Projet ANR-08-PCVI-0022 – Prion SSNMR : Atomic packing and assembly of the prion Ure2p in its fibrillar form: Insights from a synergistic biochemical and solid-state NMR approach
Programme : Programme interdiciplinaire en physique et chimie du vivant (PCV) 2008
Année de financement : 2008
Coordinateur du projet : CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-AUVERGNE (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-AUVERGNE)
Partenaires : Centre national de la recherche scientifique; Centre national de la recherche scientifique
Montant : 400000.0 euros
Date de début : 2009
Durée : 36 mois
Résumé : Protein misfolding and subsequent aggregation is at the origin of over 20 human diseases termed 'conformational' diseases. Although actively studied, the fundamental mechanism of the misfolding of a variety of polypeptides leading to their aggregation and associated diseases is far from being understood. A subset of neurodegenerative illnesses is intimately linked to the aggregation of protein termed prion. Prions are infectious, self-propagating polymers of otherwise soluble, host-encoded proteins. The yeast prion Ure2p assembles in vitro, in a manner similar to other prions, into soluble high molecular weight oligomers and fibrils. Ure2p retains within the fibrils native-like properties. Different forms of Ure2p native-like assemblies (dimers, oligomers, and fibrils) have been shown to be toxic to cells. Ure2p toxicity seems to depend on the structural properties of the flexible N-terminal prion domain, and can be considered as an inherent feature of the protein, unrelated to its aggregation state but rather associated with a basic toxic fold shared by all of the Ure2p native-like assemblies. The structural basis of prion infectivity remains elusive in the absence of a prion fibrillar structure at an atomic resolution. This issue is a technical challenge as prion oligomers are heterogeneous high molecular weight particles that are neither suitable for protein crystallography nor classical NMR studies. Solid-state NMR is one of the most promising techniques to reveal conformational details of insoluble and non-crystalline molecular assemblies. A central aspect of the most recent international effort in solid-state NMR concerns fibrillar proteins, and the last years have seen first efforts directed towards structural studies of such proteins. These studies have driven, on the other hand, many developments concerning the methodological aspects of solid-state NMR. Current studies have mostly been limited to short fragments of assembly competent proteins and were aimed at developing convenient model systems that yield detailed structural data. The proposed conformational studies of the protein Ure2 address for the first time the central questions of the packing in fibrils and dynamics of assembly of a full-length intact Q- and N-rich prion as opposed to protein fragments. Our research project is aimed to provide the first detailed structure of prion fibrils formed from intact native polypeptides under physiological conditions.The proposed combination of structural (solid state NMR) and biochemical studies will allow deciphering the molecular basis for the conversion of native polypeptides into toxic insoluble fibrillar deposits and should generate the first detailed structure of a prion.