Charge Neutralization of the Central Lysine Cluster in Prion Protein (PrP) Promotes PrPSc-like Folding of Recombinant PrP Amyloids

Groveman, Bradley R.; Kraus, Allison; Raymond, Lynne D.; Dolan, Michael; Anson, Kelsie J.; Dorward, David W.; Caughey, Byron

doi:10.1074/jbc.m114.619627

Cited by 43 publications

(55 citation statements)

References 58 publications

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“…rPrP Sen substrates were prepared, as previously described (46 (41), and stored at Ϫ80°C. For RT-QuIC analysis, brain homogenates were serially diluted in 0.1% SDS-N 2 (Gibco)-PBS, as previously reported (48); where indicated, the last dilution was performed in 0.05% SDS-N 2 -PBS.…”

Section: Methodsmentioning

confidence: 99%

Detection of Atypical H-Type Bovine Spongiform Encephalopathy and Discrimination of Bovine Prion Strains by Real-Time Quaking-Induced Conversion

et al. 2016

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Many mammalian species can be afflicted with prion diseases or transmissible spongiform encephalopathies. Prion diseases are neurodegenerative, transmissible, untreatable, and fatal (reviewed in references 1 and 2). The underlying pathogenesis of prion diseases involves the conversion of the host's normal prion protein, PrP C , to abnormal forms that are usually more protease resistant, multimeric, and insoluble. The multimeric and insoluble forms have been generically called PrP Sc (PrP-scrapie), or more functionally PrP Res (protease resistant) and PrP D (disease associated). At least some of these forms comprise the transmissible agent or prion (3-10). Multiple prion strains can be propagated within a given host species, giving consistently different clinical, pathological, and molecular phenotypes (11-16).The first prion disease to be recognized in cattle was classical bovine spongiform encephalopathy (C-BSE). C-BSE was likely caused primarily by widespread prion contamination of cattle feed (17). After peaking in the early 1990s, the incidence of C-BSE has now been greatly reduced by regulatory measures that limit its horizontal spread. C-BSE is the only known zoonotic prion disease, having caused variant Creutzfeldt-Jakob disease (vCJD) in humans who presumably consumed contaminated beef. Although new clinical cases of vCJD are rare (http://www.cjd.ed.ac .uk/documents/figs.pdf), a recent survey of appendices in the United Kingdom suggests a high incidence of subclinical vCJD infections, at ϳ1:2,000 of the population born between 1941 and 1985 (18).Since the C-BSE epidemic, two atypical strains of BSE, H-type BSE (H-BSE) and L-type BSE (L-BSE), have also been identified in cattle. PrPRes is usually composed of a mixture of glycosylated and unglycosylated molecules, and the various BSE strains can be differentiated biochemically using Western blotting of postmortem brain tissue samples by comparing the proteinase K (PK)-treated PrPRes banding patterns (19)(20)(21)(22). The H and L types of BSE are classified by their respective high and low apparent molecular masses of the unglycosylated PrP Res band. These atypical BSE strains, which are rare (Ͻ100 cases identified worldwide), tend to affect older animals (23) and appear to represent sporadic forms of bovine prion diseases (24). Despite the apparent rarity of the various types of BSE, the facts that H-and L-BSE appear to arise spontaneously and have distinct transmissibilities (20, 25-32) make it important to be able to detect and differentiate them to reduce the risk of transmission to cattle or other species, such as humans.Several in vitro methods have been developed to detect C-, L-, and H-BSE. Commercially available rapid immunochemical tests for PrP D can, in the best cases, give positive responses from 10 Ϫ3 to 10 Ϫ4 dilutions of postmortem brain tissues with high levels of PrP D (33, 34). Immunoblotting for PrP Res can detect BSE-infected tissues with similar sensitivity and also discriminate between the bovine strains based on the relative electro...

