Abstract:In a previous work, we predicted and demonstrated that the 29 -42-residue fragment of ␤-amyloid peptide (A␤ peptide) has in vitro capacities close to those of the tilted fragment of viral fusion proteins. We further demonstrated that apolipoprotein E2 and E3 but not apolipoprotein E4 can decrease the fusogenic activity of A␤(29 -42) via a direct interaction. Therefore, we suggested that this fragment is implicated in the neurotoxicity of A␤ and in the protective effects of apolipoprotein E in Alzheimer's disease. Because structurally related apolipoproteins do not interact with the A␤ C-terminal domain but inhibit viral fusion, we suggested that interactions existing between fusogenic peptides and apolipoproteins are selective and responsible for the inhibition of fusion. In this study, we simulated interactions of all amphipathic helices of apolipoproteins E and A-I with A␤ and simian immunodeficiency virus (SIV) fusogenic fragments by molecular modeling. We further calculated cross-interactions that do not inhibit fusion in vitro. The results suggest that interactions of hydrophobic residues are the major event to inhibit the fusogenic capacities of A␤(29 -42) and SIV peptides. Selectivity of those interactions is due to the steric complementarity between bulky hydrophobic residues in the fusogenic fragments and hydrophobic residues in the apolipoprotein C-terminal amphipathic helices.

The classical short-term effect (within minutes) of arginine vasopressin (AVP) consists in increasing sodium, chloride and water transport in kidney cells. More recently, long-term actions (several hours) of the hormone have been evidenced on water and sodium fluxes, due to transcriptional enhancement in the expression of their transporters. The present study demonstrates that AVP is also responsible for a long-term increase in net chloride secretion. In the RCCD 1 rat cortical collecting duct cell line, 10 38 M AVP induced, after several hours, an increase in net 36 Cl 3 secretion. This delayed effect of AVP was inhibited by basal addition of 10 34 M bumetanide and apical addition of 10 34 M glibenclamide, suggesting chloride entry at the basal membrane through a Na +

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.

Attempts to engineer enzymes with unique catalytic properties have largely focused on altering the existing specificities by reshaping the substrate binding pockets. Few experiments have aimed at modifying the configuration of the residues essential for catalysis. The difference in the topological location of the triad acids of Geotrichum candidum lipase (GCL) and the catalytic domain of human pancreatic lipase (HPL), despite great similarities in their topologies and 3-D structures, suggest that these are related enzymes whose catalytic triads have been rearranged in the course of evolution (Schrag et al., 1992). In this study we prepared a double mutant GCL in which the catalytic triad acid is shifted to the position equivalent to the location of the triad acid of HPL. The double mutant maintains approximately 10% of the wild type activity against triglycerides and the fluorogenic ester 4-methylumbelliferyl-oleate. The only significant differences between the 3-D structures of the double mutant and wild type GCL are at the mutated sites. Even the water structure in the region of the triad is unchanged. The hydrogen bonding pattern of the catalytic triad of the double mutant is very similar to that of pancreatic lipase. The acid of the double mutant is stabilized by only two hydrogen bonds, whereas three hydrogen bonds are observed in the wild type enzyme. These results strongly support the hypothesis that the pancreatic lipases are evolutionary switchpoints between the two observed arrangements of the catalytic triads supported by the alpha/beta hydrolase fold and suggest that this fold provides a stable protein core for engineering enzymes with unique catalytic properties.

Among different ab initio approaches to calculate 3D-structures of proteins out of primary sequences, a few are using restricted dihedral spaces and empirical equations of energy as is OSIRIS. All those approaches were calibrated on a few proteins or fragments of proteins. To optimize the calculation over a larger diversity of structures, we need first to define for each sequence what are good conditions of calculations in order to choose a consensus procedure fitting most 3D-structures best. This requires objective classification of calculated 3D-structures. In this work, populations of avian and bovine pancreatic polypeptides (APP, BPP) and of calcium-binding protein (CaBP) are obtained by varying the rate of the angular dynamics of the second step of OSIRIS. Then, 3D-structures are clustered using a nonhierarchical method, SICLA, using rmsd as a distance parameter. A good clustering was obtained for four subpopulations of APP, BPP and CaBP. Each subpopulation was characterized by its barycenter, relative frequency and dispersion. For the three alpha-helix proteins, after the step 1 of OSIRIS, most secondary structures were correct but molecules have a few atomic contacts. Step 2, i.e., the angular dynamics, resolves those atomic contacts and clustering demonstrates that it generates subpopulations of topological conformers as the barycenter topologies show.