Mimes peptidiques de glycosaminoglycanes : synthèses, structures et activités biologiques

Glycosaminoglycans (GAGs) are linear polysaccharides, more or less sulfated, present on the surface of cells and in the extracellular matrix. They interact with a large number of proteins (cytokines, growth factors, enzymes, etc.) and are involved in various physiological and pathological processes. As part of my thesis work, we designed and synthesized GAGs mimetic molecules: anionic peptides of 12 to 24 amino acids, carrying repeated motifs (EC'AC ')n and exhibiting the characteristic functions of natural GAGs, namely the acid functions, provided by glutamic acid, and sulfonic functions, obtained by oxidation of cysteines. A first generation of these sulfopeptides was studied, at the Collège de France, as inhibitors of the interaction of a homeodomain protein (Otx2) with the network of GAGs present around a target cell type of this protein. The peptide comprising six "EC'AC" motifs has shown very promising in vivo activity. A structural study by NMR was then carried out to analyze the interactions of these sulfopeptides with the Otx2 protein produced with a double 13C, 15N labeling. This study confirmed that the interaction takes place at the GAG binding domain of the protein. This interaction also affects the homeodomain helix 3, which is responsible for the internalization of Otx2 into cells. Finally, we completed this structural study with a Biacore-type approach, to develop a strategy for faster analysis of the activity of our analogues and allow us to develop an efficient structure-activity study.Les Glycosaminoglycanes (GAGs) sont des polysaccharides linéaires, plus ou moins sulfatés, présents à la surface des cellules et dans la matrice extracellulaire. Ils interagissent avec un grand nombre de protéines (cytokines, facteurs de croissance, enzymes ...) et sont impliqués dans divers processus physiologiques et pathologiques. Dans le cadre de mon travail de thèse, nous avons conçu et synthétisé des molécules mimes de GAGs : des peptides anioniques de 12 à 24 acides aminés, portant des motifs répétés de type (EC’AC’)n et présentant les fonctions caractéristiques des GAGs naturels, à savoir les fonctions acides, apportées par l’acide Glutamique, et sulfoniques, obtenues par oxydation des Cystéines. Une première génération de ces sulfopeptides a été étudiée, au Collège de France, comme inhibiteurs de l’interaction d’une protéine à homéodomaine (Otx2) avec le réseau de GAGs présents autour d’un type cellulaire cible de cette protéine. Le peptide comportant six motifs « EC’AC’» a montré une activité in vivo très prometteuse. Une étude structurale par RMN a ensuite été réalisée pour analyser les interactions de ces sulfopeptides avec la protéine Otx2 produite avec un double marquage 13C, 15N. Cette étude a confirmé que l’interaction se faisait au niveau du domaine de liaison aux GAGs de la protéine. Cette interaction affecte aussi l’hélice 3 de l’homéodomaine, à l’origine de l’internalisation d’Otx2 dans les cellules. Enfin nous avons complété cette étude structurale par une approche de type Biacore, pour développer une stratégie d’analyse plus rapide de l’activité de nos analogues et nous permettre de développer une étude structure-activité efficace

Peptide mimics of glycosaminoglycans : synthesis, structures and biological activities

