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Biomedical subjects

André Ménez

Publications and source records attributed to André Ménez.

At least 19 recordsLinked to original sources
PubMed2007

The venom of the snake genus Atheris contains a new class of peptides with clusters of histidine and glycine residues.

We investigated venoms from members of the genus Atheris (Serpentes, Viperidae), namely the rough scale bush viper (Atheris squamigera), the green bush viper (A. chlorechis) and the great lakes bush viper (A. nitschei), using mass spectrometry-based strategies, relying on matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionisation tandem mass spectrometry (ESI-MS/MS) with de novo peptide sequencing. We discovered a set of novel peptides with masses in the 2-3 kDa range and containing poly-His and poly-Gly segments (pHpG). Complete primary structural elucidation and confirmation of two sequences by Edman degradation indicated the consensus sequence EDDH(9)GVG(10). Bioinformatic investigations in protein sequence databanks did not show relevant homology with known peptides or proteins. However, a more extensive investigation of data in nucleic acid databases revealed some similarities to the precursor sequences of bradykinin potentiating peptides (BPP) and C-type natriuretic peptides (CNP), agents that are known to affect the cardiovascular system by acting on specific metalloproteases and receptors. The novel pHpG peptides found in Atheris venoms might also act on the cardiovascular system by inhibiting particular metalloproteases, which however remain to be identified.

Amino Acid Sequence↗
PubMed2006

Prediction of CD4(+) T cell epitopes restricted to HLA-DP4 molecules.

We have set up a method to predict peptide binding to HLA-DP4 molecules. These HLA II molecules are the most frequent worldwide and hence are an interesting target for epitope-based vaccines. The prediction is based on quantitative matrices built with binding data for peptides substituted at anchoring positions for HLA-DP4. A set of 98 peptides of various origins was used to compare the prediction with binding activity. At different prediction thresholds, the positive predictive value and the sensitivity of the prediction ranged from 50% to 80%, demonstrating its efficiency. This prediction method can be applied to the entire genomes of pathogens and large peptide sequences derived from tumor antigens.

Amino Acid Sequence↗
PubMed2006

Selective identification of HLA-DP4 binding T cell epitopes encoded by the MAGE-A gene family.

Because of the high frequency of HLA-DP4 in the Caucasian population, we have selectively delineated HLA-DP4 restricted T cell epitopes in the MAGE-A tumor antigens. We identified 12 good binders to HLA-DP4 and investigated the capacity of the seven best binders to induce in vitro specific CD4+ T cell lines from HLA-DP4 healthy donors. We found that the MAGE-A1 90-104 peptide exhibited a high and constant frequency of CD4+ T cell precursors in all the six tested donors. The MAGE-A1 268-282 peptide was found immunogenic in only two donors but with a high precursor frequency. The MAGE-A12 127-141 peptide was T cell stimulating in six different donors and induced fewer T cell lines. The peptide-specific T cell lines were stimulated by DC loaded with the lysates of cells transfected with MAGE-A1 or MAGE-A12, or loaded with the recombinant protein. We also show that the immunoreactivity of CD4+ T cell epitopes restricted to the same HLA II molecule may vary from one individual to another, as a result of inter-individual variations in the CD4+ T cell repertoire.

Animals↗
PubMed2006

Denmotoxin, a three-finger toxin from the colubrid snake Boiga dendrophila (Mangrove Catsnake) with bird-specific activity.

Boiga dendrophila (mangrove catsnake) is a colubrid snake that lives in Southeast Asian lowland rainforests and mangrove swamps and that preys primarily on birds. We have isolated, purified, and sequenced a novel toxin from its venom, which we named denmotoxin. It is a monomeric polypeptide of 77 amino acid residues with five disulfide bridges. In organ bath experiments, it displayed potent postsynaptic neuromuscular activity and irreversibly inhibited indirectly stimulated twitches in chick biventer cervicis nerve-muscle preparations. In contrast, it induced much smaller and readily reversible inhibition of electrically induced twitches in mouse hemidiaphragm nerve-muscle preparations. More precisely, the chick muscle alpha(1)betagammadelta-nicotinic acetylcholine receptor was 100-fold more susceptible compared with the mouse receptor. These data indicate that denmotoxin has a bird-specific postsynaptic activity. We chemically synthesized denmotoxin, crystallized it, and solved its crystal structure at 1.9 A by the molecular replacement method. The toxin structure adopts a non-conventional three-finger fold with an additional (fifth) disulfide bond in the first loop and seven additional residues at its N terminus, which is blocked by a pyroglutamic acid residue. This is the first crystal structure of a three-finger toxin from colubrid snake venom and the first fully characterized bird-specific toxin. Denmotoxin illustrates the relationship between toxin specificity and the primary prey type that constitutes the snake's diet.

