Date Published: October 10, 2013
Publisher: Public Library of Science
Author(s): Florent Laferrière, Philippe Tixador, Mohammed Moudjou, Jérôme Chapuis, Pierre Sibille, Laetitia Herzog, Fabienne Reine, Emilie Jaumain, Hubert Laude, Human Rezaei, Vincent Béringue, Umberto Agrimi.
Prions are proteinaceous infectious agents responsible for fatal neurodegenerative diseases in animals and humans. They are essentially composed of PrPSc, an aggregated, misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrPC). Stable variations in PrPSc conformation are assumed to encode the phenotypically tangible prion strains diversity. However the direct contribution of PrPSc quaternary structure to the strain biological information remains mostly unknown. Applying a sedimentation velocity fractionation technique to a panel of ovine prion strains, classified as fast and slow according to their incubation time in ovine PrP transgenic mice, has previously led to the observation that the relationship between prion infectivity and PrPSc quaternary structure was not univocal. For the fast strains specifically, infectivity sedimented slowly and segregated from the bulk of proteinase-K resistant PrPSc. To carefully separate the respective contributions of size and density to this hydrodynamic behavior, we performed sedimentation at the equilibrium and varied the solubilization conditions. The density profile of prion infectivity and proteinase-K resistant PrPSc tended to overlap whatever the strain, fast or slow, leaving only size as the main responsible factor for the specific velocity properties of the fast strain most infectious component. We further show that this velocity-isolable population of discrete assemblies perfectly resists limited proteolysis and that its templating activity, as assessed by protein misfolding cyclic amplification outcompetes by several orders of magnitude that of the bulk of larger size PrPSc aggregates. Together, the tight correlation between small size, conversion efficiency and duration of disease establishes PrPSc quaternary structure as a determining factor of prion replication dynamics. For certain strains, a subset of PrP assemblies appears to be the best template for prion replication. This has important implications for fundamental studies on prions.
Prion disease pathogenesis stems from the post-translational conversion of the monomeric, alpha helix-rich host-encoded prion protein (PrPC) into misfolded, β sheet-enriched PrPSc aggregates . The process is believed to be initiated by PrPSc seeds ,  acquired through infection or arising from spontaneous conversion of wild-type or mutant PrPC into PrPSc. The PrPSc seeds would template the remodeling of host PrPC to the PrPSc form . This self-sustained polymerization process, -in which polymer fragmentation is thought to play a key role , , -, leads to deposition of injurious plaques into the brain. PrPSc-templated conversion of PrPC or bacterially-derived PrP has been established in cell-free conditions using protein misfolding cyclic amplification (PMCA) assays (for reviews , ), further strengthening the conformational changes of the prion protein as the main molecular determinant of prion replication and infectivity.
Our initial SV studies revealed striking divergence in the hydrodynamic properties of the most infectious assemblies between distinct ovine prion strains from the same host species. For fast strains specifically, the most infectious assemblies sedimented slightly and were associated with low levels of PK-resistant material ( and this study). To carefully separate the respective contributions of size and density to these hydrodynamic characteristics, we varied the solubilization conditions and performed sedimentation at the equilibrium. Incidentally this is the first study that compared the density of prion particles associated with phenotypically distinct strains propagated on the same genetic background. All these experiments concurred with the view that a reduced aggregation size but not a low density accounts for the low SV properties of the fast strain most infectious component. We also provided evidence that these SV-isolated, small sized infectious species resist limited PK-proteolysis and have high templating efficiency as suggested by PMCA assay. Together, the straight relationship between small sized PrP assemblies, conversion efficacy and short incubation time observed for the fast strains establishes PrPSc quaternary structure as a determining factor of prion (strain specific) replication dynamics.