodies are powerful tools for studying the in situ localization and physiology of proteins. The prediction of epitopes by molecular modelling has been used successfully for the papilloma virus, and valuable antibodies have been raised [Müller et al. (1990) J. Gen. Virol., 71, 2709-2717]. We have improved the modelling approach to allow us to predict epitopes from the primary sequences of the cystic fibrosis transmembrane conductance regulator. The procedure involves searching for fragments of primary sequences likely to make amphipathic secondary structures, which are hydrophilic enough to be at the surface of the folded protein and thus accessible to antibodies. Amphipathic helices were predicted using the methods of Berzofsky, Eisenberg and Jähnig. Their hydrophobic-hydrophilic interface was calculated and drawn, and used to predict the orientation of the helices at the surface of the native protein. Amino acids involved in turns were selected using the algorithm of Eisenberg. Tertiary structures were calculated using 'FOLDING', a software developed by R. Brasseur for the prediction of small protein structures [Brasseur (1995) J. Mol. Graphics, in press]. We selected sequences that folded as turns with at least five protruding polar residues. One important property of antibodies is selectivity. To optimize the selectivity of the raised antibodies, each sequence was screened for similarity (FASTA) to the protein sequence from several databanks. Ubiquitous sequences were discarded. This approach led to the identification of 13 potential epitopes in the cystic fibrosis transmembrane conductance regulator: seven helices and six loops.

Algorithms↗

PubMed1995

The hydrophobic effect in protein folding.

In this review of protein folding we consider the noncovalent interactions existing between atoms or molecules at the molecular level. The electrostatic, Van der Waals, hydrogen bonding, and hydrophobic interactions are described and their contribution to protein conformation is discussed. The growing interest in the hydrophobic effect arises from its importance in the protein folding process, and a semiempirical simulation of the free energy of solvation is proposed. In most proteins, the different forces we describe contribute to the stability of the protein conformation in a complex way. However, in the case of the apolipoproteins and cytochrome C551, the energetic contributions are easily distinguished. For this reason, these proteins are used to illustrate the importance of the different energy fields.

Apolipoproteins↗

PubMed1995

Molecular cloning and characterization of a soluble inorganic pyrophosphatase in potato.

A cDNA clone encoding a soluble inorganic pyrophosphatase (EC 3.6.1.1) of potato (Solanum tuberosum L.) was isolated by screening a developing tuber library with a heterologous probe. The central domain of the encoded polypeptide is nearly identical at the sequence level with its Arabidopsis homolog (J.J. Kieber and E.R. Signer [1991] Plant Mol Biol 16: 345-348). Computer-assisted analysis of the potato, Arabidopsis, and Escherichia coli soluble pyrophosphatases indicated a remarkably conserved organization of the hydrophobic protein domains. The enzymatic function of the potato protein could be deduced from the presence of amino acid residues highly conserved in soluble pyrophosphatases and was confirmed by its capacity to complement a thermosensitive pyrophosphatase mutation in E. coli. The potato polypeptide was purified from complemented bacterial cells and its pyrophosphatase activity was shown to be strictly dependent on Mg2+ and strongly inhibited by Ca2+. The subcellular location of the potato pyrophosphatase is unknown. Structure analysis of the N-terminal protein domain failed to recognize typical transit peptides and the calculated molecular mass of the polypeptide (24 kD) is significantly inferior to the values reported for the plastidic (alkaline) or mitochondrial pyrophosphatases in plants (28-42 kD). Two unlinked loci could be mapped by restriction fragment length polymorphism analysis in the potato genome using the full-length cDNA as probe.

Amino Acid Sequence↗

PubMed1995

Association of synthetic peptide fragments of human apolipoprotein A-I with phospholipids.

