Alain J. P. Alix

Predictive estimation of protein linear epitopes by using the program PEOPLE

A single small segment (sequence recognition) or domain (conformation recognition) of a protein could act as an antigen (antigenic determinant) vs an antibody. Epitopes of the first kind being a continuous segment along the sequence (linear), generally bent with a typical non-ordered structure (turns and/or loops), can be predicted from the only knowledge of the primary structure. After reviewing the different algorithms, we present PEOPLE (Predictive Estimation Of Protein Linear Epitopes) which uses combined prediction methods, taking into account the basic fundamental properties corresponding to what should be an ideal epitope: bent (secondary structure mainly beta-turns), surface accessible, hydrophilic and mobile and/or flexible. Four classes of basic biophysical parameters are considered for the determination of an antigenic index AG - secondary structure; hydrophilicity; surface accessibility; flexibility. The AG index is finally defined as a linear combination of the four class profiles. Typical applications are presented.

Fast determination of the quantitative secondary structure of proteins by using some parameters of the Raman Amide I band

Abstract A very simple and fast method is presented for the quantitative determination of the secondary structural contents of a protein by Raman spectroscopy. We performed a statistical analysis of the correlations between the structural data on the one hand, obtained from X-ray crystallography, and the spectroscopic Raman data on the other, this has been for a large set of reference proteins. Such multi-parametric analysis permits one to express the percentages of structural contents of a protein in terms of some simple parameters of the experimental Raman Amide I band of that protein (maximum intensity frequency, width …). For instance, in a single parameter analysis, it was shown that the secondary structures can be estimated with accuracy from the knowledge of only the frequency of the peak of the Raman Amide I band. This, by using equations of the type: % structure = a ν(maximum) + b. a and b are coefficients calculated for each class of structure by using the least squares method. The best agreement between observed and calculated structures is obtained when using as parameters the frequency of the peak and both the left and right half band widths of the Amide I band.

High accuracy prediction of β-turns and their types using propensities and multiple alignments

We have developed a method that predicts both the presence and the type of β‐turns, using a straightforward approach based on propensities and multiple alignments. The propensities were calculated classically, but the way to use them for prediction was completely new: starting from a tetrapeptide sequence on which one wants to evaluate the presence of a β‐turn, the propensity for a given residue is modified by taking into account all the residues present in the multiple alignment at this position. The evaluation of a score is then done by weighting these propensities by the use of Position‐specific score matrices generated by PSI‐BLAST. The introduction of secondary structure information predicted by PSIPRED or SSPRO2 as well as taking into account the flanking residues around the tetrapeptide improved the accuracy greatly. This latter evaluated on a database of 426 reference proteins (previously used on other studies) by a sevenfold crossvalidation gave very good results with a Matthews Correlation Coefficient (MCC) of 0.42 and an overall prediction accuracy of 74.8%; this places our method among the best ones. A jackknife test was also done, which gave results within the same range. This shows that it is possible to reach neural networks accuracy with considerably less computional cost and complexity. Furthermore, propensities remain excellent descriptors of amino acid tendencies to belong to β‐turns, which can be useful for peptide or protein engineering and design. For β‐turn type prediction, we reached the best accuracy ever published in terms of MCC (except for the irregular type IV) in the range of 0.25–0.30 for types I, II, and I′ and 0.13–0.15 for types VIII, II′, and IV. To our knowledge, our method is the only one available on the Web that predicts types I′ and II′. The accuracy evaluated on two larger databases of 547 and 823 proteins was not improved significantly. All of this was implemented into a Web server called COUDES (French acronym for: Chercher Où Une Déviation Existe Sûrement), which is available at the following URL: http://bioserv.rpbs.jussieu.fr/Coudes/index.html within the new bioinformatics platform RPBS. Proteins 2005;59.

