Development of a new largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies
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Author
Eraña, Hasier
Charco, Jorge M.
Di Bar, Michele A.
Díaz-Domínguez, Carlos M.
López-Moreno, Rafael
González-Miranda, Ezequiel
Pérez-Castro, Miguel A.
García-Martínez, Sandra
Bravo, Susana
Fernández-Borges, Natalia
Geijo, Mariví
D’Agostino, Claudia
Garrido, Joseba
Bian, Jifeng
König, Anna
Uluca-Yazgi, Boran
Sabate, Raimon
Khaychuk, Vadim
Vanni, Ilaria
Telling, Glenn C.
Heise, Henrike
Nonno, Romolo
Requena, Jesús R.
Castilla, Joaquín
Publication date
2019-10-23ISSN
1553-7366
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.
Document Type
Article
Document version
Published version
Language
English
Subject (CDU)
619 - Veterinary science
Pages
33
Publisher
Public Library of Science
Is part of
PLoS Pathogens
Citation
Eraña, Hasier, Jorge M. Charco, Michele A. Di Bari, Carlos M. Díaz-Domínguez, Rafael López-Moreno, Enric Vidal, and Ezequiel González-Miranda et al. 2019. "Development Of A New Largely Scalable In Vitro Prion Propagation Method For The Production Of Infectious Recombinant Prions For High Resolution Structural Studies". PLOS Pathogens 15 (10): e1008117. Public Library of Science (PLoS). doi:10.1371/journal.ppat.1008117.
Grant agreement number
MINECO/Programa Estatal de I+D+I orientada a los retos de la sociedad/AGL2015-65046-C2-1-R/ES/Estudio de los mecanismos moleculares implicados en la diversidad y estabilidad de las cepas priónicas/
MINECO/Programa Estatal de fomento de la investigación científica y técnica de excelencia/BFU2013-48436-C2-1-P/ES/Decodificando los priones: elucidación de la base estructural de la infectividad de proteínas/
MINECO-FEDER/Programa Estatal de fomento de la investigación científica y técnica de excelencia/BFU2017-86692-P/ES/ELUCIDACION DE LA ESTRUCTURA DE LOS PRIONES PRPSC RECOMBINANTES/
EC/INTERREG-POCTEFA/EU/EFA-148-16/ /REDPRION
Program
Sanitat Animal
This item appears in the following Collection(s)
- ARTICLES CIENTÍFICS [2831]
Except where otherwise noted, this item's license is described as http://creativecommons.org/licenses/by/4.0/