Development of a new largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies
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...
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creator | Eraña, Hasier Charco, Jorge M Di Bari, Michele A Díaz-Domínguez, Carlos M López-Moreno, Rafael Vidal, Enric 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 |
description | 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. |
doi_str_mv | 10.1371/journal.ppat.1008117 |
format | Article |
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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.</description><identifier>ISSN: 1553-7374</identifier><identifier>ISSN: 1553-7366</identifier><identifier>EISSN: 1553-7374</identifier><identifier>DOI: 10.1371/journal.ppat.1008117</identifier><identifier>PMID: 31644574</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>3D printing ; Amplification ; Animal diseases ; Animals ; Arvicolinae ; Atoms ; Biology ; Biology and Life Sciences ; Central Nervous System - pathology ; Cofactors ; Dextran ; Dextran sulfate ; Dextran Sulfate - pharmacology ; Dextrans ; Diagnostic imaging ; Disease ; Disease Models, Animal ; Food safety ; Heterogeneity ; High resolution ; High resolution spectroscopy ; In vitro methods and tests ; Magnetic resonance imaging ; Mammals ; Medicine and Health Sciences ; Mice, Transgenic ; Molecular modelling ; Neuroimaging ; NMR ; Novels ; Nuclear magnetic resonance ; Nuclear Magnetic Resonance, Biomolecular - methods ; Particles ; Partícules (Matèria) ; Pathogenic microorganisms ; Pharmacy ; Physical Sciences ; Polysaccharides ; Prion diseases ; Prion Diseases - pathology ; Prion protein ; Prion Proteins - metabolism ; Prions ; Prions (Proteins) ; Prions - metabolism ; Propagation ; Protein folding ; Protein purification ; Protein Structure, Tertiary ; Proteins ; Proteostasis Deficiencies - pathology ; Public health ; Research and Analysis Methods ; Shaking ; Sonication ; Sulfates ; Supervision ; Transmissible spongiform encephalopathy ; Àtoms</subject><ispartof>PLoS pathogens, 2019-10, Vol.15 (10), p.e1008117-e1008117</ispartof><rights>COPYRIGHT 2019 Public Library of Science</rights><rights>2019 Eraña et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>cc-by (c) Eraña, Hasier et al., 2019 info:eu-repo/semantics/openAccess <a href="http://creativecommons.org/licenses/by/3.0/es">http://creativecommons.org/licenses/by/3.0/es</a></rights><rights>2019 Eraña et al 2019 Eraña et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c664t-1950874cfcda28df22e14d0d8e14bb90213e7a3ec89245143491cc72529a87ba3</citedby><cites>FETCH-LOGICAL-c664t-1950874cfcda28df22e14d0d8e14bb90213e7a3ec89245143491cc72529a87ba3</cites><orcidid>0000-0003-3894-2362 ; 0000-0001-8776-4211 ; 0000-0001-7615-3079 ; 0000-0002-0410-1963 ; 0000-0001-7556-1564 ; 0000-0002-4965-3286 ; 0000-0002-3120-1038 ; 0000-0002-2216-1361 ; 0000-0003-2617-4263 ; 0000-0002-9081-3894 ; 0000-0003-2850-3470 ; 0000-0003-3476-1855 ; 0000-0003-0943-6823 ; 0000-0002-6910-9202</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827918/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827918/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,26951,27901,27902,53766,53768,79569,79570</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31644574$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Eraña, Hasier</creatorcontrib><creatorcontrib>Charco, Jorge M</creatorcontrib><creatorcontrib>Di Bari, Michele A</creatorcontrib><creatorcontrib>Díaz-Domínguez, Carlos M</creatorcontrib><creatorcontrib>López-Moreno, Rafael</creatorcontrib><creatorcontrib>Vidal, Enric</creatorcontrib><creatorcontrib>González-Miranda, Ezequiel</creatorcontrib><creatorcontrib>Pérez-Castro, Miguel A</creatorcontrib><creatorcontrib>García-Martínez, Sandra</creatorcontrib><creatorcontrib>Bravo, Susana</creatorcontrib><creatorcontrib>Fernández-Borges, Natalia</creatorcontrib><creatorcontrib>Geijo, Mariví</creatorcontrib><creatorcontrib>D'Agostino, Claudia</creatorcontrib><creatorcontrib>Garrido, Joseba</creatorcontrib><creatorcontrib>Bian, Jifeng</creatorcontrib><creatorcontrib>König, Anna</creatorcontrib><creatorcontrib>Uluca-Yazgi, Boran</creatorcontrib><creatorcontrib>Sabate, Raimon</creatorcontrib><creatorcontrib>Khaychuk, Vadim</creatorcontrib><creatorcontrib>Vanni, Ilaria</creatorcontrib><creatorcontrib>Telling, Glenn