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A J Alix

Publications and source records attributed to A J Alix.

At least 19 recordsLinked to original sources
PubMed2001

[A turning point in the knowledge of the structure-function-activity relations of elastin].

In this review are presented the last new results of our research group dealing with the molecular structures (atomic level) of tropoelastin, elastin and elastin derived peptides studied by using essentially methods of bioinformatics (theoretical predictions and molecular modelling) linked to experimental circular dichroism spectroscopic studies. We already had characterized both the local secondary structure and some parts of the tertiary structure of the tropoelastin and elastin molecules (human, bovine...), by using either theoretical predictions (local secondary structure, linear epitopes...) and/or experimental data (optical spectroscopic methods: Raman scattering, infrared absorption, circular dichroism). Except the cross-linking regions which are in helical conformations, the whole tropoelastin structure displays a lot of beta-reverse turns which usually belong to irregular structures in proteins. These turns play a key role in other regularly structures orientation (alpha-helix, beta-strand), thus they are very important in the native protein 3D architecture. It is particularly true for human tropoelastin, because its sequence is rich in glycines and prolines, and these residues are frequently met in beta-turns (a beta-turn is made of four consecutive residues which are stabilized by an hydrogen bond). Several types of beta-turns can be defined with the dihedral angles values phi and psi of the two central residues. Thus, by using a very recent updated set of propensities for the amino acid residues to belong to given types of reverse beta-turns (extracted from a reference set of known 3-D structures of globular proteins), we have determined, (by using our home made software COUDES), for all possible tetrapeptides of the human tropoelastin sequence, the distribution and the characterization of the possible type of turns. Thus, it is shown that the locations and/or the types of these reverse beta-turns reveal a regularity and are not all random. This confirms our hypothesis that intra-molecular elasticity of tropoelastin could be explained by the possibility of transitions between conformations involving short beta-strands and beta-turns. This result is of great interest in the construction (by using molecular biology) of elastic biomaterials derived from the elastin sequence (particularly, the elastin derived peptides corresponding to the sequence exon 21--(exon 24--exon 24...). Our study permit also to predict the conformations of specific elastin derived peptides which could have interesting biological activity. Peptides resulting from the degradation of elastin, the insoluble polymer of tropoelastin and responsible for the elasticity of vertebrate tissues, can induce biological effects and notably the regulation of matrix metalloproteinases (MMP-s) activity. Recently, it was proposed that some elastin derived hexapeptides resulting from circular permutations of VGVAPG (a three fold repetition sequence in exon 24 of human tropoelastin) possess MMP-1 production and activation regulation properties. This effect depends on the presence of the tropoelastin specific membraneous receptor 67 KDa EBP (Elastin Binding Protein). Our results obtained by using both circular dichroism spectroscopy and linear predictions confirmed the hypothesis of a structure dependent mechanism with a possibly occurring type VIII beta-turn on the first four residues of the GXXPG sequence consensus which is only present among all active peptides. Thus, we have performed extensive molecular dynamics studies, in both implicit and explicit solvent, on these active and inactive elastin derived hexapeptides. Using our own analysis method of pattern recognition of the types of the beta-reverse-turns followed during the molecular dynamics trajectory, we found that active and inactive peptides effectively form two well distinct conformational groups in which active peptides preferentially adopt conformation close to type VIII GXXP (beta-reverse-turn. The structural role of the C terminal G residue could also be explained. Additional molecular simulations on (VGVAPG)2 and (VGVAPG)3 show the formation of two or three GXXP tetrapeptides adopting a structure close to type VIII beta-reverse-turn, suggesting a local conformational preference for this motif. This observation of a specific structural single and/or repeated motif is in agreement with the circular dichroism spectra of the involved (VGVAPG)1, (VGVAPG)2 and (VGVAPG)3 peptides and then it can be proposed that their biological activities have to be linear. The final aim of this type of work is to understand more about the sequence/structure/function/activity relationships of those structured peptides in order to propose specific sequences (corresponding to specific structures) for best biological activity results.

Amino Acid Sequence↗
PubMed2000

Conformational dependence of collagenase (matrix metalloproteinase-1) up-regulation by elastin peptides in cultured fibroblasts.

