Cargando…
Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity
During prion infection, the normal, protease-sensitive conformation of prion protein (PrP(C)) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrP(Sc)). In vitro, protein misfolding cyclic amplification (PMCA) uses PrP(Sc) in...
Autores principales: | , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2013
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3728029/ https://www.ncbi.nlm.nih.gov/pubmed/23936256 http://dx.doi.org/10.1371/journal.pone.0071081 |
_version_ | 1782278795247484928 |
---|---|
author | Timmes, Andrew G. Moore, Roger A. Fischer, Elizabeth R. Priola, Suzette A. |
author_facet | Timmes, Andrew G. Moore, Roger A. Fischer, Elizabeth R. Priola, Suzette A. |
author_sort | Timmes, Andrew G. |
collection | PubMed |
description | During prion infection, the normal, protease-sensitive conformation of prion protein (PrP(C)) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrP(Sc)). In vitro, protein misfolding cyclic amplification (PMCA) uses PrP(Sc) in prion-infected brain homogenates as an initiating seed to convert PrP(C) and trigger the self-propagation of PrP(Sc) over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrP(Sc). More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrP(Sc) seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrP(Sc). However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res. |
format | Online Article Text |
id | pubmed-3728029 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-37280292013-08-09 Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity Timmes, Andrew G. Moore, Roger A. Fischer, Elizabeth R. Priola, Suzette A. PLoS One Research Article During prion infection, the normal, protease-sensitive conformation of prion protein (PrP(C)) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrP(Sc)). In vitro, protein misfolding cyclic amplification (PMCA) uses PrP(Sc) in prion-infected brain homogenates as an initiating seed to convert PrP(C) and trigger the self-propagation of PrP(Sc) over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrP(Sc). More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrP(Sc) seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrP(Sc). However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res. Public Library of Science 2013-07-30 /pmc/articles/PMC3728029/ /pubmed/23936256 http://dx.doi.org/10.1371/journal.pone.0071081 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. |
spellingShingle | Research Article Timmes, Andrew G. Moore, Roger A. Fischer, Elizabeth R. Priola, Suzette A. Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity |
title | Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity |
title_full | Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity |
title_fullStr | Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity |
title_full_unstemmed | Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity |
title_short | Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity |
title_sort | recombinant prion protein refolded with lipid and rna has the biochemical hallmarks of a prion but lacks in vivo infectivity |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3728029/ https://www.ncbi.nlm.nih.gov/pubmed/23936256 http://dx.doi.org/10.1371/journal.pone.0071081 |
work_keys_str_mv | AT timmesandrewg recombinantprionproteinrefoldedwithlipidandrnahasthebiochemicalhallmarksofaprionbutlacksinvivoinfectivity AT moorerogera recombinantprionproteinrefoldedwithlipidandrnahasthebiochemicalhallmarksofaprionbutlacksinvivoinfectivity AT fischerelizabethr recombinantprionproteinrefoldedwithlipidandrnahasthebiochemicalhallmarksofaprionbutlacksinvivoinfectivity AT priolasuzettea recombinantprionproteinrefoldedwithlipidandrnahasthebiochemicalhallmarksofaprionbutlacksinvivoinfectivity |