Les Glycosaminoglycanes (GAGs) sont des polysaccharides linéaires, plus ou moins sulfatés, présents à la surface des cellules et dans la matrice extracellulaire. Ils interagissent avec un grand nombre de protéines (cytokines, facteurs de croissance, enzymes ...) et sont impliqués dans divers processus physiologiques et pathologiques. Dans le cadre de mon travail de thèse, nous avons conçu et synthétisé des molécules mimes de GAGs : des peptides anioniques de 12 à 24 acides aminés, portant des motifs répétés de type (EC’AC’)n et présentant les fonctions caractéristiques des GAGs naturels, à savoir les fonctions acides, apportées par l’acide Glutamique, et sulfoniques, obtenues par oxydation des Cystéines. Une première génération de ces sulfopeptides a été étudiée, au Collège de France, comme inhibiteurs de l’interaction d’une protéine à homéodomaine (Otx2) avec le réseau de GAGs présents autour d’un type cellulaire cible de cette protéine. Le peptide comportant six motifs « EC’AC’» a montré une activité in vivo très prometteuse. Une étude structurale par RMN a ensuite été réalisée pour analyser les interactions de ces sulfopeptides avec la protéine Otx2 produite avec un double marquage 13C, 15N. Cette étude a confirmé que l’interaction se faisait au niveau du domaine de liaison aux GAGs de la protéine. Cette interaction affecte aussi l’hélice 3 de l’homéodomaine, à l’origine de l’internalisation d’Otx2 dans les cellules. Enfin nous avons complété cette étude structurale par une approche de type Biacore, pour développer une stratégie d’analyse plus rapide de l’activité de nos analogues et nous permettre de développer une étude structure-activité efficace.Glycosaminoglycans (GAGs) are linear polysaccharides, more or less sulfated, present on the surface of cells and in the extracellular matrix. They interact with a large number of proteins (cytokines, growth factors, enzymes, etc.) and are involved in various physiological and pathological processes. As part of my thesis work, we designed and synthesized GAGs mimetic molecules: anionic peptides of 12 to 24 amino acids, carrying repeated motifs (EC'AC ')n and exhibiting the characteristic functions of natural GAGs, namely the acid functions, provided by glutamic acid, and sulfonic functions, obtained by oxidation of cysteines. A first generation of these sulfopeptides was studied, at the Collège de France, as inhibitors of the interaction of a homeodomain protein (Otx2) with the network of GAGs present around a target cell type of this protein. The peptide comprising six "EC'AC" motifs has shown very promising in vivo activity. A structural study by NMR was then carried out to analyze the interactions of these sulfopeptides with the Otx2 protein produced with a double 13C, 15N labeling. This study confirmed that the interaction takes place at the GAG binding domain of the protein. This interaction also affects the homeodomain helix 3, which is responsible for the internalization of Otx2 into cells. Finally, we completed this structural study with a Biacore-type approach, to develop a strategy for faster analysis of the activity of our analogues and allow us to develop an efficient structure-activity study

Mimes peptidiques de glycosaminoglycanes : synthèses, structures et activités biologiques

Glycosaminoglycans (GAGs) are linear polysaccharides, more or less sulfated, present on the surface of cells and in the extracellular matrix. They interact with a large number of proteins (cytokines, growth factors, enzymes, etc.) and are involved in various physiological and pathological processes. As part of my thesis work, we designed and synthesized GAGs mimetic molecules: anionic peptides of 12 to 24 amino acids, carrying repeated motifs (EC'AC ')n and exhibiting the characteristic functions of natural GAGs, namely the acid functions, provided by glutamic acid, and sulfonic functions, obtained by oxidation of cysteines. A first generation of these sulfopeptides was studied, at the Collège de France, as inhibitors of the interaction of a homeodomain protein (Otx2) with the network of GAGs present around a target cell type of this protein. The peptide comprising six "EC'AC" motifs has shown very promising in vivo activity. A structural study by NMR was then carried out to analyze the interactions of these sulfopeptides with the Otx2 protein produced with a double 13C, 15N labeling. This study confirmed that the interaction takes place at the GAG binding domain of the protein. This interaction also affects the homeodomain helix 3, which is responsible for the internalization of Otx2 into cells. Finally, we completed this structural study with a Biacore-type approach, to develop a strategy for faster analysis of the activity of our analogues and allow us to develop an efficient structure-activity study.Les Glycosaminoglycanes (GAGs) sont des polysaccharides linéaires, plus ou moins sulfatés, présents à la surface des cellules et dans la matrice extracellulaire. Ils interagissent avec un grand nombre de protéines (cytokines, facteurs de croissance, enzymes ...) et sont impliqués dans divers processus physiologiques et pathologiques. Dans le cadre de mon travail de thèse, nous avons conçu et synthétisé des molécules mimes de GAGs : des peptides anioniques de 12 à 24 acides aminés, portant des motifs répétés de type (EC’AC’)n et présentant les fonctions caractéristiques des GAGs naturels, à savoir les fonctions acides, apportées par l’acide Glutamique, et sulfoniques, obtenues par oxydation des Cystéines. Une première génération de ces sulfopeptides a été étudiée, au Collège de France, comme inhibiteurs de l’interaction d’une protéine à homéodomaine (Otx2) avec le réseau de GAGs présents autour d’un type cellulaire cible de cette protéine. Le peptide comportant six motifs « EC’AC’» a montré une activité in vivo très prometteuse. Une étude structurale par RMN a ensuite été réalisée pour analyser les interactions de ces sulfopeptides avec la protéine Otx2 produite avec un double marquage 13C, 15N. Cette étude a confirmé que l’interaction se faisait au niveau du domaine de liaison aux GAGs de la protéine. Cette interaction affecte aussi l’hélice 3 de l’homéodomaine, à l’origine de l’internalisation d’Otx2 dans les cellules. Enfin nous avons complété cette étude structurale par une approche de type Biacore, pour développer une stratégie d’analyse plus rapide de l’activité de nos analogues et nous permettre de développer une étude structure-activité efficace

