Abstract
The resolution of the three-dimensional structure of infectious prions at the atomic level is pivotal to understand the pathobiology of Transmissible Spongiform Encephalopathies (TSE), but has been long hindered due to certain particularities of these proteinaceous pathogens. Difficulties related to their purification from brain homogenates of disease-affected animals were resolved almost a decade ago by the development of in vitro recombinant prion propagation systems giving rise to highly infectious recombinant prions. However, lack of knowledge about the molecular mechanisms of the misfolding event and the complexity of systems such as the Protein Misfolding Cyclic Amplification (PMCA), have limited generating the large amounts of homogeneous recombinant prion preparations required for high-resolution techniques such as solid state Nuclear Magnetic Resonance (ssNMR) imaging. Herein, we present a novel recombinant prion propagation system based on PMCA that substitutes sonication with shaking thereby allowing the production of unprecedented amounts of multi-labeled, infectious recombinant prions. The use of specific cofactors, such as dextran sulfate, limit the structural heterogeneity of the in vitro propagated prions and makes possible, for the first time, the generation of infectious and likely homogeneous samples in sufficient quantities for studies with high-resolution structural techniques as demonstrated by the preliminary ssNMR spectrum presented here. Overall, we consider that this new method named Protein Misfolding Shaking Amplification (PMSA), opens new avenues to finally elucidate the three-dimensional structure of infectious prions.
Highlights
IntroductionThe generation of recombinant prions in vitro able to cause Transmissible Spongiform Encephalopathy (TSE) in vivo has been one of the greatest advances of the last decades in the field [1,2,3,4,5,6,7,8,9,10,11,12] and has been successfully achieved by several research groups, there are still several unsolved issues that make this process of particular interest: 1) The recombinant prions generated by different research groups applying similar techniques yielded highly variable results
The molecular mechanisms by which this proteinaceous pathogen is able to propagate in the central nervous system and cause neuronal death are poorly understood, partially due to the difficulties elucidating the three-dimensional structure of the aggregation-prone aberrant isoform or prion
We present a novel method for the production of large amounts of highly infectious recombinant prions suitable for solid state Nuclear Magnetic Resonance imaging, which could help to unveil the molecular pathogenesis of these particular pathogens
Summary
The generation of recombinant prions in vitro able to cause Transmissible Spongiform Encephalopathy (TSE) in vivo has been one of the greatest advances of the last decades in the field [1,2,3,4,5,6,7,8,9,10,11,12] and has been successfully achieved by several research groups, there are still several unsolved issues that make this process of particular interest: 1) The recombinant prions generated by different research groups applying similar techniques yielded highly variable results. There are several methods capable of generating misfolded self-propagating recombinant PrP but given the disparity of infective abilities, it is impossible to clarify which is the best to generate infectious recombinant prions and which may yield only non-infectious misfolded recombinant PrPs. 3) these techniques could help to determine the critical difference between infectious and non-infectious self-propagating, protease-resistant misfolded recombinant PrPs. The vast majority of studies for in vitro recombinant prion generation have been performed using PrPs from well-known rodent species, mainly mouse (Mus musculus) [1, 8] and hamster (Mesocricetus auratus) [5]. The vast majority of studies for in vitro recombinant prion generation have been performed using PrPs from well-known rodent species, mainly mouse (Mus musculus) [1, 8] and hamster (Mesocricetus auratus) [5] Both are some of the best-characterized models of TSE and are the preferred ones for evaluating in vivo infectivity due to short incubation period and ease of handling. The bank vole is considered to be an almost universal acceptor of prions since can be infected with a large diversity of prion strains from different donor species [17] and when infected with
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