Amino Acid Sequence↗
PubMed2006

Scanning the HIV genome for CD4+ T cell epitopes restricted to HLA-DP4, the most prevalent HLA class II molecule.

HLA-DP4 alleles are carried by 75% of individuals and are the most frequent HLA II alleles worldwide. Because we have recently characterized the peptide-binding specificity of HLA-DP4 molecules, we developed a peptide-binding prediction method to identify HLA-DP4-restricted peptides in multiple Ags. CD4(+) T cell response plays a key role in the immune control of HIV infection, but few HIV-specific T cell epitopes with multi-individual specificity have been identified. They are mostly restricted to HLA-DR molecules, which are very polymorphic molecules. We therefore looked for HLA-DP4-restricted CD4(+) T cell epitopes in the whole genome of HIV. Twenty-one peptides were selected from the HXB2 HIV genome based on the prediction of binding to HLA-DP4 molecules. They were submitted to HLA-DP4-binding assays. Seventeen peptides bound to the HLA-DP401 molecule, whereas 15 peptides bound to HLA-DP402. Six peptides bound very tightly to HLA-DP401 and were investigated for their capacity to induce specific CD4(+) T cell lines in vitro using dendritic cells and CD4(+) T cells collected from eight seronegative HLA-DP4(+) donors. Four peptides from env and reverse transcriptase proteins induced in vitro-specific T cell lines restricted to HLA-DP4 molecules. Peptide-induced T cells recognized variants other than the HXB2 sequence and were stimulated by native Ags processed by immature dendritic cells. The reverse transcriptase peptide is present in 65% of the isolated HIV variants. To our knowledge, we describe the first HIV epitopes restricted to HLA-DP4 molecules.

Amino Acid Sequence↗
PubMed2006

Identification of various allosteric interaction sites on M1 muscarinic receptor using 125I-Met35-oxidized muscarinic toxin 7.

Monoiodinated, Met35-oxidized muscarinic toxin 7 (MT7ox) was synthesized, and its affinity constants for free or N-methyl scopolamine (NMS)-occupied hM1 receptor were measured directly by equilibrium and kinetic binding experiments. Identical values were obtained with the two types of assay methods, 14 pM and 0.9 nM in free or NMS-liganded receptor states, respectively, highlighting a strong negative cooperativity between this allosteric toxin and NMS. Identical results were obtained with indirect binding experiments with [3H]NMS using the ternary complex model, clearly demonstrating the reciprocal nature of this cooperativity. Furthermore, the effects of various orthosteric and allosteric agents on the dissociation kinetic of 125I-MT7ox were measured and show that, except for the MT1 toxin, all of the ligands studied [NMS, atropine, gallamine, brucine, tacrine, staurosporine, and (9S,10S,12R)-2,3,9,10,11-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT5720)] interact allosterically with muscarinic toxin 7. Equilibrium binding experiments with 125I-MT7ox and [3H]NMS were conducted to reveal the effects of these ligands on the free receptor, and affinity constants (pKx values) were calculated using the allosteric ternary complex model. Our results suggest that MT7 toxin interacts with hM1 receptor at a specific allosteric site, which may partially overlap those identified previously for "classic" or "atypical" allosteric agents and highlight the potential of this new allosteric tracer in studying allosterism at muscarinic receptors.

Allosteric Regulation↗
PubMed2006

Structural studies of human alkaline phosphatase in complex with strontium: implication for its secondary effect in bones.

Strontium is used in the treatment of osteoporosis as a ranelate compound, and in the treatment of painful scattered bone metastases as isotope. At very high doses and in certain conditions, it can lead to osteomalacia characterized by impairment of bone mineralization. The osteomalacia symptoms resemble those of hypophosphatasia, a rare inherited disorder associated with mutations in the gene encoding for tissue-nonspecific alkaline phosphatase (TNAP). Human alkaline phosphatases have four metal binding sites--two for zinc, one for magnesium, and one for calcium ion--that can be substituted by strontium. Here we present the crystal structure of strontium-substituted human placental alkaline phosphatase (PLAP), a related isozyme of TNAP, in which such replacement can have important physiological implications. The structure shows that strontium substitutes the calcium ion with concomitant modification of the metal coordination. The use of the flexible and polarizable force-field TCPEp (topological and classical polarization effects for proteins) predicts that calcium or strontium has similar interaction energies at the calcium-binding site of PLAP. Since calcium helps stabilize a large area that includes loops 210-228 and 250-297, its substitution by strontium could affect the stability of this region. Energy calculations suggest that only at high doses of strontium, comparable to those found for calcium, can strontium substitute for calcium. Since osteomalacia is observed after ingestion of high doses of strontium, alkaline phosphatase is likely to be one of the targets of strontium, and thus this enzyme might be involved in this disease.

Alkaline Phosphatase↗
PubMed2006

How three-finger-fold toxins interact with various cholinergic receptors.

