Synthèse, structure et dynamique des peptides membranaires impliqués dans le cancer de l'apoptose. Une approche par modélisation moléculaire,diochroisme circulaire et résonnance magnétique nucléaire des solides.

Le récepteur Neu/ErbB2 est un dimère dont la fonction est de transmettre au sein de la cellule les signaux de croissance cellulaire. Le récepteur est à l’origine de nombreux cancers provoqués par la mutation ponctuelle Val/Glu dans le segment transmembranaire. Cette mutation stabiliserait les dimères en absence de ligand induisant ainsi la prolifération non contrôlée de cellules. La détermination de la structure et de la dynamique du récepteur natif (Neu/erbB-2) et muté (Neu*/erbB-2) représentent donc une étape capitale dans la compréhension des mécanismes de transduction du signal. La synthèse des peptides hydrophobes transmembranaires a été entreprise. Leurs structures ont été déterminées par dichroïsme circulaire en solution et en milieu membranaire. L’hélice π, présente dans le domaine transmembranaire, a été modélisée afin de mieux appréhender les mécanismes de dimérisation. La RMN a permis de caractériser la dynamique des lipides, afin d’étudier la flexibilité des peptides ou l’adaptation de la membrane. Une méthode innovante a été développée pour extraire les peptides du milieu membranaire.The Neu/ErbB2 receptor is a dimer whose function is to transmit the signals of cell multiplication in the cell. The receptor is involved in several cancers induced by the Val to Glu point mutation in the transmembrane domain. This mutation would stabilize dimers in the absence of ligand and thus induce the non controlled proliferation of cells. Therefore the determination of the structure and the dynamics of the native (Neu/erbB-2) receptor and mutated (Neu*/erbB-2) represents a capital step in the comprehension of the mechanisms of the signal transduction. We performed the solid phase synthesis of the transmembrane peptides. The structure was determined by circular dichroism in solution and in the membrane. The π-Helix, found in the transmembrane domain, was studied by molecular dynamics simulations to better understand the mechanism of the dimerization. The solid state NMR allowed characterizing the dynamics of the lipids, in order to study the flexibility of peptides or the adaptation of the membrane. An innovating method was developed to separate peptides from the membrane

Membrane-Catalyzed Aggregation of Islet Amyloid Polypeptide Is Dominated by Secondary Nucleation

Type II diabetes is characterized by the loss of pancreatic β-cells. This loss is thought to be a consequence of membrane disruption, caused by the aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils. However, the molecular mechanisms of IAPP aggregation in the presence of membranes have remained unclear. Here, we use kinetic analysis to elucidate the aggregation mechanism of IAPP in the presence of mixed zwitterionic and anionic lipid membranes. The results converge to a model in which aggregation on the membrane is strongly dominated by secondary nucleation, that is, the formation of new nuclei on the surface of existing fibrils. The critical nucleus consists of a single IAPP molecule, and anionic lipids catalyze both primary and secondary nucleation, but not elongation. The fact that anionic lipids promote secondary nucleation implies that these events take place at the interface between the membrane and existing fibrils, demonstrating that fibril growth occurs at least to some extent on the membrane surface. These new insights into the mechanism of IAPP aggregation on membranes may help to understand IAPP toxicity and will be important for the development of therapeutics to prevent β-cell death in type II diabetes

Fibril elongation by human islet amyloid polypeptide is the main event linking aggregation to membrane damage

The aggregation of human islet amyloid polypeptide (hIAPP) is linked to the death of pancreatic β-cells in type II diabetes. The process of fibril formation by hIAPP is thought to cause membrane damage, but the precise mechanisms are still unclear. Previously, we showed that the aggregation of hIAPP in the presence of membranes containing anionic lipids is dominated by secondary nucleation events, which occur at the interface between existing fibrils and the membrane surface. Here, we used vesicles with different lipid composition to explore the connection between hIAPP aggregation and vesicle leakage. We found that different anionic lipids promote hIAPP aggregation to the same extent, whereas remarkably stochastic behaviour is observed on purely zwitterionic membranes. Vesicle leakage induced by hIAPP consists of two distinct phases for any of the used membrane compositions: (i) an initial phase in which hIAPP binding causes a certain level of leakage that is strongly dependent on osmotic conditions, membrane composition and the used dye, and (ii) a main leakage event that we attribute to elongation of hIAPP fibrils, based on seeded experiments. Altogether, our results shed more light on the relationship between hIAPP fibril formation and membrane damage, and strongly suggest that oligomeric intermediates do not considerably contribute to vesicle leakage

