Development of a new largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies

Hasier Eraña, Jorge M Charco, Michele A Di Bari, Carlos M Díaz-Domínguez, Rafael López-Moreno, Enric Vidal, Ezequiel González-Miranda, Miguel A Pérez-Castro, Sandra García-Martínez, Susana Bravo, Natalia Fernández-Borges, Mariví Geijo, Claudia D'Agostino, Joseba Garrido, Jifeng Bian, Anna König, Boran Uluca-Yazgi, Raimon Sabate, Vadim Khaychuk, Ilaria VanniGlenn C Telling, Henrike Heise, Romolo Nonno, Jesús R Requena, Joaquín Castilla

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)e1008117
JournalPLoS Pathogens
Volume15
Issue number10
DOIs
Publication statusPublished - Oct 2019

Keywords

  • Animals
  • Arvicolinae
  • Central Nervous System/pathology
  • Dextran Sulfate/pharmacology
  • Disease Models, Animal
  • Mice, Transgenic
  • Nuclear Magnetic Resonance, Biomolecular/methods
  • Prion Diseases/pathology
  • Prion Proteins/metabolism
  • Prions/metabolism
  • Protein Structure, Tertiary
  • Proteostasis Deficiencies/pathology

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