Étude numérique des premières étapes d'agrégation du peptide amyloïde GNNQQNY, impliqué dans une maladie à prion

Les protéines amyloïdes sont impliquées dans les maladies neurodégénératives comme Alzheimer, Parkinson et les maladies à prions et forment des structures complexes, les fibres amyloïdes. Le mécanisme de formation de ces fibres est un processus complexe qui implique plusieurs espèces d’agrégats intermédiaires. Parmi ces espèces, des petits agrégats, les oligomères, sont reconnus comme étant l’espèce amyloïde toxique, mais leur mécanisme de toxicité et d’agrégation sont mal compris. Cette thèse présente les résultats d’une étude numérique des premières étapes d’oligomérisation d’un peptide modèle GNNQQNY, issu d’une protéine prion, pour des systèmes allant du trimère au 50-mère, par le biais de simulations de dynamique moléculaire couplée au potentiel gros-grain OPEP. Nous trouvons que le mécanisme d’agrégation du peptide GNNQQNY suit un processus complexe de nucléation, tel qu’observé expérimentalement pour plusieurs protéines amyloïdes. Nous observons aussi que plusieurs chemins de formation sont accessibles à l’échelle du 20-mère et du 50-mère, ce qui confère aux structures un certain degré de polymorphisme et nous sommes capable de reproduire, dans nos simulations, des oligomères protofibrillaires qui présentent des caractéristiques structurelles observées expérimentalement chez les fibres amyloïdes.Amyloid proteins are involved in neurodegenerative diseases such as Alzheimer’s, Parkinson’s and prion diseases and form complex structures called amyloid fibrils. The fibril formation mechanism is a complex process, which involves several intermediary species. Among these species, small early aggregates, called oligomers, are thought to be the toxic amyloid species but their toxicity and aggregation mechanisms are poorly understood. This thesis aims at presenting the results of a numerical study of the first oligomerization steps of the model peptide GNNQQNY, from a prion protein, for system sizes ranging from the trimer to the 50-mer, via molecular dynamics simulations using the OPEP coarse-grained potential. We find that GNNQQNY’s assembly follows a complex nucleation process, as observed experimentally for numerous amyloid proteins. We also observe that the 20-mer and 50-mer systems form polymorphic structures that are the byproducts of different formation pathways. We further report the spontaneous formation of protofibrillar oligomers with structural characteristics typical of experimentally determined amyloid fibril structures

A Multiscale Approach to Characterize the Early Aggregation Steps of the Amyloid-Forming Peptide GNNQQNY from the Yeast Prion Sup-35

The self-organization of peptides into amyloidogenic oligomers is one of the key events for a wide range of molecular and degenerative diseases. Atomic-resolution characterization of the mechanisms responsible for the aggregation process and the resulting structures is thus a necessary step to improve our understanding of the determinants of these pathologies. To address this issue, we combine the accelerated sampling properties of replica exchange molecular dynamics simulations based on the OPEP coarse-grained potential with the atomic resolution description of interactions provided by all-atom MD simulations, and investigate the oligomerization process of the GNNQQNY for three system sizes: 3-mers, 12-mers and 20-mers. Results for our integrated simulations show a rich variety of structural arrangements for aggregates of all sizes. Elongated fibril-like structures can form transiently in the 20-mer case, but they are not stable and easily interconvert in more globular and disordered forms. Our extensive characterization of the intermediate structures and their physico-chemical determinants points to a high degree of polymorphism for the GNNQQNY sequence that can be reflected at the macroscopic scale. Detailed mechanisms and structures that underlie amyloid aggregation are also provided

A multiscale approach to characterize the early aggregation steps of the amyloid-forming peptide GNNQQNY from the yeast prion sup-35

ABSTRACT: The self-organization of peptides into amyloidogenic oligomers is one of the key events for a wide range of molecular and degenerative diseases. Atomic-resolution characterization of the mechanisms responsible for the aggregation process and the resulting structures is thus a necessary step to improve our understanding of the determinants of these pathologies. To address this issue, we combine the accelerated sampling properties of replica exchange molecular dynamics simulations based on the OPEP coarse-grained potential with the atomic resolution description of interactions provided by all-atom MD simulations, and investigate the oligomerization process of the GNNQQNY for three system sizes: 3-mers, 12-mers and 20-mers. Results for our integrated simulations show a rich variety of structural arrangements for aggregates of all sizes. Elongated fibril-like structures can form transiently in the 20-mer case, but they are not stable and easily interconvert in more globular and disordered forms. Our extensive characterization of the intermediate structures and their physico-chemical determinants points to a high degree of polymorphism for the GNNQQNY sequence that can be reflected at the macroscopic scale. Detailed mechanisms and structures that underlie amyloid aggregation are also provided

Critical nucleus characterization.