The Role of Membranes in the Organization of HIV-1 Gag p6 and Vpr: p6 Shows High Affinity for Membrane Bilayers Which Substantially Increases the Interaction between p6 and Vpr.

International audienceThe molecular mechanism by which HIV-1 Gag proteins are targeted and transported to the plasma membrane after ribosomal synthesis is unknown. In this work, we investigated the potential interaction of p6 and Vpr with model membranes and have determined their binding constants. Plasmon waveguide resonance (PWR) experiments showed that p6 strongly interacts with membranes (K(d) approximately 40 nM), which may help explaining in part why Gag is targeted to and assembles into membranes by coating itself with lipids. Moreover, a substantial increased affinity of Vpr for p6 was observed while in a membrane environment. In order to further investigate the molecular properties behind the high affinity to model membranes, molecular dynamics simulations were carried out for p6 with a dodecylphosphocholine (DPC) micelle. The results indicate an integration route model for Vpr into virions and may help explain why previous reports failed to detect p6 in virion core preparations

Structural Studies of HIV-1 Gag p6ct and Its Interaction with Vpr Determined by Solution Nuclear Magnetic Resonance † , ‡

International audienceThe ability of human immunodeficiency virus type 1 (HIV-1) to egress from human cells by budding with the cell membrane remains a complex phenomenon of unclear steps. HIV-1 viral protein R (Vpr) incorporation in sorting virions relies greatly on the interaction with the group-specific antigen (Gag) C-terminal region, which encompasses protein p6. The complete role of p6 is still undetermined; however, it is thought that p6 interacts with protein core elements from the endosomal sorting complex ESCRT-1, known to sort ubiquitinated cargo into multivesicular bodies (MVB). The three-dimensional structure of the p6 C-terminus (p6ct) comprising amino acids 32-52, determined in this study using NMR methods, includes the region thought to interact with Vpr, i.e., the LXXLF sequence. Here we present new results indicating that the region which interacts with Vpr is the ELY(36) sequence, in the same region where mutational studies revealed that replacing Y36 with a phenylalanine would increase the infectivity of virions by 300-fold. The interaction of Vpr with an egg PC bilayer in the presence of p6ct measured by plasmon waveguide resonance (PWR) is approximately 0.8 microM, approximately 100 times stronger in the absence of p6ct. Our results suggests an interaction based on an ELYP(37) sequence bearing similarities with recently published results, which elegantly demonstrated that the HIV-1 Gag LYPx(n)LxxL motif interacts with Alix 364-702. Moreover, we performed a 60 ns molecular dynamics (MD) simulation of p6ct in DPC micelles. The MD results, supported by differential scanning calorimetry measurements in DMPC, show that p6ct adsorbs onto the DPC micelle surface by adopting a rather stable alpha-helix. Our results provide insights regarding the HIV-1 virion sorting mechanism, specifically concerning the interaction between p6 and Vpr. We also suggest that Gag p6 may adsorb onto the surface of membranes during the sorting process, a property so far only attributed to the N-terminal portion of Gag matrix (MA), which is myristylated. The implications of such a novel event provide an alternative direction toward understanding the assembly and escape mechanisms of virions, which have been undetected so far

Dynamic Amino Acid Side-Chains Grafting on Folded Peptide Backbone

International audienceAn efficient strategy for the synthesis of large libraries of conformationally defined peptides is reported, using dynamic combinatorial chemistry as a tool to graft amino acid side chains on a well-ordered 3D (3-dimension) peptide backbone. Combining rationally designed scaffolds with combinatorial side chains selection represents an alternative method to access peptide libraries for structures that are not genetically encodable. This method would allow a breakthrough for the discovery of protein mimetic for unconventional targets for which little is known