C</creatorcontrib><creatorcontrib>Heise, Henrike</creatorcontrib><creatorcontrib>Nonno, Romolo</creatorcontrib><creatorcontrib>Requena, Jesús R</creatorcontrib><creatorcontrib>Castilla, Joaquín</creatorcontrib><title>Development of a new largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies</title><title>PLoS pathogens</title><addtitle>PLoS Pathog</addtitle><description>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.</description><subject>3D printing</subject><subject>Amplification</subject><subject>Animal diseases</subject><subject>Animals</subject><subject>Arvicolinae</subject><subject>Atoms</subject><subject>Biology</subject><subject>Biology and Life Sciences</subject><subject>Central Nervous System - pathology</subject><subject>Cofactors</subject><subject>Dextran</subject><subject>Dextran sulfate</subject><subject>Dextran Sulfate - pharmacology</subject><subject>Dextrans</subject><subject>Diagnostic imaging</subject><subject>Disease</subject><subject>Disease Models, Animal</subject><subject>Food safety</subject><subject>Heterogeneity</subject><subject>High resolution</subject><subject>High resolution spectroscopy</subject><subject>In vitro methods and tests</subject><subject>Magnetic resonance imaging</subject><subject>Mammals</subject><subject>Medicine and Health Sciences</subject><subject>Mice, Transgenic</subject><subject>Molecular modelling</subject><subject>Neuroimaging</subject><subject>NMR</subject><subject>Novels</subject><subject>Nuclear magnetic resonance</subject><subject>Nuclear Magnetic Resonance, Biomolecular - methods</subject><subject>Particles</subject><subject>Partícules (Matèria)</subject><subject>Pathogenic microorganisms</subject><subject>Pharmacy</subject><subject>Physical Sciences</subject><subject>Polysaccharides</subject><subject>Prion diseases</subject><subject>Prion Diseases - pathology</subject><subject>Prion protein</subject><subject>Prion Proteins - metabolism</subject><subject>Prions</subject><subject>Prions (Proteins)</subject><subject>Prions - metabolism</subject><subject>Propagation</subject><subject>Protein folding</subject><subject>Protein purification</subject><subject>Protein Structure, Tertiary</subject><subject>Proteins</subject><subject>Proteostasis Deficiencies - pathology</subject><subject>Public health</subject><subject>Research and Analysis Methods</subject><subject>Shaking</subject><subject>Sonication</subject><subject>Sulfates</subject><subject>Supervision</subject><subject>Transmissible spongiform 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of a new largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies</title><author>Eraña, Hasier ; Charco, Jorge M ; Di Bari, Michele A ; Díaz-Domínguez, Carlos M ; López-Moreno, Rafael ; Vidal, Enric ; 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</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c664t-1950874cfcda28df22e14d0d8e14bb90213e7a3ec89245143491cc72529a87ba3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>3D printing</topic><topic>Amplification</topic><topic>Animal diseases</topic><topic>Animals</topic><topic>Arvicolinae</topic><topic>Atoms</topic><topic>Biology</topic><topic>Biology and Life Sciences</topic><topic>Central Nervous System - pathology</topic><topic>Cofactors</topic><topic>Dextran</topic><topic>Dextran sulfate</topic><topic>Dextran Sulfate - pharmacology</topic><topic>Dextrans</topic><topic>Diagnostic imaging</topic><topic>Disease</topic><topic>Disease Models, Animal</topic><topic>Food safety</topic><topic>Heterogeneity</topic><topic>High resolution</topic><topic>High resolution spectroscopy</topic><topic>In vitro methods and tests</topic><topic>Magnetic resonance imaging</topic><topic>Mammals</topic><topic>Medicine and Health Sciences</topic><topic>Mice, Transgenic</topic><topic>Molecular