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

Autoři: Hasier Eraña aff001;  Jorge M. Charco aff001;  Michele A. Di Bari aff003;  Carlos M. Díaz-Domínguez aff001;  Rafael López-Moreno aff001;  Enric Vidal aff004;  Ezequiel González-Miranda aff001;  Miguel A. Pérez-Castro aff001;  Sandra García-Martínez aff001;  Susana Bravo aff005;  Natalia Fernández-Borges aff001;  Mariví Geijo aff006;  Claudia D’Agostino aff003;  Joseba Garrido aff006;  Jifeng Bian aff007;  Anna König aff008;  Boran Uluca-Yazgi aff008;  Raimon Sabate aff010;  Vadim Khaychuk aff007;  Ilaria Vanni aff003;  Glenn C. Telling aff007;  Henrike Heise aff008;  Romolo Nonno aff003;  Jesús R. Requena aff012;  Joaquín Castilla aff001
Působiště autorů: CIC bioGUNE, Derio (Bizkaia), Spain aff001;  ATLAS Molecular Pharma S. L. Derio (Bizkaia), Spain aff002;  Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy aff003;  Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Barcelona, Spain aff004;  Proteomics Lab, IDIS, Santiago de Compostela, Spain aff005;  Animal Health Department, NEIKER-Instituto Vasco de Investigación y Desarrollo Agrario, Derio (Bizkaia), Spain aff006;  Prion Research Center (PRC), Colorado State University, Fort Collins, Colorado, United States of America aff007;  Institute of Complex Systems (ICS-6) and Jülich Center for Structural Biology (JuStruct), Forschungszentrum Jülich, Jülich, Germany aff008;  Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany aff009;  Department of Pharmacy and Pharmaceutical Technology and Physical-Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona, Spain aff010;  Institute of Nanoscience and Nanotechnology (IN2UB), University of Barcelona, Spain aff011;  CIMUS Biomedical Research Institute, University of Santiago de Compostela-IDIS, Spain aff012;  IKERBasque, Basque Foundation for Science, Bilbao (Bizkaia), Spain aff013
Vyšlo v časopise: Development of a new largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies. PLoS Pathog 15(10): e32767. doi:10.1371/journal.ppat.1008117
Kategorie: Research Article
doi: 10.1371/journal.ppat.1008117


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.

Klíčová slova:

Mouse models – Prion diseases – Proteases – Recombinant proteins – Sulfates – Voles – Dextran – Electrophoretic staining


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2019 Číslo 10

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