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      Prion infectivity is encoded exclusively within the structure of proteinase K-resistant fragments of synthetically generated recombinant PrP Sc

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          Abstract

          Transmissible spongiform encephalopathies, also known as prion diseases, are a group of fatal neurodegenerative disorders affecting both humans and animals. The central pathogenic event in prion disease is the misfolding of normal prion protein (PrP C) into the pathogenic conformer, PrP Sc, which self-replicates by converting PrP C to more of itself. The biochemical hallmark of PrP Sc is its C-terminal resistance to proteinase K (PK) digestion, which has been historically used to define PrP Sc and is still the most widely used characteristic for prion detection. We used PK-resistance as a biochemical measure for the generation of recombinant prion from bacterially expressed recombinant PrP. However, the existence of both PK- resistant and -sensitive PrP Sc forms in animal and human prion disease led to the question of whether the in vitro-generated recombinant prion infectivity is due to the PK-resistant or -sensitive recombinant PrP forms. In this study, we compared undigested and PK-digested recombinant prions for their infectivity using both the classical rodent bioassay and the cell-based prion infectivity assay. Similar levels of infectivity were detected in PK-digested and -undigested samples by both assays. A time course study of recombinant prion propagation showed that the increased capability to seed the conversion of endogenous PrP in cultured cells coincided with an increase of the PK-resistant form of recombinant PrP. Moreover, prion infectivity diminished when recombinant prion was subjected to an extremely harsh PK digestion. These results demonstrated that the infectivity of recombinant prion is encoded within the structure of the PK-resistant PrP fragments. This characteristic of recombinant prion, that a simple PK digestion is able to eliminate all PK-sensitive (non-infectious) PrP species, makes possible a more homogenous material that will be ideal for dissecting the molecular basis of prion infectivity.

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          Generating a prion with bacterially expressed recombinant prion protein.

          The prion hypothesis posits that a misfolded form of prion protein (PrP) is responsible for the infectivity of prion disease. Using recombinant murine PrP purified from Escherichia coli, we created a recombinant prion with the attributes of the pathogenic PrP isoform: aggregated, protease-resistant, and self-perpetuating. After intracerebral injection of the recombinant prion, wild-type mice developed neurological signs in approximately 130 days and reached the terminal stage of disease in approximately 150 days. Characterization of diseased mice revealed classic neuropathology of prion disease, the presence of protease-resistant PrP, and the capability of serially transmitting the disease; these findings confirmed that the mice succumbed to prion disease. Thus, as postulated by the prion hypothesis, the infectivity in mammalian prion disease results from an altered conformation of PrP.
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            Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie.

            The 'protein only' hypothesis postulates that the prion, the agent causing transmissible spongiform encephalopathies, is PrP(Sc), an isoform of the host protein PrP(C). Protease treatment of prion preparations cleaves off approximately 60 N-terminal residues of PrP(Sc) but does not abrogate infectivity. Disruption of the PrP gene in the mouse abolishes susceptibility to scrapie and prion replication. We have introduced into PrP knockout mice transgenes encoding wild-type PrP or PrP lacking 26 or 49 amino-proximal amino acids which are protease susceptible in PrP(Sc). Inoculation with prions led to fatal disease, prion propagation and accumulation of PrP(Sc) in mice expressing both wild-type and truncated PrPs. Within the framework of the 'protein only' hypothesis, this means that the amino-proximal segment of PrP(C) is not required either for its susceptibility to conversion into the pathogenic, infectious form of PrP or for the generation of PrP(Sc).
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              The prion's elusive reason for being.

              The protein-only hypothesis posits that the infectious agent causing transmissible spongiform encephalopathies consists of protein and lacks any informational nucleic acids. This agent, termed prion by Stanley Prusiner, is thought to consist partly of PrP(Sc), a conformational isoform of a normal cellular protein termed PrP(C). Scientists and lay persons have been fascinated by the prion concept, and it has been subjected to passionate critique and intense experimental scrutiny. As a result, PrP(C) and its isoforms rank among the most intensively studied proteins encoded by the mammalian genome. Despite all this research, both the physiological function of PrP(C) and the molecular pathways leading to neurodegeneration in prion disease remain unknown. Here we review the salient traits of those diseases ascribed to improper behavior of the prion protein and highlight how the physiological functions of PrP(C) may help explain the toxic phenotypes observed in prion disease.
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                Author and article information

                Contributors
                Fei.Wang@vai.org
                Xinhe.Wang@vai.org
                rnn2222@yahoo.com
                Jiyan.Ma@vai.org
                Journal
                Acta Neuropathol Commun
                Acta Neuropathol Commun
                Acta Neuropathologica Communications
                BioMed Central (London )
                2051-5960
                24 April 2018
                24 April 2018
                2018
                : 6
                : 30
                Affiliations
                [1 ]ISNI 0000 0004 0406 2057, GRID grid.251017.0, Center for Neurodegenerative Science, Van Andel Research Institute, ; 333 Bostwick Avenue N.E., Grand Rapids, MI 49503 USA
                [2 ]ISNI 0000 0004 0404 7762, GRID grid.419615.e, National Institute of Oceanography and Fisheries (NIOF), ; Cairo, 11516 Egypt
                Author information
                http://orcid.org/0000-0001-7150-1898
                Article
                534
                10.1186/s40478-018-0534-0
                5921397
                29699569
                fa830b0e-b0fa-43f3-9c07-2a21d39ceeaa
                © The Author(s). 2018

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 12 March 2018
                : 11 April 2018
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000065, National Institute of Neurological Disorders and Stroke;
                Award ID: R01NS060729
                Award ID: R01NS071035
                Award Recipient :
                Categories
                Research
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                © The Author(s) 2018

                prion disease,prion,prpsc,recombinant prion,proteinase k-resistant,prion infectivity,protein conformation

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