The sequences of the plasma apolipoproteins have a high degree of internal homology as they contain several 22-mer internal repeats. These amphipathic helical repeats are considered as the structural and functional units of this class of proteins. We proposed that the 22-mer repeats of the plasma apolipoproteins consist of 17-mer helical segments separated by extended beta-strands comprising five amino acid residues with a proline in the center of this segment. These beta-strand segments help reverse the orientation of the consecutive helices of apoA-I, A-IV, and E in a discoidal apolipoprotein-phospholipid complex. In order to support this hypothesis, we synthesized apoA-I fragments consisting of, respectively, one putative helix (residues 166-183), one helix plus a beta-strand (residues 161-183), and a pair of helices separated by a beta-strand (residues 145-183). The structural and lipid-binding properties of these peptides were investigated by turbidity, fluorescence, binding studies with unilamellar phospholipid vesicles, electron microscopy, and circular dichroism measurements. Our data show that one single putative helical segment or one helical segment plus one extended beta-strand do not form stable complexes with phospholipids. The addition of a second adjacent helix has no influence on the lipid affinity of the apoA-I 145-183 peptide compared to the shorter segments but substantially improves the stability of the complexes. The helical content of the peptide increases upon lipid association as observed with apoA-I. The complexes generated with the apoA-I 145-183 peptide appear as discoidal particles by negative staining electron microscopy, with heterogeneous sizes ranging between 250 and 450 A. The relative orientation of the peptide and the phospholipid is the same as in a DMPC/apoA-I complex as the helices are oriented parallel to the acyl chains of the phospholipid. However, the stability of these complexes is significantly lower than that of the corresponding DMPC/apoA-I complexes. The transition temperature, fluidity, and cooperativity of the phospholipid bilayer are only weakly affected by the association with the apoA-I 145-183 peptide. These data suggest that a pair of helical peptides linked through a beta-strand associates more tightly with lipids and can form discoidal lipid-peptide complexes, than a single helix. A comparison with the properties of native apoA-I suggests, however, that the cooperativity between pairs of helices in native apoA-I further contributes to strengthen the lipid-protein association.

Amino Acids↗

PubMed1995

Conformational analysis of the calcium complexes formed by meglitinide analogs.

The conformation of calcium complexes formed with meglitinide and its hypoglycemic analogs KAD-1229, A-4166, S 3075 and repaglinide was analyzed by a semi-empirical procedure, which includes calculation of the molecular hydrophobicity potential. The calcium complexes formed with KAD-1229, A-4166, repaglinide or its enantiomer, displayed favourable attributes to act as ionophores. Such was not the case for the poorly efficient insulinotropic agent meglitinide and the biologically inactive enantiomer of A-4166. The configuration of the calcium complex formed with S 3075 also failed to display the features required for a high ionophoretic capacity.

Benzamides↗

PubMed1994

Orientation of the alpha-helices of apocytochrome c and derived fragments at membrane interfaces, as studied by circular dichroism.

The orientation of the different helical regions of the mitochondrial precursor protein apocytochrome c has been studied using circular dichroism on isolated fragments of this protein associated with oriented films composed of various phospholipids [de Jongh, H. H. J., Goormaghtigh, E., & Killian, J. A. (1994) Biochemistry (preceding article in this issue)]. Both the N and C terminus adopt helical structures in a membrane environment. The middle region can also be helical, but only in the presence of the N-terminal domain of the protein. In the presence of the unsaturated lipids dioleoylphosphatidylcholine and dioleoylphosphatidylglycerol, all three helices are found to have a preferred orientation perpendicular to the membrane normal, whereas in the presence of the saturated lipids dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol, the terminal helices are preferentially oriented parallel to the membrane normal. In films composed of dioleoylphosphatidylserine, it is found that the N-terminal helix is oriented preferentially perpendicular, whereas the C-terminal helix is aligned more parallel to the membrane normal. The differences in preferred orientation between the terminal helices are demonstrated by molecular modeling of the helices at a water-lipid interface. The results are discussed in light of the translocation of apocytochrome c over the outer mitochondrial membrane, an important step in the import process of this protein in mitochondria.

Animals↗

PubMed1994

Identification of specific amphipathic alpha-helical sequence of human apolipoprotein A-IV involved in lecithin:cholesterol acyltransferase activation.

To investigate the structure-function relationship of human apolipoprotein A-IV (apoA-IV), several deletion mutants of this protein were constructed by sequentially removing pairs of 22-residue repeats, potentially having an amphipathic alpha-helical conformation. The mutants, produced as recombinant poly-histidine-tagged apolipoproteins (t-apo) in Escherichia coli, assembled with phosphatidylcholine (i.e. dimyristoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine, or egg lecithin) as did native apoA-IV. Lecithin:cholesterol acyltransferase (LCAT) cofactor function, measured as cholesterol esterification occurring when t-apo-phosphatidylcholine-cholesterol complexes were incubated with purified enzyme, decreased significantly when pairs of repeats between residues 117 and 248 were deleted and most markedly when residues 117-160 were deleted. LCAT cofactor activity decreased by 90 and 75%, respectively, when egg lecithin or palmitoyloleoylphosphatidylcholine was used to form the particles with the delta aa 117-160 mutant. Thus, on the basis of deletion scanning of t-apo, residues 117-160 seem to be involved in the LCAT cofactor function of apoA-IV.