The structures of elastins and their function

Elastin structures and their significance towards elastic recoil properties have been reviewed. Starting from the initial hypothesis that elastin conformation is conditioned by that of its monomer, the structure of tropoelastin was first described using theoretical and experimental methods and a beta class folding type was evidenced for the isolated unbound tropoelastin molecules. The structure of elastin in the solid state was consistent with that of its monomer and consequently, fibrous elastin appeared constituted of globular tropoelastin molecules. Finally, theoretical and experimental considerations have led us to the conclusion that the functional form of the elastomer, water swollen elastin, could be a triphasic system comprising the protein chains, hydration water and solvent water. Following this description, the dynamic structural equilibria occurring within elastin hydrophobic domains and the plasticizing effect of water could explain elastin elasticity, in keeping with a classical entropic mechanism.

BOVINE ELASTIN AND KAPPA -ELASTIN SECONDARY STRUCTURE DETERMINATION BY OPTICAL SPECTROSCOPIES

Elastin is the macromolecular polymer of tropoelastin molecules responsible for the elastic properties of tissues. The understanding of its specific elasticity is uncertain because its structure is still unknown. Here, we report the first experimental quantitative determination of bovine elastin secondary structures as well as those of its corresponding soluble κ-elastin. Using circular dichroism and Fourier transform infrared and near infrared Fourier transform Raman spectroscopic data, we estimated the secondary structure contents of elastin to be ∼10% α-helices, ∼45% β-sheets, and ∼45% undefined conformations. These values were very close to those we had previously determined for the free monomeric tropoelastin molecule, suggesting thus that elastin would be constituted of a closely packed assembly of globular β structural class tropoelastin molecules cross-linked to form the elastic network (liquid drop model of elastin architecture). The presence of a strong hydration shell is demonstrated for elastin, and its possible contribution to elasticity is discussed.

The Antitumor Properties of the α3(IV)-(185-203) Peptide from the NC1 Domain of Type IV Collagen (Tumstatin) Are Conformation-dependent

Tumor progression may be controlled by various fragments derived from noncollagenous 1 (NC1) C-terminal domains of type IV collagen. We demonstrated previously that a peptide sequence from the NC1 domain of the α3(IV) collagen chain inhibits the in vitro expression of matrix metalloproteinases in human melanoma cells through RGD-independent binding to αvβ3 integrin. In the present paper, we demonstrate that in a mouse melanoma model, the NC1 α3(IV)-(185-203) peptide inhibits in vivo tumor growth in a conformation-dependent manner. The decrease of tumor growth is the result of an inhibition of cell proliferation and a decrease of cell invasive properties by down-regulation of proteolytic cascades, mainly matrix metalloproteinases and the plasminogen activation system. A shorter peptide comprising the seven N-terminal residues 185-191 (CNYYSNS) shares the same inhibitory profile. The three-dimensional structures of the CNYYSNS and NC1 α3(IV)-(185-203) peptides show a β-turn at the YSNS (188-191) sequence level, which is crucial for biological activity. As well, the homologous MNYYSNS heptapeptide keeps the β-turn and the inhibitory activity. In contrast, the DNYYSNS heptapeptide, which does not form the β-turn at the YSNS level, is devoid of inhibitory activity. Structural studies indicate a strong structure-function relationship of the peptides and point to the YSNS turn as necessary for biological activity. These peptides could act as potent and specific antitumor antagonists of αvβ3 integrin in melanoma progression.

Spectroscopic and Biochemical Characterisation of Self-Aggregates Formed by Antitumor Drugs of the Camptothecin Family: Their Possible Role In the Unique Mode of Drug Action

