scholarly journals The effects of prion protein proteolysis and disaggregation on the strain properties of hamster scrapie

2008 ◽  
Vol 89 (10) ◽  
pp. 2642-2650 ◽  
Author(s):  
Andrea M. Deleault ◽  
Nathan R. Deleault ◽  
Brent T. Harris ◽  
Judy R. Rees ◽  
Surachai Supattapone
Keyword(s):  
Physical Properties ◽  
Conformational Stability ◽  
Proteinase K ◽  
Prion Strain ◽  
Biochemical Characteristics ◽  
Strain Diversity ◽  
Protease Digestion ◽  
Scrapie Strains ◽  
Strain Dependent ◽  
Mammalian Prions

Native mammalian prions exist in self-propagating strains that exhibit distinctive clinical, pathological and biochemical characteristics. Prion strain diversity is associated with variations in PrPSc conformation, but it remains unknown precisely which physical properties of the PrPSc molecules are required to encipher mammalian prion strain phenotypes. In this study, we subjected prion-infected brain homogenates derived from three different hamster scrapie strains to either (i) proteinase K digestion or (ii) sonication, and inoculated the modified samples into normal hamsters. The results show that the strain-specific clinical features and neuropathological profiles of inoculated animals were not affected by either treatment. Similarly, the strain-dependent biochemical characteristics of the PrPSc molecules (including electrophoretic mobility, glycoform composition, conformational stability and susceptibility to protease digestion) in infected animals were unaffected by either proteolysis or sonication of the original inocula. These results indicate that the infectious strain properties of native prions do not appear to be altered by PrPSc disaggregation, and that maintenance of such properties does not require the N-domain (approximately residues 23–90) of the protease-resistant PrPSc molecules or protease-sensitive PrPSc molecules.

scholarly journals The Region Approximately between Amino Acids 81 and 137 of Proteinase K-Resistant PrPSc Is Critical for the Infectivity of the Chandler Prion Strain

2009 ◽  
Vol 83 (8) ◽  
pp. 3852-3860 ◽  
Author(s):  
Ryo Shindoh ◽  
Chan-Lan Kim ◽  
Chang-Hyun Song ◽  
Rie Hasebe ◽  
Motohiro Horiuchi
Keyword(s):  
Amino Acids ◽  
Guanidine Hydrochloride ◽  
Conformational Stability ◽  
Terminal Region ◽  
Proteinase K ◽  
Prion Strain ◽  
Prion Infectivity ◽  
Prion Strains ◽  
Incubation Periods ◽  
The Relationship

ABSTRACT Although the major component of the prion is believed to be the oligomer of PrPSc, little information is available concerning regions on the PrPSc molecule that affect prion infectivity. During the analysis of PrPSc molecules from various prion strains, we found that PrPSc of the Chandler strain showed a unique property in the conformational-stability assay, and this property appeared to be useful for studying the relationship between regions of the PrPSc molecule and prion infectivity. Thus, we analyzed PrPSc of the Chandler strain in detail and analyzed the infectivities of the N-terminally denatured and truncated forms of proteinase K-resistant PrP. The N-terminal region of PrPSc of the Chandler strain showed region-dependent resistance to guanidine hydrochloride (GdnHCl) treatment. The region approximately between amino acids (aa) 81 and 137 began to be denatured by treatment with 1.5 M GdnHCl. Within this stretch, the region comprising approximately aa 81 to 90 was denatured almost completely by 2 M GdnHCl. Furthermore, the region approximately between aa 90 and 137 was denatured completely by 3 M GdnHCl. However, the C-terminal region thereafter was extremely resistant to the GdnHCl treatment. This property was not observed in PrPSc molecules of other prion strains. Denaturation of the region between aa 81 and 137 by 3 M GdnHCl significantly prolonged the incubation periods in mice compared to that for the untreated control. More strikingly, the denaturation and removal of this region nearly abolished the infectivity. This finding suggests that the conformation of the region between aa 81 and 137 of the Chandler strain PrPSc molecule is directly associated with prion infectivity.