We have established that treatment of cultured human skin fibroblasts with tropoelastin or with heterogenic peptides, obtained after organo-alkaline or leukocyte elastase hydrolysis of insoluble elastin, induces a high expression of pro-collagenase-1 (pro-matrix metalloproteinase-1 (pro-MMP-1)). The identical effect was achieved after stimulation with a VGVAPG synthetic peptide, reflecting the elastin-derived domain known to bind to the 67-kDa elastin-binding protein. This clearly indicated involvement of this receptor in the described phenomenon. This notion was further reinforced by the fact that elastin peptides-dependent MMP-1 up-regulation has not been demonstrated in cultures preincubated with 1 mm lactose, which causes shedding of the elastin-binding protein and with pertussis toxin, which blocks the elastin-binding protein-dependent signaling pathway involving G protein, phospholipase C, and protein kinase C. Moreover, we demonstrated that diverse peptides maintaining GXXPG sequences can also induce similar cellular effects as a "principal" VGVAPG ligand of the elastin receptor. Results of our biophysical studies suggest that this peculiar consensus sequence stabilizes a type VIII beta-turn in several similar, but not identical, peptides that maintain a sufficient conformation to be recognized by the elastin receptor. We have also established that GXXPG elastin-derived peptides, in addition to pro-MMP-1, cause up-regulation of pro-matrix metalloproteinase-3 (pro-stromelysin 1). Furthermore, we found that the presence of plasmin in the culture medium activated these MMP proenzymes, leading to a consequent degradation of collagen substrate. Our results may be, therefore, relevant to pathobiology of inflammation, in which elastin-derived peptides bearing the GXXPG conformation (created after leukocyte-dependent proteolysis) bind to the elastin receptor of local fibroblasts and trigger signals leading to expression and activation of MMP-1 and MMP-3, which in turn exacerbate local connective tissue damage.

Cells, Cultured↗
PubMed2000

Molecular determinants of site-specific inhibition of human DNA topoisomerase I by fagaronine and ethoxidine. Relation to DNA binding.

DNA topoisomerase (top) I inhibition activity of the natural alkaloid fagaronine (NSC157995) and its new synthetic derivative ethoxidine (12-ethoxy-benzo[c]phenanthridine) has been correlated with their molecular interactions and sequence specificity within the DNA complexes. Flow linear dichroism shows that ethoxidine exhibits the same inhibition of DNA relaxation as fagaronine at the 10-fold lower concentration. The patterns of DNA cleavage by top I show linear enhancement of CPT-dependent sites at the 0.016-50 microM concentrations of fagaronine, whereas ethoxidine suppress both top I-specific and CPT-dependent sites. Suppression of top I-mediated cleavage by ethoxidine is found to be specific for the sites, including strand cut between A and T. Fagaronine and ethoxidine are DNA major groove intercalators. Ethoxidine intercalates DNA in A-T sequences and its 12-ethoxy-moiety (absent in fagaronine) extends into the DNA minor groove. These findings may explain specificity of suppression by ethoxidine of the strong top I cleavage sites with the A(+1), T(-1) immediately adjacent to the strand cut. Fagaronine does not show any sequence specificity of DNA intercalation, but its highly electronegative oxygen of hydroxy group (absent in ethoxidine) is shown to be an acceptor of the hydrogen bond with the NH(2) group of G base of DNA. Ability of fagaronine to stabilize top I-mediated ternary complex is proposed to be determined by interaction of its hydroxy group with the guanine at position (+1) of the DNA cleavage site and of quaternary nitrogen interaction with top I. The model proposed provides a guidance for screening new top I-targeted drugs in terms of identification of molecular determinants responsible for their top I inhibition effects.

Alkaloids↗
PubMed1999

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.

Animals↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1998

The secondary structure and architecture of human elastin.

The presented work constitutes the first structural characterization of both insoluble human elastin and its solubilized form, kappa-elastin. Structural data were reached following the use of Fourier transform infrared, near infrared Fourier transform Raman and circular dichroism optical spectroscopic methods and their quantitative analysis permitted us to estimate approximately 10% alpha-helices, approximately 35% beta-strands and approximately 55% undefined conformations in the global secondary structure of insoluble human elastin in the solid state. Following the use of the LINK method, the probable local distribution of the secondary-structure elements along the sequence was determined and compared to that obtained for bovine elastin, the historical standard of elastin. This comparison led us to propose a globular architecture for the human elastomer and permitted us to delineate some elements of its structure-elasticity relationship.