modelling</topic><topic>Neuroimaging</topic><topic>NMR</topic><topic>Novels</topic><topic>Nuclear magnetic resonance</topic><topic>Nuclear Magnetic Resonance, Biomolecular - 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Edition</collection><collection>ProQuest Central China</collection><collection>MEDLINE - Academic</collection><collection>Recercat</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PLoS pathogens</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Eraña, Hasier</au><au>Charco, Jorge M</au><au>Di Bari, Michele A</au><au>Díaz-Domínguez, Carlos M</au><au>López-Moreno, Rafael</au><au>Vidal, Enric</au><au>González-Miranda, Ezequiel</au><au>Pérez-Castro, Miguel A</au><au>García-Martínez, Sandra</au><au>Bravo, Susana</au><au>Fernández-Borges, Natalia</au><au>Geijo, Mariví</au><au>D'Agostino, Claudia</au><au>Garrido, Joseba</au><au>Bian, Jifeng</au><au>König, Anna</au><au>Uluca-Yazgi, Boran</au><au>Sabate, Raimon</au><au>Khaychuk, Vadim</au><au>Vanni, Ilaria</au><au>Telling, Glenn C</au><au>Heise, Henrike</au><au>Nonno, Romolo</au><au>Requena, Jesús R</au><au>Castilla, Joaquín</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development of a new largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies</atitle><jtitle>PLoS pathogens</jtitle><addtitle>PLoS Pathog</addtitle><date>2019-10-23</date><risdate>2019</risdate><volume>15</volume><issue>10</issue><spage>e1008117</spage><epage>e1008117</epage><pages>e1008117-e1008117</pages><issn>1553-7374</issn><issn>1553-7366</issn><eissn>1553-7374</eissn><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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>31644574</pmid><doi>10.1371/journal.ppat.1008117</doi><tpages>33</tpages><orcidid>https://orcid.org/0000-0003-3894-2362</orcidid><orcidid>https://orcid.org/0000-0001-8776-4211</orcidid><orcidid>https://orcid.org/0000-0001-7615-3079</orcidid><orcidid>https://orcid.org/0000-0002-0410-1963</orcidid><orcidid>https://orcid.org/0000-0001-7556-1564</orcidid><orcidid>https://orcid.org/0000-0002-4965-3286</orcidid><orcidid>https://orcid.org/0000-0002-3120-1038</orcidid><orcidid>https://orcid.org/0000-0002-2216-1361</orcidid><orcidid>https://orcid.org/0000-0003-2617-4263</orcidid><orcidid>https://orcid.org/0000-0002-9081-3894</orcidid><orcidid>https://orcid.org/0000-0003-2850-3470</orcidid><orcidid>https://orcid.org/0000-0003-3476-1855</orcidid><orcidid>https://orcid.org/0000-0003-0943-6823</orcidid><orcidid>https://orcid.org/0000-0002-6910-9202</orcidid><oa>free_for_read</oa></addata></record> |
fulltext | fulltext |
identifier | ISSN: 1553-7374 |
ispartof | PLoS pathogens, 2019-10, Vol.15 (10), p.e1008117-e1008117 |
issn | 1553-7374 1553-7366 1553-7374 |
language | eng |
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source | MEDLINE; DOAJ Directory of Open Access Journals; Recercat; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; PubMed Central Open Access; Public Library of Science (PLoS) |
subjects | 3D printing Amplification Animal diseases Animals Arvicolinae Atoms Biology Biology and Life Sciences Central Nervous System - pathology Cofactors Dextran Dextran sulfate Dextran Sulfate - pharmacology Dextrans Diagnostic imaging Disease Disease Models, Animal Food safety Heterogeneity High resolution High resolution spectroscopy In vitro methods and tests Magnetic resonance imaging Mammals Medicine and Health Sciences Mice, Transgenic Molecular modelling Neuroimaging NMR Novels Nuclear magnetic resonance Nuclear Magnetic Resonance, Biomolecular - methods Particles Partícules (Matèria) Pathogenic microorganisms Pharmacy Physical Sciences Polysaccharides Prion diseases Prion Diseases - pathology Prion protein Prion Proteins - metabolism Prions Prions (Proteins) Prions - metabolism Propagation Protein folding Protein purification Protein Structure, Tertiary Proteins Proteostasis Deficiencies - pathology Public health Research and Analysis Methods Shaking Sonication Sulfates Supervision Transmissible spongiform encephalopathy Àtoms |
title | 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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