scholarly journals Mouse-Adapted Ovine Scrapie Prion Strains Are Characterized by Different Conformers of PrPSc

2007 ◽  
Vol 81 (22) ◽  
pp. 12119-12127 ◽  
Author(s):  
Alana M. Thackray ◽  
Lee Hopkins ◽  
Michael A. Klein ◽  
Raymond Bujdoso
Keyword(s):  
Biological Diversity ◽  
Guanidine Hydrochloride ◽  
Serial Passage ◽  
Biological Properties ◽  
Proteinase K ◽  
Specific Information ◽  
Strain Typing ◽  
Prion Strain ◽  
Prion Strains ◽  
Scrapie Strains

ABSTRACT The agent responsible for prion disease may exist in different forms, commonly referred to as strains, with each carrying the specific information that determines its own distinct biological properties, such as incubation period and lesion profile. Biological strain typing of ovine scrapie isolates by serial passage in conventional mice has shown some diversity in ovine prion strains. However, this biological diversity remains poorly supported by biochemical prion strain typing. The protein-only hypothesis predicts that variation between different prion strains in the same host is manifest in different conformations adopted by PrPSc. Here we have investigated the molecular properties of PrPSc associated with two principal Prnp a mouse-adapted ovine scrapie strains, namely, RML and ME7, in order to establish biochemical prion strain typing strategies that may subsequently be used to discriminate field cases of mouse-passaged ovine scrapie isolates. We used a conformation-dependent immunoassay and a conformational stability assay, together with Western blot analysis, to demonstrate that RML and ME7 PrPSc proteins show distinct biochemical and physicochemical properties. Although RML and ME7 PrPSc proteins showed similar resistance to proteolytic digestion, they differed in their glycoform profiles and levels of proteinase K (PK)-sensitive and PK-resistant isoforms. In addition, the PK-resistant core (PrP27-30) of ME7 was conformationally more stable following exposure to guanidine hydrochloride or Sarkosyl than was RML PrP27-30. Our data show that mouse-adapted ovine scrapie strains can be discriminated by their distinct conformers of PrPSc, which provides a basis to investigate their diversity at the molecular level.

scholarly journals Synergistic and strain-specific effects of bovine spongiform encephalopathy and scrapie prions in the cell-free conversion of recombinant prion protein

2006 ◽  
Vol 87 (12) ◽  
pp. 3753-3761 ◽  
Author(s):  
Martin Eiden ◽  
Gottfried J. Palm ◽  
Winfried Hinrichs ◽  
Ulrich Matthey ◽  
Ralph Zahn ◽  
...  
Keyword(s):  
Bovine Spongiform Encephalopathy ◽  
Prion Protein ◽  
De Novo ◽  
Proteinase K ◽  
Spongiform Encephalopathy ◽  
Cooperative Action ◽  
Specific Effects ◽  
Scrapie Strains ◽  
Recombinant Prion Protein

This study describes the conversion of murine PrPC by PrPSc from three different mouse scrapie strains (ME7, 87V and 22A) and from a mouse-passaged bovine spongiform encephalopathy (BSE) strain (BSE/Bl6). This was demonstrated by a modified, non-radioactive, cell-free conversion assay using bacterial prion protein, which was converted into a proteinase K (PK)-resistant fragment designated PrPres. Using this assay, newly formed PrPres could be detected by an antibody that discriminated de novo PrPres and the original PrPSc seed. The results suggested that PrPres formation occurs in three phases: the first 48 h when PrPres formation is delayed, followed by a period of substantially accelerated PrPres formation and a plateau phase when a maximum concentration of PrPres is reached after 72 h. The conversion of prokaryotically expressed PrPC by ME7 and BSE prions led to unglycosylated, PK-digested, abnormal PrPres fragments, which differed in molecular mass by 1 kDa. Therefore, prion strain phenotypes were retained in the cell-free conversion, even when recombinant PrPC was used as the substrate. Moreover, co-incubation of ME7 and BSE prions resulted in equal amounts of both ME7- and BSE-derived PrPres fragments (as distinguished by their different molecular sizes) and also in a significantly increased total amount of de novo-generated PrPres. This was found to be more than twice the amount of either strain when incubated separately. This result indicates a synergistic effect of both strains during cell-free conversion. It is not yet known whether such a cooperative action between BSE and scrapie prions also occurs in vivo.