We describe the effect strongly influencing the biological activity of some camptothecin (CPT) drugs, the inhibitors of DNA topoisomerase I (topo I), namely, the formation of J-type aggregates in an aqueous buffer solution. These aggregates were built up under certain dilution conditions of the stock DMSO solutions of 20-S-camptothecin (20(S)CPT), 10,11-methylenedioxy-CPT (10,11-CPT) and 7-ethyl-10-hydroxy-CPT (SN38). The aggregates were found to be stereospecific, not being detectable for the 20(R)-stereoisomer of CPT. They were formed by the stacking interaction between quinoline rings of CPT chromophores with the inverse position of the nitrogen atoms. The aggregates were stable at acidic and neutral pHs, but dissociated at basic pHs. Self-aggregation prevented hydrolysis of the lactone ring at neutral pHs, thus preserving the drugs in a biologically active form. Addition of BSA did not induce either disaggregation or hydrolysis of the lactone ring, whereas the monomeric form of the drugs was shown to undergo rapid conversion to an inactive carboxylate form in the presence of human serum albumin [5]. The drugs did not form the aggregates in the presence of topo I. Moreover, rapid dissociation of the aggregates was observed if a self-aggregated drug solution was added to topo I alone or to the DNA-topo I cleavage assay. Neither DNA alone nor oligonucleotides derived from the sequences of the CPT-enhanced or topo I-induced cleavage sites in SV40 plasmid DNA induces changes in the aggregation state of the drugs. These observations are indicative of interaction between the aggregates and topo I. The aggregates were found to penetrate within the cells with much higher efficiency than a monomeric form of the drugs. Cellular uptake of aggregated and nonaggregated species correlated well with cytotoxic effects produced by the drug. In this manner, CPTs self-aggregation should be regarded as a favourable phenomenon producing species with a more stable biologically active structure of the lactone ring and exhibiting enhanced cellular uptake levels relative to the monomeric forms of medications.

Camptothecin-binding site in human serum albumin and protein transformations induced by drug binding

Circular dichroism (CD) and Raman spectroscopy were employed in order to locate a camptothecin (CPT)‐binding site within human serum albumin (HSA) and to identify protein structural transformations induced by CPT binding. A competitive binding of CPT and 3′‐azido‐3′‐deoxythymidine (a ligand occupying IIIA structural sub‐domain of the protein) to HSA does not show any competition and demonstrates that the ligands are located in the different binding sites, whereas a HSA‐bound CPT may be replaced by warfarin, occupying IIA structural sub‐domain of the protein. Raman and CD spectra of HSA and HSA/CPT complexes show that the CPT‐binding does not induce changes of the global protein secondary structure. On the other hand, Raman spectra reveal pronounced CPT‐induced local structural modifications of the HSA molecule, involving changes in configuration of the two disulfide bonds and transfer of a single Trp‐residue to hydrophilic environment. These data suggest that CPT is bound in the region of inter‐domain connections within the IIA structural domain of HSA and it induces relative movement of the protein structural domains.

Predictions of the secondary structure and antigenicity of human and bovine tropoelastins

Secondary structure and antigenicity predictive methods have been applied to the sequences of human and bovine tropoelastins in order to have some insight into the molecular structure of its insoluble counterpart, i.e., elastin. For both tropoelastins, all the predictions yielded 11 major regions, in which the pleated conformation was predominant, separated by 10 strong helical segments of various lengths located within alanyl rich regions of the chains. The overall conformations of human and bovine tropoelastins were estimated to contain 18 ± 5% α-helices, 63 ± 17% β-sheets, 13 ± 13% β-turns and 6 ± 6% random coil. For both tropoelastins, antigenicity predictions indicated the presence of seven synthetic decapeptides corresponding to continuous linear epitopes of the molecule. Some of the predicted epitopes are located in the same regions in both species while others are not. These predictions have allowed us to propose an α/β conformation for tropoelastin. Therefore this extracellular matrix macromolecule might be more structured (10 helical segments for about 18% of the overall structure) than previously suggested.

Optical spectroscopic determination of bovine tropoelastin molecular model

Abstract Aqueous solution (CD in water) and solid state (FT-IR on a ZnSe window) spectra of bovine tropoelastin (BTPE) have been recorded and analysed in order to get additional insights into the molecular structure of its elastic elastin polymer. Conformational analyses evidenced high levels of both ordered and unordered secondary structures in the solid and solution states. The structural contents of BTPE were estimated to : α -helices ∼ 10 %, β -sheets ∼ 30 %, β -turns ∼ 20 % and coil ∼ 40 % (Kabsch and Sander definition). Moreover, CD experiments showed the possibility for the molecule to be folded in an all-β configuration with short and / or distorted sheets. As this structural features were formerly predicted for BTPE, our experimental data allowed us to suggest a similar folding for monomeric elastin. Our all-β structural proposal for BTPE is discussed according to bovine elastin structure-elasticity relationship.