Amino Acid Sequence↗
PubMed1998

Raman and CD spectroscopy of recombinant 68-kDa DNA human topoisomerase I and its complex with suicide DNA-substrate.

N-terminally truncated recombinant 68-kDa human topoisomerase (topo) I exhibits the same DNA-driving activities as the wild-type protein. In the present study, Raman and circular dichroism techniques were employed for detailed structural characterization of the 68-kDa human topo I and its transformations induced by the suicide sequence-specific oligonucleotide (solig) binding and cleavage. Spectroscopic data combined with statistical prediction techniques were employed to construct a model of the secondary structure distribution along the primary protein structure in solution. The 68-kDa topo I was found to consist of ca. 59% alpha-helix, 24% beta-strand and/or sheets, and 17% other structures. A secondary structure transition of the 68-kDa topo I was found to accompany solig binding and cleavage. Nearly 15% of the alpha-helix of 68-kDa topo I is transferred within the other structures when in the complex with its DNA substrate. Raman spectroscopy analysis also shows redistribution of the structural rotamers of the 68-kDa topo I disulfide bonds and significant changes in the H-bonding of the Tyr residues and in the microenvironment/conformation of the Trp side chains. No structural modifications of the DNA substrate were detected by spectroscopic techniques. The data presented provide the first direct experimental evidence of the human topo I conformational transition after the cleavage step in the reaction of binding and cleavage of DNA substrate by the enzyme. This evidence supports the model of the enzyme function requiring the protein conformational transition. The most probable location of the enzyme transformations was the core and the C-terminal conservative 68-kDa topo I structural domains. By contrast, the linker domain was found to have an extremely low potential for solig-induced structural transformations. The pattern of redistribution of protein secondary structures induced by solig binding and covalent suicide complex formation supports the model of an intramolecular bipartite mode of topo I/DNA interaction in the substrate binding and cleavage reaction.

Algorithms↗
PubMed1998

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, CPT's 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.

Antineoplastic Agents, Phytogenic↗
PubMed1998

Cloning and expression of an iron-containing superoxide dismutase in the parasitic protist, Trichomonas vaginalis.

A superoxide dismutase (SOD) gene of the parasitic protist Trichomonas vaginalis was cloned, sequenced, expressed in Escherichia coli, and its gene product characterized. It is an iron-containing dimeric protein with a monomeric mass of 22,067 Da. Southern blots analyses suggested the presence of seven iron-containing (FeSOD) gene copies. Hydrophobic cluster analysis revealed some peculiarities in the 2D structure of the FeSOD from T. vaginalis and a strong structural conservation between prokaryotic and eukaryotic FeSODs. Phylogenetic reconstruction of the SOD sequences confirmed the dichotomy between FeSODs and manganese-containing SODs. FeSODs of protists appeared to group together with homologous proteobacterial enzymes suggesting a possible origin of eukaryotic FeSODs through an endosymbiotic event.

Amino Acid Sequence↗
PubMed1997

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 interdomain connections within the IIA structural domain of HSA and it induces relative movement of the protein structural domains.

Binding Sites↗
PubMed1997

Solution structure of R-elafin, a specific inhibitor of elastase.

The solution structure of r-elafin, a specific elastase inhibitor, has been determined using NMR spectroscopy. Characterized by a flat core and a flexible N-terminal extremity, the three-dimensional structure is formed by a central twisted beta-hairpin accompanied by two external segments linked by the proteinase binding loop. A cluster of three disulfide bridges connects the external segments to the central beta-sheet and a single fourth disulfide bridge links the binding loop to the central beta-turn. The same spatial distribution of disulfide bridges can be observed in both domains of the secretory leukocyte protease inhibitor (SLPI), another elastase inhibitor. The structural homology between r-elafin and the C-terminal domain of SLPI confirms the former as a second member of the chelonianin family of proteinase inhibitors. Based on the homology between the two proteins and recent results obtained for elastase binding mutants of the bovine pancreatic trypsin inhibitor (BPTI), we define the segment 22 to 27 as the binding loop of elafin, with the scissile peptide bond between Ala24 and Met25. In our solution structures, this loop is extended and solvent-exposed, and exhibits a large degree of flexibility. This mobility, already observed for the binding loop in other protease inhibitors in solution, might be an important feature for the interaction with the corresponding protease.