Amino Acid Sequence↗

PubMed1994

The topology of the S protein in the yeast-derived hepatitis B surface antigen particles.

Hepatitis B surface antigen particles are highly immunogenic and have been shown to provide a suitable support for the presentation of foreign epitopes. More information about the topology of their constitutive protein, the S (small envelope) protein, is a prerequisite to any rational attempt to replace region of this protein with foreign epitopes without modifying the assembly of the particle. The topology of the S protein within the lipid membrane was investigated here by combining extensive proteolysis of the external protein domains with proteinase K and (FTIR-ATR). The proteolytic hydrolysis of the S protein and the identification of the digestion products allowed characterization of the membrane-protected regions of the protein. FTIR spectra of the digested hepatitis B particles revealed that the peptides associated with the particles are rich in alpha-helix structure. The kinetic of 2H/H exchange provided evidence that a large fraction of the native S protein is poorly accessible to the aqueous medium.

Aminopeptidases↗

PubMed1994

Human apolipoprotein B signal sequence variants confer a secretion-defective phenotype when expressed in yeast.

Hyperlipidemia arises from a disturbance in the balance between production and degradation of lipoprotein particles. Variation in the secretion of human apolipoprotein B (apoB), the major protein component of triglyceride-rich lipoproteins, directly affects this homeostasis. Naturally occurring apoB signal peptide variants (associated with hypertriglyceridemia, altered postprandial lipid metabolism, or atherosclerosis) were investigated for their ability to direct transit through the secretion pathway. Three apoB signal peptide isoforms were fused to the secretory protein, invertase, and expressed in yeast. A deletion or insertion in the hydrophobic core of the signal peptide mediated inefficient translocation into the endoplasmic reticulum and was secretion-defective, relative to the common 27-residue isoform. Additionally, the insertion apoB isoform was observed in yeast to confer a defect in export from the endoplasmic reticulum. Secretion of the apoB signal peptide-invertase fusions responded positively to an inhibitor of calpain type I proteases. These observations suggest that the apoB signal peptide plays a role in determining the levels of apoB degradation and secretion and, thus, hyperlipidemia.

Amino Acid Sequence↗

PubMed1994

Structural domain of apolipoprotein A-I involved in its interaction with cells.

Apolipoprotein A-I (apo A-I) is the major protein constituent of high-density lipoprotein (HDL), the lipoprotein fraction which mediates the reverse cholesterol transport. This apolipoprotein plays an important role in the binding of HDL to cells and participates in the efflux of cellular cholesterol. We have recently compared six different genetic variants of apo A-I and found that the apo A-I (Pro 165-->Arg) mutant is defective in promoting cellular cholesterol efflux from murine adipocytes and peritoneal macrophages and we have proposed that this region of apo A-I may be involved in their interaction with cells. To confirm this hypothesis, four monoclonal antibodies (mAbs) specific for apo A-I were used to study the inhibition of the interaction of palmitoyloleoylphosphatidylcholine (POPC): apoA-I complexes with HeLa cells and adipocytes. Among these antibodies, the apo A-I epitope recognized by the A44 mAb lies in the COOH terminal region (amino acid residues 149-186) including the proposed region. The antibodies A05, and A03 react with residues 25-82, 135-140, respectively and the A11 mAb corresponds to a discontinuous epitope at residues 99-105 and 126-132. Our results show clearly that the A44 and A05 mAbs reduce both the binding to HeLa cells and the cholesterol efflux from adipocytes. The inhibition of POPC: apoA-I complexes binding to both cell types is more strictly observed with the Fab fragments of monoclonal antibodies A44 and A05. Partial cotitration curves of these mAbs in a solid phase assay (RIA), indicated partial competition between these two antibodies. We propose a structural model for the POPC: apoA-I complexes where the N-terminal domain of one apo A-I molecule is in close spatial relationship with the C-terminal domain of the adjacent apo A-I molecule. We therefore suggest that the domain around amino acid 165 of apo A-I and which is recognized by mAb A44 (149-186) forms or contains some specific regions which mediate selectively the interaction with the binding site of cells and is involved in the efflux of cellular cholesterol.

Adipocytes↗

PubMed1994

Common prevalence of alanine and glycine in mobile reactive centre loops of serpins and viral fusion peptides: do prions possess a fusion peptide?