Three-finger-fold toxins, isolated from various snake venoms, are recognized by high affinity and various specificities by different nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs, respectively) present in peripheral, as well as central, nervous systems (Karlsson et al., 2000; Servent and Ménez, 2001; Nirthanan and Gwee, 2004). The goal of our studies is (1) to identify, at the molecular level, the functional determinants involved in the various interaction profiles of nicotinic or muscarinic toxins on their respective receptors subtypes, (2) to model some of these toxin-receptor complexes using distance constraints obtained from cycle-mutant experiments, and (3) to understand how a unique scaffold (the three-finger fold) is able to support these different functional profiles and how molecular determinants have been selected during the evolution process to create these different specific properties. Finally, these structure/function analyses should be exploited to engineer non-natural peptides with new binding and functional properties useful as pharmacological tools or therapeutic agents.

Animals↗
PubMed2005

HIV-1 Tat raises an adjuvant-free humoral immune response controlled by its core region and its ability to form cysteine-mediated oligomers.

Proteins are poor immunogens that require an adjuvant to raise an immune response. Here we show that the human immunodeficiency virus, type 1 Tat protein possesses an autoadjuvant property, and we have identified the determinants and the molecular events that are associated with this unusual property. Using a series of chemically synthesized Tat101 derivatives, we show that the core region controls the autoadjuvant phenomenon independently of the B-cell recognition and T-cell stimulation that are associated with epitopes respectively located on the N-terminal region and the cysteine-rich region. We also show that cysteine-mediated oligomerization is a key molecular event of the adjuvant-free antibody response. In particular, a Tat dimer formed by the oxidation of two cysteine residues, at position 34 only, raises an adjuvant-free antibody response that is comparable with that observed with the wild-type protein. Unlike the parent protein, the Tat dimer has no transactivating activity and remains homogeneous for several weeks in solution. This construct might be of value for the design of an adjuvant-free Tat-based vaccine. Furthermore, we suggest that the specific autoadjuvanticity determinant of Tat could be used to provide other proteins with adjuvant-free immunogenicity.

Adjuvants, Immunologic↗
PubMed2005

Structural studies of human placental alkaline phosphatase in complex with functional ligands.

The activity of human placental alkaline phosphatase (PLAP) is downregulated by a number of effectors such as l-phenylalanine, an uncompetitive inhibitor, 5'-AMP, an antagonist of the effects of PLAP on fibroblast proliferation and by p-nitrophenyl-phosphonate (PNPPate), a non-hydrolysable substrate analogue. For the first two, such regulation may be linked to its biological function that requires a reduced and better-regulated hydrolytic rate. To understand how such disparate ligands are able to inhibit the enzyme, we solved the structure of the complexes at 1.6A, 1.9A and 1.9A resolution, respectively. These crystal structures are the first of an alkaline phosphatase in complex with organic inhibitors. Of the three inhibitors, only l-Phe and PNPPate bind at the active site hydrophobic pocket, providing structural data on the uncompetitive inhibition process. In contrast, all three ligands interact at a remote peripheral site located 28A from the active site. In order to extend these observations to the other members of the human alkaline phosphatase family, we have modelled the structures of the other human isozymes and compared them to PLAP. This comparison highlights the crucial role played by position 429 at the active site in the modulation of the catalytic process, and suggests that the peripheral binding site may be involved in the functional specialization of the PLAP isozyme.

Adenosine Monophosphate↗
PubMed2005

A variable residue in the pore of Kv1 channels is critical for the high affinity of blockers from sea anemones and scorpions.

Animal toxins are associated with well defined selectivity profiles; however the molecular basis for this property is not understood. To address this issue we refined our previous three-dimensional models of the complex between the sea anemone toxin BgK and the S5-S6 region of Kv1.1 (Gilquin, B., Racape, J., Wrisch, A., Visan, V., Lecoq, A., Grissmer, S., Ménez, A., and Gasparini, S. (2002) J. Biol. Chem. 277, 37406-37413) using a docking procedure that scores and ranks the structures by comparing experimental and back-calculated values of coupling free energies DeltaDeltaGint obtained from double-mutant cycles. These models further highlight the interaction between residue 379 of Kv1.1 and the conserved dyad tyrosine residue of BgK. Because the nature of the residue at position 379 varies from one channel subtype to another, we explored how these natural mutations influence the sensitivity of Kv1 channel subtypes to BgK using binding and electrophysiology experiments. We demonstrated that mutations at this single position indeed suffice to abolish or enhance the sensitivity of Kv1 channels for BgK and other sea anemone and scorpion toxins. Altogether, our data suggest that the residue at position 379 of Kv1 channels controls the affinity of a number of blocking toxins.

Amino Acid Substitution↗
PubMed2005

Fine tuning of the specificity of an anti-progesterone antibody by first and second sphere residue engineering.