scholarly journals Access of proteinase K to partially translocated nascent polypeptides in intact and detergent-solubilized membranes.

1989 ◽  
Vol 108 (2) ◽  
pp. 299-307 ◽  
Author(s):  
T Connolly ◽  
P Collins ◽  
R Gilmore
Keyword(s):  
Amino Acid ◽  
Proteinase K ◽  
Low Molecular Weight ◽  
Amino Acid Residues ◽  
Detergent Solubilization ◽  
Protease Digestion ◽  
Cytoplasmic Face ◽  
Nascent Chain ◽  
Microsomal Membranes ◽  
Vesicular Stomatitis

We have used proteinase K as a probe to detect cytoplasmically and luminally exposed segments of nascent polypeptides undergoing transport across mammalian microsomal membranes. A series of translocation intermediates consisting of discrete-sized nascent chains was prepared by including microsomal membranes in cell-free translations of mRNAs lacking termination codons. The truncated mRNAs were derived from preprolactin and the G protein of vesicular stomatitis virus and encoded nascent chains ranging between 64 and 200 amino acid residues long. Partially translocated nascent chains of 100 amino acid residues or less were insensitive to protease digestion from the external surface of the membrane while longer nascent chains were susceptible to digestion by externally added protease. We conclude that the increased protease sensitivity of larger nascent chains is due to the exposure of a segment of the nascent polypeptide on the cytoplasmic face of the membrane. In contrast, low molecular weight nascent chains were remarkably resistant to protease digestion even after detergent solubilization of the membrane. The protease resistant behaviour of detergent solubilized nascent chains could be abolished by release of the polypeptide from the ribosome or by the addition of protein denaturants. We propose that the protease resistance of partially translocated nascent chains can be ascribed to components of the translocation apparatus that remain bound to the nascent chain after detergent solubilization of the membrane.

scholarly journals PrPSc-Specific Antibody Reveals C-Terminal Conformational Differences between Prion Strains

2016 ◽  
Vol 90 (10) ◽  
pp. 4905-4913 ◽  
Author(s):  
Eri Saijo ◽  
Andrew G. Hughson ◽  
Gregory J. Raymond ◽  
Akio Suzuki ◽  
Motohiro Horiuchi ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Enzyme Linked Immunosorbent Assay ◽  
Strain Variation ◽  
Prion Strains ◽  
Disease Biology ◽  
Folded Structure ◽  
C Terminus ◽  
Wide Range ◽  
Scrapie Strains ◽  
Strain Dependent

ABSTRACTUnderstanding the structure of PrPScand its strain variation has been one of the major challenges in prion disease biology. To study the strain-dependent conformations of PrPSc, we purified proteinase-resistant PrPSc(PrPRES) from mouse brains with three different murine-adapted scrapie strains (Chandler, 22L, and Me7) and systematically tested the accessibility of epitopes of a wide range of anti-PrP and anti-PrPScspecific antibodies by indirect enzyme-linked immunosorbent assay (ELISA). We found that epitopes of most anti-PrP antibodies were hidden in the folded structure of PrPRES, even though these epitopes are revealed with guanidine denaturation. However, reactivities to a PrPSc-specific conformational C-terminal antibody showed significant differences among the three different prion strains. Our results provide evidence for strain-dependent conformational variation near the C termini of molecules within PrPScmultimers.IMPORTANCEIt has long been apparent that prion strains can have different conformations near the N terminus of the PrPScprotease-resistant core. Here, we show that a C-terminal conformational PrPSc-specific antibody reacts differently to three murine-adapted scrapie strains. These results suggest, in turn, that conformational differences in the C terminus of PrPScalso contribute to the phenotypic distinction between prion strains.