Recent Insight in Islet Amyloid Polypeptide Morphology, Structure, Membrane Interaction, and Toxicity in Type 2 Diabetes

International audienceThe formation of protein amyloid deposits is associated with major human diseases including Alzheimer’s disease, Parkinson’s disease, Spongiform Encephalopathy, and type 2 diabetes mellitus (T2DM). Today, 382 million people live with diabetes. Diabetes is on the rise all over the world and countries are struggling to keep pace treating all these patients. Worldwide, one person dies of the consequences of diabetes (cardiovascular disease, kidney failure, and lower limb amputation) every 6 seconds, more than AIDS and malaria combined

Synthèse, structure et dynamique de peptides membranaires impliqués dans le cancer et l'apoptose (une approche par modélisation moléculaire, dichroïsme circulaire et résonance magnétique nucléaire des solides)

Le récepteur Neu/ErbB2 est un dimére dont la fonction est de transmettre au sein de la cellule les signaux de croissance cellulaire. Le récepteur est à l'origine de nombreux cancers provoqués par la mutation ponctuelle Val/Glu dans le segment transmembranaire. Cette mutation stabiliserait les dimères en absence de ligand induisant ainsi la prolifération non contrpolée de cellules. La détermination de la structure et de la dynamqie du récepteur natif (Neu/erbB-2) et muté (Neu*/erbB-2) représentent donc une étape capitale dans la compréhension des mécanismes de tranduction du signal. La synthèse des peptides hydrophobes transmembranaires a été entreprise. Leurs structures ont été déterminées par dichroïsme circulaire en solution et en milieu membranaire. L'hélice p, présente dans le domaine transmembranaire, a été modélisée afin de mieux apprégender les mécanismes de dimérisation. La RMN a permis de caractériser la dynamique de lipides, afin d'étudier la flexibilité des peptides ou l'adaptation de la membrane. Une méthode innovante a été développée pour extraire les peptides du milieu membranaire.BORDEAUX1-BU Sciences-Talence (335222101) / SudocSudocFranceF

Targeting hIAPP fibrillation: A new paradigm to prevent β-cell death?

International audienceLoss of pancreatic β-cell mass is deleterious for type 2 diabetes patients since it reduces insulin production, critical for glucose homeostasis. The main research axis developed over the last few years was to generate new pancreatic β-cells or to transplant pancreatic islets as occurring for some specific type 1 diabetes patients. We evaluate here a new paradigm consisting in preservation of β-cells by prevention of human islet amyloid polypeptide (hIAPP) oligomers and fibrils formation leading to pancreatic β-cell death. We review the hIAPP physiology and the pathology that contributes to β-cell destruction, deciphering the various cellular steps that could be involved. Recent progress in understanding other amyloidosis such as Aβ, Tau, α-synuclein or prion, involved in neurodegenerative processes linked with inflammation, has opened new research lines of investigations to preserve neuronal cells. We evaluate and estimate their transposition to the pancreatic β-cells preservation. Among them is the control of reactive oxygen species (ROS) production occurring with inflammation and the possible implication of the mitochondrial translocator protein as a diagnostic and therapeutic target. The present review also focuses on other amyloid forming proteins from molecular to physiological and physiopathological points of view that could help to better decipher hIAPP-induced β-cell death mechanisms and to prevent hIAPP fibril formation

Etude biophysique de peptides amyloïdes en présence de membranes (caractérisation de leurs interactions et détermination de leurs structures)

