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

English version

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: https://doi.org/10.1371/journal.ppat.1008117

Souhrn

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

Zdroje

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