Dynamic Amino Acid Side-Chains Grafting on Folded Peptide Backbone

An efficient strategy for the synthesis of large libraries of conformationnally defined peptides is reported, using dynamic combinatorial chemistry as a tool to graft amino acid side chains on a well-ordered 3D peptide backbone. Combining rationnally designed scaffolds with combinatorial side chains selection represents an alternative method to access peptide libraries for structures that are not genetically encodable. This method would allow a breakthrough for the discovery of protein mimetic for unconventional targets for which little is known

Inviting Trifluoromethylated Pseudoprolines into Collagen Model Peptides

Numerous Collagen Model Peptides (CMPs) have been engineered using proline derivatives substituted at their C(3) and/or C(4) position in order to stabilize or to functionalize collagen triple helix mimics. However, no example has been reported so far with C(5) substitutions. Here, we introduce a fluorinated CMP incorporating trifluoromethyl groups at the C(5) position of pseudoproline residues. In tripeptide models, our NMR and Molecular Dynamics (MD) studies have shown that, when properly arranged, these residues meet the structural requirements for triple helix assembly. A host-guest CMP could be synthesized and its NMR analysis in solution confirmed the presence of structured homotrimers that we interpret as triple helices. MD calculations showed that the triple helix model remained stable throughout the simulation, with all six trifluoromethyl groups pointing outwards from the triple helix. Pseudoprolines substituted at the C(5) positions appeared as valuable tools for the design of new fluorinated collagen mimicking peptides

Structure and Dynamics of an Intrinsically Disordered Protein Region That Partially Folds upon Binding by Chemical-Exchange NMR

International audienceMany intrinsically disordered proteins (IDPs) and protein regions (IDRs) engage in transient, yet specific, interactions with a variety of protein partners. Often, if not always, interactions with a protein partner lead to partial folding of the IDR. Characterizing the conformational space of such complexes is challenging: in solution-­‐state NMR, signals of the IDR in the interacting region become broad, weak and often invisible; while X-­‐ray crystallography only provides information on fully ordered regions. There is thus a need for a simple method to characterize both fully and partially ordered regions in the bound state of IDPs. Here, we introduce an approach based on monitoring chemical ex-­‐ change by NMR to investigate the state of an IDR that folds upon binding through the observation of the free state of the protein. Structural constraints for the bound state are obtained from chemical shifts and site-­‐specific dynamics of the bound state are characterized by relaxation rates. The conformation of the interacting part of the IDR was determined and subsequently docked onto the structure of the folded partner. We apply the method to investigate the interaction between the disordered C-­‐terminal region of Artemis and the DNA binding domain of Ligase IV. We show that we can accurately reproduce the structure of the core of the complex determined by X-­‐ray crystallography and identify a broader interface. The method is widely applicable to the biophysical investigation of complexes of disordered proteins and folded proteins

Peptidoglycan potentiates the membrane disrupting effect of the carboxyamidated form of DMS-DA6, a Gram-positive selective antimicrobial peptide isolated from Pachymedusa dacnicolor skin.

The occurrence of nosocomial infections has been on the rise for the past twenty years. Notably, infections caused by the Gram-positive bacteria Staphylococcus aureus represent a major clinical problem, as an increase in antibiotic multi-resistant strains has accompanied this rise. There is thus a crucial need to find and characterize new antibiotics against Gram-positive bacteria, and against antibiotic-resistant strains in general. We identified a new dermaseptin, DMS-DA6, produced by the skin of the Mexican frog Pachymedusa dacnicolor, with specific antibacterial activity against Gram-positive bacteria. This peptide is particularly effective against two multiple drug-resistant strains Enterococcus faecium BM4147 and Staphylococcus aureus DAR5829, and has no hemolytic activity. DMS-DA6 is naturally produced with the C-terminal carboxyl group in either the free or amide forms. By using Gram-positive model membranes and different experimental approaches, we showed that both forms of the peptide adopt an α-helical fold and have the same ability to insert into, and to disorganize a membrane composed of anionic lipids. However, the bactericidal capacity of DMS-DA6-NH2 was consistently more potent than that of DMS-DA6-OH. Remarkably, rather than resulting from the interaction with the negatively charged lipids of the membrane, or from a more stable conformation towards proteolysis, the increased capacity to permeabilize the membrane of Gram-positive bacteria of the carboxyamidated form of DMS-DA6 was found to result from its enhanced ability to interact with peptidoglycan