<p>(a) Free energy as a function of the aggregate size at 280 K. (b) Same at 300 K. In both (a) and (b), the maximum of free energy corresponds to a critical nucleus size of 5 monomers. (c) Free energy as a function of the number of residues in a conformation at 280 K. (d) Same at 300 K. In both (c) and (d), the first maximum in free energy represents the critical amount of secondary structure necessary for a nucleus to be stable and trigger aggregation. (e) Typical structure at 280 K of a pentamer nucleus with the critical amount of secondary structure shown in panel (c). (f) Typical structure at 300 K of a pentamer nucleus with the critical amount of secondary structure shown in figure (d).</p

Kinetics of Amyloid Aggregation: A Study of the GNNQQNY Prion Sequence

<div><p>The small amyloid-forming GNNQQNY fragment of the prion sequence has been the subject of extensive experimental and numerical studies over the last few years. Using unbiased molecular dynamics with the OPEP coarse-grained potential, we focus here on the onset of aggregation in a 20-mer system. With a total of 16.9 of simulations at 280 K and 300 K, we show that the GNNQQNY aggregation follows the classical nucleation theory (CNT) in that the number of monomers in the aggregate is a very reliable descriptor of aggregation. We find that the critical nucleus size in this finite-size system is between 4 and 5 monomers at 280 K and 5 and 6 at 300 K, in overall agreement with experiment. The kinetics of growth cannot be fully accounted for by the CNT, however. For example, we observe considerable rearrangements after the nucleus is formed, as the system attempts to optimize its organization. We also clearly identify two large families of structures that are selected at the onset of aggregation demonstrating the presence of well-defined polymorphism, a signature of amyloid growth, already in the 20-mer aggregate.</p> </div

Evolution of the structural properties for the GNNQQNY 20-mer at 280 K as a function of the number of hydrogen bonds and of the number of side chain contacts.

<p>(a) Time evolution map of the system. Black regions indicate the beginning of the simulation while yellow regions indicate the end. (b) Density map representing the probability of having a configuration lie in a specific region. Yellow is the highest density and red the lowest. (c) Proportion of parallel -strands. Yellow regions indicate that 100% of the strands are in parallel orientation while black regions indicate that none of the strands are in parallel orientation thus meaning that they all are in antiparallel orientation. Since cluster determination is based on the presence of hydrogen bonds, the percentage of antiparallel orientation of the strands is equal to 1 minus the percentage of parallel orientation. Regions 1 and 2 indicate the two regions of highest parallel orientation. The discontinuities in the maps (a) and (c) is a plotting artifact in low-density regions where the system is rapidly changing during aggregation and there are therefore not enough points to fill the map regions.</p

Aggregation mechanism.

<p>Time evolution of association and dissociation events before during and after nucleation by either single monomer events (meaning one at a time), by oligomer fusion/fragmentation, or by formation/destruction of oligomers from/into monomers at (a) 280 K and (b) 300 K. The dashed grey line indicates the beginning of nucleation. In (a), at 280 K, the aggregate stops growing in size after t = 9 ns while in (b), at 300 K, the aggregate stops growing just before t = 8 ns. For ease of reading, each point in the graphs is the sum of events in (5).</p

Diversity of the morphologies.

<p>(a) Oligomer displaying an extremely high amount of contacts (simulation R1). (b) Protofibril-like structure displaying a high number of hydrogen bonds (simulation R2); (c) typical 3-plus sheet structure often generated in our simulations; (d) typical 2-sheet structure.</p

Competition between the globular oligomer (R1) and the protofibril (R2) at 280 K.

<p>(a) Low energy profile displaying a low amount of hydrogen bonds for the globular oligomer; (b) corresponding time map as a function of the number of hydrogen bonds and of the side chain contacts; (c) corresponding amount of parallel -strands as a function of the same parameters; (d) kinetic profile displaying a particularly high number of hydrogen bonds in the protofibril structure; (e) corresponding time map as a function of the number of hydrogen bonds and of the side chain contacts. f) corresponding amount of parallel beta-strands as a function of the same parameters. These graphs demonstrate the existence of a competition between the globular structure with a low amount of hydrogen bonds and a high amount of contacts and the protofibril structure with a high amount of hydrogen bonds and a low amount of contacts. The actual structures are shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002782#pcbi-1002782-g006" target="_blank">Figure 6 (a) and (b)</a>.</p