scholarly journals Prion strain-dependent tropism is maintained between spleen and granuloma and relies on lymphofollicular structures

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Iman Al-Dybiat ◽  
Mohammed Moudjou ◽  
Davy Martin ◽  
Fabienne Reine ◽  
Laetitia Herzog ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Lymphoid Tissue ◽  
Detection Method ◽  
Prion Diseases ◽  
Follicular Dendritic Cells ◽  
Prion Strain ◽  
Strain Type ◽  
Lymphoid Structures ◽  
Nonlymphoid Tissue ◽  
Strain Dependent

Abstract In peripherally acquired prion diseases, prions move through several tissues of the infected host, notably in the lymphoid tissue, long before the occurrence of neuroinvasion. Accumulation can even be restricted to the lymphoid tissue without neuroinvasion and clinical disease. Several experimental observations indicated that the presence of differentiated follicular dendritic cells (FDCs) in the lymphoid structures and the strain type are critical determinants of prion extraneural replication. In this context, the report that granulomatous structures apparently devoid of FDCs could support prion replication raised the question of the requirements for prion lymphotropism. The report also raised the possibility that nonlymphoid tissue-tropic prions could actually target these inflammatory structures. To investigate these issues, we examined the capacity of closely related prions, albeit with opposite lymphotropism (or FDC dependency), for establishment in experimentally-induced granuloma in ovine PrP transgenic mice. We found a positive correlation between the prion capacity to accumulate in the lymphoid tissue and granuloma, regardless of the prion detection method used. Surprisingly, we also revealed that the accumulation of prions in granulomas involved lymphoid-like structures associated with the granulomas and containing cells that stain positive for PrP, Mfge-8 but not CD45 that strongly suggest FDCs. These results suggest that the FDC requirement for prion replication in lymphoid/inflammatory tissues may be strain-dependent.

scholarly journals Alteration of Prion Strain Emergence by Nonhost Factors

mSphere ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Sara A. M. Holec ◽  
Qi Yuan ◽  
Jason C. Bartz
Keyword(s):  
Protein Misfolding ◽  
Cumulative Effect ◽  
Brain Homogenate ◽  
Proteinase K ◽  
Silty Clay ◽  
Surface Binding ◽  
Prion Strain ◽  
Weathering Processes ◽  
Mechanism Of Inhibition ◽  
Environmental Treatment

ABSTRACT Prions can persist in the environment for extended periods of time after adsorption to surfaces, including soils, feeding troughs, or fences. Prion strain- and soil-specific differences in prion adsorption, infectivity, and response to inactivation may be involved in strain maintenance or emergence of new strains in a population. Extensive proteinase K (PK) digestion of Hyper (HY) and Drowsy (DY) PrPSc resulted in a greater reduction in the level of DY PrPSc than of HY PrPSc. Use of the PK-digested material in protein misfolding cyclic amplification strain interference (PMCAsi) resulted in earlier emergence of HY PrPSc than of undigested controls. This result established that strain-specific alteration of the starting ratios of conversion-competent HY and DY PrPSc can alter strain emergence. We next investigated whether environmentally relevant factors such as surface binding and weathering could alter strain emergence. Adsorption of HY and DY PrPSc to silty clay loam (SCL), both separately and combined, resulted in DY interfering with the emergence of HY in PMCAsi in a manner similar to that seen with unbound controls. Similarly, repeated cycles of wetting and drying of SCL-bound HY and DY PrPSc did not alter the emergence of HY PrPSc compared to untreated controls. Importantly, these data indicate that prion strain interference can occur when prions are bound to surfaces. Interestingly, we found that drying of adsorbed brain homogenate on SCL could restore its ability to interfere with the emergence of HY, suggesting a novel strain interference mechanism. Overall, these data provide evidence that the emergence of a strain from a mixture can be influenced by nonhost factors. IMPORTANCE The prion strain, surface type, and matrix containing PrPSc can influence PrPSc surface adsorption. The cumulative effect of these factors can result in strain- and soil-specific differences in prion bioavailability. Environmental weathering processes can result in decreases in PrPSc conversion efficiency and infectivity. Little is known about how incomplete inactivation of surface-bound PrPSc affects transmission and prion strain emergence. Here, we show that strain interference occurs with soil-bound prions and that altering the ratios of prion strains by strain-specific inactivation can affect strain emergence. Additionally, we identify a novel mechanism of inhibition of prion conversion by environmental treatment-induced changes at the soil-protein interface altering strain emergence. These novel findings suggest that environmental factors can influence strain emergence of surface-bound prions.