Le peptide amyloïde IAPP, impliqué dans le diabète de type 2, possède la propriété de s agréger, passant d un état monomérique initial à des fibres amyloïdes matures, via des espèces oligomériques. Ce processus d agrégation, qui se produit au contact de la membrane, a été étudié par fluorescence, microscopie électronique, dichroïsme circulaire et RMN. Tout d abord, l influence du modèle membranaire a été mise en évidence, en termes de forme, taille (micelles, bicelles, SUV, LUV) et composition lipidique (chaînes et têtes différentes) sur les cinétiques d agrégation et de changement conformationnel et sur la morphologie des fibres. Nous avons cherché à comprendre le rôle du cholestérol dans les interactions peptide/membranes, du point de vue du peptide et de la membrane, en utilisant des vésicules contenant entre 0 et 30% de cholestérol. Il a ainsi été observé qu un pourcentage élevé de cholestérol semble accélérer la cinétique d agrégation. De plus, des expériences de RMN liquide ont été réalisées dans le but de déterminer la structure du peptide IAPP en présence de bicelles. Les premiers résultats montrent que l extrémité C terminale ne s insère pas dans la membrane et possède une flexibilité importante. Enfin, le peptide IAPP a également été comparé à un peptide antimicrobien aux propriétés amyloïdes, la dermaseptine S9. Ces travaux indiquent que les mécanismes de fibrillation et de perméabilisation membranaire ne sont pas reliés et que le mode d action de la dermaseptine S9 repose sur la formation de pores transitoires impliquant des espèces oligomériques.The amyloid peptide IAPP, which is implicated in type 2 diabetes mellitus, aggregates from an initial monomeric state to amyloid fibrils, via oligomeric species. Peptide aggregation, which takes place through membrane contact, was studied using fluorescence, electron microscopy, circular dichroism and NMR. The effect of membrane model was highlighted, in terms of shape, size (micelles, bicelles, SUV, LUV) and composition (lipid headgroups and acyl chains), on aggregation kinetics, conformational change kinetics and fibril morphology. Next, we wanted to elucidate the role of cholesterol in peptide/membranes interactions using vesicles composed of 0 to 30% cholesterol. High cholesterol content was shown to increase aggregation kinetics. Furthermore, IAPP in the presence of bicelles was studied by liquid state NMR in order to solve its structure under these conditions. First results indicate that the C terminus does not insert into the membrane and has an important flexibility. Finally, IAPP was compared with an antimicrobial and amyloid-like peptide, dermaseptin S9. This study shows that fibril formation and membrane permeabilisation mechanisms are not linked and that dermaseptin S9 binds to membrane in an aggregated state, maybe leading to the formation of a transient pore.PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Molecular Structure, Membrane Interactions, and Toxicity of the Islet Amyloid Polypeptide in Type 2 Diabetes Mellitus

Human islet amyloid polypeptide (hIAPP) is the major component of the amyloid deposits found in the pancreatic islets of patients with type 2 diabetes mellitus (T2DM). Mature hIAPP, a 37-aa peptide, is natively unfolded in its monomeric state but forms islet amyloid in T2DM. In common with other misfolded and aggregated proteins, amyloid formation involves aggregation of monomers of hIAPP into oligomers, fibrils, and ultimately mature amyloid deposits. hIAPP is coproduced and stored with insulin by the pancreatic islet β-cells and is released in response to the stimuli that lead to insulin secretion. Accumulating evidence suggests that hIAPP amyloid deposits that accompany T2DM are not just an insignificant phenomenon derived from the disease progression but that hIAPP aggregation induces processes that impair the functionality and the viability of β-cells. In this review, we particularly focus on hIAPP structure, hIAPP aggregation, and hIAPP-membrane interactions. We will also discuss recent findings on the mechanism of hIAPP-membrane damage and on hIAPP-induced cell death. Finally, the development of successful antiamyloidogenic agents that prevent hIAPP fibril formation will be examined