The specificity of anti-progesterone P15G12C12G11 antibody was improved by combination of in vitro scanning saturation mutagenesis and error-prone PCR. The most evolved mutant is able to discriminate against 5beta- or 5alpha-dihydroprogesterone, 23 and 15 times better than the starting antibody, while maintaining the affinity for progesterone that remains in the picomolar range. The high level of homology with anti-progesterone monoclonal antibody DB3 allowed the construction of three-dimensional models of P15G12C12G11 based on the structures of DB3 in complex with various steroids. These models together with binding data, derived from site-directed mutagenesis, were used to build a phage library in which five first sphere positions in complementarity-determining regions 2H and 3L were varied. Variants selected by an initial screening in competition against a large excess of 5beta- or 5alpha-dihydroprogesterone were characterized by a convergent amino acid signature different from that of the wild-type antibody and had lower cross-reactivity. Binding properties of this first set of mutants were further improved by the addition of second sphere mutations selected independently from an error-prone library. The three-dimensional models of the best variant show changes in the antigen binding site that explain well the increase in selectivity. The improvements are partly linked to a change in the canonical class of the light chain third hypervariable loop.

5-alpha-Dihydroprogesterone↗
PubMed2005

Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide.

We report the crystal structure of a soluble form of human urokinase-type plasminogen activator receptor (uPAR/CD87), which is expressed at the invasive areas of the tumor-stromal microenvironment in many human cancers. The structure was solved at 2.7 A in association with a competitive peptide inhibitor of the urokinase-type plasminogen activator (uPA)-uPAR interaction. uPAR is composed of three consecutive three-finger domains organized in an almost circular manner, which generates both a deep internal cavity where the peptide binds in a helical conformation, and a large external surface. This knowledge combined with the discovery of a convergent binding motif shared by the antagonist peptide and uPA allowed us to build a model of the human uPA-uPAR complex. This model reveals that the receptor-binding module of uPA engages the uPAR central cavity, thus leaving the external receptor surface accessible for other protein interactions (vitronectin and integrins). By this unique structural assembly, uPAR can orchestrate the fine interplay with the partners that are required to guide uPA-focalized proteolysis on the cell surface and control cell adhesion and migration.

Crystallization↗
PubMed2005

Cross-presentation of a CMV pp65 epitope by human dendritic cells using bee venom PLA2 as a membrane-binding vector.

We have used bee venom phospholipase A2 as a vector to load human dendritic cells ex vivo with a major histocompatibility complex (MHC) class I-restricted epitope fused to its C-terminus. The fusion protein bound to human monocyte-derived dendritic cells and was internalized into early endosomes. In vitro immunization experiments showed that these dendritic cells were able to generate specific CD8 T cell lines against the epitope carried by the fusion protein. Cross-presentation did not require proteasome, transporter associated with antigen processing, or endosome proteases, but required newly synthesized MHC molecules. Comparison of the antigen presentation pathway observed in this study to that followed by other toxins used as vectors is discussed.

Antigen Presentation↗
PubMed2005

Structural features common to intracellularly acting toxins from bacteria.

This mini-review focuses on structural features shared by bacterial intracellularly-acting toxins. These complex proteins adopt an A(n)B(m) assembly. B(m) is a cellular-uptake machinery that delivers the enzymatic A(n) component, where it specifically modifies an intracellular eukaryotic cell target. In this nomenclature, the m index reflects the mono- or oligomeric (homo or hetero) state of the B component and the n index indicates the number of A molecules that concomitantly bind to B(m). A structural analysis of the available 3D structures suggests that each of the A molecules that constitute the A(n) component can be divided into A(link) and A(enz) sub-domains, with A(link) specifically linking the enzymatically active A(enz) domain to a given B(m). This module-based A(n)B(m) assembly seems decisive for natural intracellularly-acting toxins to be potent and for the success of engineered toxins.

Bacterial Toxins↗
PubMed2005

[Critical role of peptidic toxins in the functional and structural analysis of nicotinic acetylcholine receptors].

Animal toxins which interact on various receptors and channels have been often used in the studies of the functional roles of these targets. Nicotinic toxins have been purified from snake and cone venoms and are characterized by high affinity and various selectivity of interactions on the different nicotinic receptors subtypes. Since 30 years they have been used as molecular probes to identify, localize and purify these receptors. Furthermore, they have played a crucial role in the better understanding of their functional properties and have been useful in their structural studies. These peptidic toxins could be chemically synthetized or recombinantly expressed and nonnatural residues could be introduced in their sequences in order to delineate their functional interaction sites. The structural modelisation of toxin-nAChR interaction allows us to understand the antagonistic property of these toxins and open the way to the design of engineered ligands with predetermined specificity, useful as pharmacological tools or therapeutic agents in the numerous diseases involving this receptor family.

Animals↗