scholarly journals Characterization of goat prions demonstrates geographical variation of scrapie strains in Europe and reveals the composite nature of prion strains

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Romolo Nonno ◽  
Alba Marin-Moreno ◽  
Juan Carlos Espinosa ◽  
Christine Fast ◽  
Lucien Van Keulen ◽  
...  
Keyword(s):  
Small Ruminants ◽  
Direct Interaction ◽  
Biological Properties ◽  
Spongiform Encephalopathy ◽  
Strain Diversity ◽  
Wide Range ◽  
Strain Components ◽  
Composite Strain ◽  
Composite Nature ◽  
Scrapie Strains

AbstractBovine Spongiform Encephalopathy (BSE) is the only animal prion which has been recognized as a zoonotic agent so far. The identification of BSE in two goats raised the need to reliably identify BSE in small ruminants. However, our understanding of scrapie strain diversity in small ruminants remains ill-defined, thus limiting the accuracy of BSE surveillance and spreading fear that BSE might lurk unrecognized in goats. We investigated prion strain diversity in a large panel of European goats by a novel experimental approach that, instead of assessing the neuropathological profile after serial transmissions in a single animal model, was based on the direct interaction of prion isolates with several recipient rodent models expressing small ruminants or heterologous prion proteins. The findings show that the biological properties of scrapie isolates display different patterns of geographical distribution in Europe and suggest that goat BSE could be reliably discriminated from a wide range of biologically and geographically diverse goat prion isolates. Finally, most field prion isolates showed composite strain features, with discrete strain components or sub-strains being present in different proportions in individual goats or tissues. This has important implications for understanding the nature and evolution of scrapie strains and their transmissibility to other species, including humans.

scholarly journals Successful Transmission of Three Mouse-Adapted Scrapie Strains to Murine Neuroblastoma Cell Lines Overexpressing Wild-Type Mouse Prion Protein

2000 ◽  
Vol 74 (1) ◽  
pp. 320-325 ◽  
Author(s):  
Noriyuki Nishida ◽  
David A. Harris ◽  
Didier Vilette ◽  
Hubert Laude ◽  
Yveline Frobert ◽  
...  
Keyword(s):  
Prion Protein ◽  
Cell Lines ◽  
Ex Vivo ◽  
Neuroblastoma Cell ◽  
Wild Type Mouse ◽  
Proteinase K ◽  
Transmission Model ◽  
Successful Transmission ◽  
Scrapie Strains ◽  
Neuroblastoma Cell Lines