Amino Acid Sequence↗
PubMed1995

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 kappa-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 approximately 10% alpha-helices, approximately 45% beta-sheets, and approximately 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 beta 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.

Amino Acids↗
PubMed1995

Detailed assessment of spatial hydrophobic and electrostatic properties of 2D NMR-derived models of neurotoxin II.

2D NMR-derived spatial structures of neurotoxin II (NtII) and several homologous toxins in solution were assessed by comparison with their own amino acid sequences using a three-dimensional (3D) profile method. 3D profiles of all the toxin models match the sequences well and, therefore, the method of 3D profile was demonstrated to work correctly for these well-resolved NMR structures in aqueous solution. At the same time, the profile window plots reveal low scores in the bottom tip of loop II (residues 22-34 in NtII) and in beta-strand of loop III (residues 49-52). Some residues in the first poor-scoring region are of functional importance being involved in binding with nicotinic acetylcholine receptor (AChR). Furthermore, the second segment participates in intermolecular hydrogen bonding upon dimerization of postsynaptic neurotoxins in solution resulting in increasing of the 3D-1D score for residues at the interface between monomers. Therefore, the 3D profile method can be useful for detection functionally-important regions in well-resolved protein structures.

Algorithms↗
PubMed1994

Conformational changes of lipoxygenase (LOX) in modified environments. Contribution to the variation in specificity of soybean LOX type 1.

The addition of water-soluble cosolvents in the reaction medium of type 1 soybean lipoxygenase can modify the selectivity of the enzyme in the hydroperoxide synthesis reaction. This also results in changes in secondary reactions such as carbonyl compound formation. The possibility of a conformational change of the enzyme due to variations in its microenvironment was considered. Using enzyme immobilization and laser visible Raman spectroscopy, both indirect and direct observations of such a protein conformational rearrangement are described. Subtle modifications in the secondary and/or tertiary structures, for example in microenvironments of Tyr and Trp residues, in orientations of lateral side chains were evidenced, and their importance to enzyme specificity is discussed.

Linoleic Acid↗
PubMed1994

Toxoplasma gondii-structure variations of the antigen P30.

The major surface immunodominant antigen (P30) of Toxoplasma gondii was purified by two methods (i) SDS-PAGE and (ii) immunoaffinity chromatography. The secondary elements within this protein were assessed by circular dichroism and spectra obtained were compared to those proposed by Manavalan & Johnson (1983). The results allowed us to determine an all beta protein status for this antigen. This experimental result was in agreement with the predicted secondary structures deduced from the P30 primary sequence. Modifications in conformation according to pH and temperature were recorded without any change in immunoactivity. The epitope, which was always recognized by a monoclonal antibody against P30, could be a linear epitope.

Amino Acid Sequence↗
PubMed1993

Environmental characteristics of residues in proteins: three-dimensional molecular hydrophobicity potential approach.

A new method for quantitative estimation of surrounding hydrophobicity for residues in proteins is proposed. It is based on the formalism of three-dimensional molecular hydrophobicity potential (3D MHP) and includes calculation of 3D MHP created by all atoms in a system "molecule+surrounding water". We calculated 3D MHP in the geometrical centers of residues for 23 proteins (from Protein Data Bank) surrounded by water layers 12-15 A thick. These data, plotted along the protein sequence (1D MHP profiles) contain comprehensive information on protein, water, prosthetic groups and neighbouring domains contributions to residue environment. 1D MHP profiles generated by water molecules strongly correlate with standard accessibility profiles based on calculations of exposed surface areas. The results confirm recently observed interrelation between the resonance Raman cross sections of tryptophan residues and polarity of their microenvironment. 3D MHP contributions (and related 1D MHP profiles) to residue environment from protein atoms, heme in myoglobin, flavin in flavodoxin as well as neighbouring domains in pyruvate kinase were characterized. 3D MHP data for rubredoxin in native and totally extended conformations reveal striking deviations reflecting the influence of long-range interactions between residues far removed in the sequence. Statistical analysis of 3D MHP data yields a new hydrophobicity scale (MHPS) that correlates with several commonly used scales obtained by a variety of complementary techniques.

Algorithms↗
PubMed1992

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% alpha-helices, 63 +/- 17% beta-sheets, 13 +/- 13% beta-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 alpha/beta 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.

Algorithms↗