Development of a new largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies
Eraña, Hasier (ATLAS Molecular Pharma S. L.)
Charco, Jorge M. (CIC bioGUNE)
Di Bari, Michele Angelo (CIC bioGUNE)
Díaz-Domínguez, Carlos M. (Istituto Superiore di Sanità. Department of Veterinary Public Health and Food Safety.)
López-Moreno, R. (CIC bioGUNE)
Vidal Barba, Enric (Institut de Recerca i Tecnologia Agroalimentàries. Centre de Recerca en Sanitat Animal)
González-Miranda, E. (CIC bioGUNE)
Pérez-Castro, Miguel A. (CIC bioGUNE)
García-Martínez, Sandra (ATLAS Molecular Pharma S. L.)
Bravo Lopez, Susana Belén (Proteomics Lab. IDIS)
Fernández-Borges, Natalia (CIC bioGUNE)
Geijo, Maria V. (NEIKER-Instituto Vasco de Investigación y Desarrollo Agrario)
D'Agostino, C. (Department of Veterinary Public Health and Food Safety. Istituto Superiore di Sanità)
Garrido, Joseba M (NEIKER-Instituto Vasco de Investigación y Desarrollo Agrario)
Bian, J. (Colorado State University. Prion Research Center (PRC).)
König, A. (Heinrich-Heine-Universität Düsseldorf. Physikalische Biologie.)
Uluca-Yazgi, B. (Heinrich-Heine-Universität Düsseldorf. Physikalische Biologie.)
Sabate, R. (Universitat de Barcelona. Institut de Nanociència i Nanotecnologia)
Khaychuk, V. (Colorado State University. Prion Research Center (PRC).)
Vanni, Ilaria (Istituto Superiore di Sanità. Department of Veterinary Public Health and Food Safety.)
Telling, G. C. (Colorado State University. Prion Research Center (PRC).)
Heise, Henrike (Heinrich-Heine-Universität Düsseldorf. Physikalische Biologie.)
Nonno, Romolo (Istituto Superiore di Sanità. Department of Veterinary Public Health and Food Safety.)
Requena, Jesús R (Universidade de Santiago de Compostela)
Castilla, Joaquín (IKERBasque. Basque Foundation for Science)

Data:	2019
Resum:	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.
Ajuts:	Ministerio de Economía y Competitividad AGL2015-65046-C2-1-R Ministerio de Economía y Competitividad BFU2013-48436-C2-1-P Ministerio de Economía y Competitividad BFU2017-86692-P
Nota:	This work was supported financially by several Spanish grants awarded to JC (AGL2015-65046-C2-1-R and BFU2013-48436-C2-1-P) and JRR (BFU2017-86692-P) by MINECO/FEDER, as well as an Interreg (POCTEFA EFA148/16) grant awarded to JC by FEDER. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Nota:	Altres ajuts: FEDER/POCTEFA EFA148/16
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	Animals ; Arvicolinae ; Central Nervous System ; Dextran Sulfate ; Disease Models, Animal ; Mice, Transgenic ; Nuclear Magnetic Resonance, Biomolecular ; Prion Diseases ; Prion Proteins ; Prions ; Protein Structure, Tertiary ; Proteostasis Deficiencies
Publicat a:	PLOS pathogens, Vol. 15 Núm. 10 (2019) , p. e1008117, ISSN 1553-7374

DOI: 10.1371/journal.ppat.1008117
PMID: 31644574

33 p, 2.7 MB

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