Serpin reactive centre loops and fusion peptides released by proteolytic cleavage are particularly mobile. Their amino acid compositions reveal a common and unusual abundance of alanine, accompanied by high levels of glycine. These two small residues, which are not simultaneously abundant in stable helices (standard or transmembrane), probably play an important role in mobility. Threonine and valine (also relatively small amino acids) are also abundant in these two kinds of peptides. Moreover, the known 3D structures of an uncleaved serpin reactive centre and a fusion peptide are strikingly similar. Such sequences possess many small residues and are found in several signal peptides and in PrP, a protein associated with spongiform encephalopathies and resembling virus envelope proteins. These properties may be related to the infection mechanisms of these diseases.

Alanine↗

PubMed1994

Structure and orientation of apo B-100 peptides into a lipid bilayer.

Peptides corresponding to lipid binding domains of Apo B-100 were synthesized, purified, and incubated with dimyristoylphosphatidylcholine (DMPC) liposomes. The secondary structure of the apo B-100 peptide-lipid complexes was evaluated by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Those peptides belonging to the hydrophobic "core" domain of apo B-100 when associated with phospholipids were rich in beta sheet structure; a predominant alpha helical conformation was shown to be associated with one peptide located in a surface region of apo B-100. IR dichroic spectra revealed, in the case of the "core" peptides, that the beta sheet component is the only oriented structure with respect to the phospholipid acyl chains. This orientation of the beta sheet was recently found in LDL particles after proteolytic digestion by trypsin (Goormaghtigh, E., Cabiaux, V., De Meutter, J., Rosseneu, M., and Ruysschaert, J. M., 1993, Biochemistry 32, 6104-6110). Altogether, the data suggest that beta sheet, present in a high proportion in the native apo B-100, is probably another protein structure in addition to the amphipathic helix which strongly interacts with the lipid outer layer surrounding the LDL particle.

Apolipoprotein B-100↗

PubMed1994

WinMGM: a fast CPK molecular graphics program for analyzing molecular structure.

A molecular modeling program is presented which has been written for Microsoft windows 3.1 and Windows NT operating systems. The program permits interactive molecular manipulation and also provides analytical tools such as energy computations and solvent accessible surfaces. An extremely fast algorithm is used which generates realistic space-filling CPK images in addition to wire frame, ribbons, MIDAS, labels, and points. An important feature of this algorithm is a highly optimized Z-buffer, which is described.

Algorithms↗

PubMed1994

Topology of diphtheria toxin B fragment inserted in lipid vesicles.

Diphtheria toxin (DT) is a bacterial protein that crosses the membrane of endosomes of target cells in response to the low endosomal pH. In this paper, we have inserted diphtheria toxin in asolectin vesicles at pH 5.0 and treated the reconstituted system with pronase. The peptides that were protected from digestion were separated by gel electrophoresis, transferred to a membrane and their N-terminal sequences were determined. All peptides belong to the B fragment of DT and cover residues 194-223, 265-375 and 429-528. The secondary structures of the peptides inserted in the membrane, determined by Fourier-transformed infrared spectroscopy, were shown to be mostly alpha-helices and beta-sheets (44% and 53%, respectively). On the basis of these data and the recently published X-ray structure of DT, we are proposing a topology for the DTB fragment in the membrane.

Amino Acid Sequence↗

PubMed1994

Correlation between fusogenicity of synthetic modified peptides corresponding to the NH2-terminal extremity of simian immunodeficiency virus gp32 and their mode of insertion into the lipid bilayer: an infrared spectroscopy study.

The amino-terminal extremity of the simian immunodeficiency virus (SIV) transmembrane protein (gp32) has been shown to play a pivotal role in cell-virus fusion and syncytium formation. We provide here evidence of a correlation between the structure and orientation of the modified SIV fusion peptide after insertion into the lipid membrane and its fusogenic activity. The sequence of the wild-type SIV peptide has been modified in such a way that the calculated angles of insertion correspond to an oblique, parallel, or normal orientation with respect to the lipid-water interface. Fourier transform infrared spectroscopy was used to gain experimental informations about the structures and orientations, of the membrane-inserted peptides with respect to the lipid acyl chains. The peptides adopt mainly a beta-sheet conformation in the absence of lipids. After interaction with large unilamellar liposomes, this beta sheet is partly converted into alpha helix. The ability of the modified peptides to promote lipid mixing was assessed by a fluorescence energy transfer assay. The data provide evidence that alpha-helix formation is not sufficient to induce lipid mixing and that the fusogenic activity of the peptide depends on its orientation in the lipid bilayer.

Amino Acid Sequence↗