ABSTRACT Propagation of the agents responsible for transmissible spongiform encephalopathies (TSEs) in cultured cells has been achieved for only a few cell lines. To establish efficient and versatile models for transmission, we developed neuroblastoma cell lines overexpressing type A mouse prion protein, MoPrPC-A, and then tested the susceptibility of the cells to several different mouse-adapted scrapie strains. The transfected cell clones expressed up to sixfold-higher levels of PrPC than the untransfected cells. Even after 30 passages, we were able to detect an abnormal proteinase K-resistant form of prion protein, PrPSc, in the agent-inoculated PrP-overexpressing cells, while no PrPSc was detectable in the untransfected cells after 3 passages. Production of PrPSc in these cells was also higher and more stable than that seen in scrapie-infected neuroblastoma cells (ScN2a). The transfected cells were susceptible to PrPSc-A strains Chandler, 139A, and 22L but not to PrPSc-B strains 87V and 22A. We further demonstrate the successful transmission of PrPSc from infected cells to other uninfected cells. Our results corroborate the hypothesis that the successful transmission of agents ex vivo depends on both expression levels of host PrPC and the sequence of PrPSc. This new ex vivo transmission model will facilitate research into the mechanism of host-agent interactions, such as the species barrier and strain diversity, and provides a basis for the development of highly susceptible cell lines that could be used in diagnostic and therapeutic approaches to the TSEs.

scholarly journals Host Determinants of Prion Strain Diversity Independent of Prion Protein Genotype

2015 ◽  
Vol 89 (20) ◽  
pp. 10427-10441 ◽  
Author(s):  
Jenna Crowell ◽  
Andrew Hughson ◽  
Byron Caughey ◽  
Richard A. Bessen
Keyword(s):  
Transgenic Mice ◽  
Prion Protein ◽  
Protein Gene ◽  
Phenotypic Diversity ◽  
Prion Diseases ◽  
Conformational Stability ◽  
Epigenetic Mechanism ◽  
Prion Strain ◽  
Disease Phenotypes ◽  
Prion Strains

ABSTRACTPhenotypic diversity in prion diseases can be specified by prion strains in which biological traits are propagated through an epigenetic mechanism mediated by distinct PrPScconformations. We investigated the role of host-dependent factors on phenotypic diversity of chronic wasting disease (CWD) in different host species that express the same prion protein gene (Prnp). Two CWD strains that have distinct biological, biochemical, and pathological features were identified in transgenic mice that express the Syrian golden hamster (SGH)Prnp. The CKY strain of CWD had a shorter incubation period than the WST strain of CWD, but after transmission to SGH, the incubation period of CKY CWD was ∼150 days longer than WST CWD. Limited proteinase K digestion revealed strain-specific PrPScpolypeptide patterns that were maintained in both hosts, but the solubility and conformational stability of PrPScdiffered for the CWD strains in a host-dependent manner. WST CWD produced PrPScamyloid plaques in the brain of the SGH that were partially insoluble and stable at a high concentration of protein denaturant. However, in transgenic mice, PrPScfrom WST CWD did not assemble into plaques, was highly soluble, and had low conformational stability. Similar studies using the HY and DY strains of transmissible mink encephalopathy resulted in minor differences in prion biological and PrPScproperties between transgenic mice and SGH. These findings indicate that host-specific pathways that are independent ofPrnpcan alter the PrPScconformation of certain prion strains, leading to changes in the biophysical properties of PrPSc, neuropathology, and clinical prion disease.IMPORTANCEPrions are misfolded pathogenic proteins that cause neurodegeneration in humans and animals. Transmissible prion diseases exhibit a spectrum of disease phenotypes and the basis of this diversity is encoded in the structure of the pathogenic prion protein and propagated by an epigenetic mechanism. In the present study, we investigated prion diversity in two hosts species that express the same prion protein gene. While prior reports have demonstrated that prion strain properties are stable upon infection of the same host species and prion protein genotype, our findings indicate that certain prion strains can undergo dramatic changes in biological properties that are not dependent on the prion protein. Therefore, host factors independent of the prion protein can affect prion diversity. Understanding how host pathways can modify prion disease phenotypes may provide clues on how to alter prion formation and lead to treatments for prion, and other, human neurodegenerative diseases of protein misfolding.

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