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Section: Methodsmentioning

confidence: 99%

Detection of Atypical H-Type Bovine Spongiform Encephalopathy and Discrimination of Bovine Prion Strains by Real-Time Quaking-Induced Conversion

et al. 2016

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“…Although the conformation of PrP Sc remains enigmatic, considerable evidence indicates that synthetic amyloid fibrils of PrP that are produced under physiologically compatible conditions in vitro, with (62,63) or without RES was treated with PK and several possible PK cleavage sites are at the C terminus of PrP, the C-terminal linear-epitope antibody R1 was used to examine whether the C-terminal epitopes were still present in PrP RES . OD 450 readings with background subtracted are indicated as the means and SD from triplicate wells.…”

Section: Discussionmentioning

confidence: 99%

PrP^Sc-Specific Antibody Reveals C-Terminal Conformational Differences between Prion Strains

Saijo

Hughson

Raymond

et al. 2016

J Virol

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Understanding the structure of PrPSc and its strain variation has been one of the major challenges in prion disease biology. To study the strain-dependent conformations of PrP Sc , we purified proteinase-resistant PrP Sc (PrP RES ) 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-PrP Sc specific antibodies by indirect enzyme-linked immunosorbent assay (ELISA). We found that epitopes of most anti-PrP antibodies were hidden in the folded structure of PrP RES , even though these epitopes are revealed with guanidine denaturation. However, reactivities to a PrP Sc -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 PrP Sc multimers. IMPORTANCEIt has long been apparent that prion strains can have different conformations near the N terminus of the PrP Sc protease-resistant core. Here, we show that a C-terminal conformational PrP Sc -specific antibody reacts differently to three murine-adapted scrapie strains. These results suggest, in turn, that conformational differences in the C terminus of PrP Sc also contribute to the phenotypic distinction between prion strains. T ransmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative diseases resulting in the accumulation of the misfolded form of prion protein (PrP) in the brain (1). Prions are disease-causing infectious agents that lack agent-coding nucleic acids (1). The normal cellular glycoprotein PrP (PrP C ), which is typically bound to a carboxyl-terminal glycosylphosphatidylinositol (GPI) anchor, can undergo major conformational changes into pathogenic disease-causing forms of PrP (PrP Sc ). This conversion is induced by the binding and templating effects of preexisting PrP Sc (2). Relative to PrP C , PrP Sc tends to be rich in ␤-sheets, detergent insoluble, oligomeric or fibrillar, and partially resistant to proteinase K (PK) digestion. PK treatment of PrP Sc typically produces a PK-resistant carboxyl-terminal core referred to as PrP RES or PrP(27-30) (3, 4). Although there is increasing evidence that protease-sensitive forms of disease-associated PrP can also exist in the brains of humans and animals affected with prion diseases (5-8), the presence of PrP RES is a major diagnostic indicator of prion diseases. However, the detailed threedimensional structures of PrP Sc and its variants remain a mystery. One approach to probing prion structures and studying prion pathogenesis has been the development of PrP Sc -and/or PrP RESselective antibodies (9-21). Despite some successes, the development of PrP Sc -specific antibodies with diverse epitopes has been limited by the fact that PrP Sc has the same primary sequence as PrP C . This requires PrP Sc -specific epitopes to be conformational. However, such potentially...

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“…The best characterized forms of PrP D are amyloid fibrils that have tightly packed and proteinase K (PK)-resistant (PrP Res ) cores ranging from residues ϳ80 -90 to ϳ231 (20 -23). However, in attempting to reconstitute the PrP C -toPrP D conversion under chemically defined cell-free conditions with purified recombinant PrP C , many researchers have observed that the C-terminal domain of residues ϳ160 -231 is much more easily converted to a PK-resistant, parallel in-register intermolecular ␤-sheet amyloid core than the more N-terminal residues that include prolines 102 and 105 and the CLC (12,20,24). Recently, we reported that the CLC strongly impedes the incorporation of residues ϳ90 to ϳ159 into a PrP D -like amyloid core (12).…”

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confidence: 99%

“…However, although the pathogenic potential of the P102L and P105L mutants is evident, Pro-102 and Pro-105 are contained within an intrinsically disordered region of PrP C , and the structural constraints imposed by these proline residues in PrP are not clear. Furthermore, the field has not ascertained the structural role that Pro-102 and Pro-105 play within the central lysine cluster (CLC), a highly conserved region encompassing amino acids 101-110 that modulates cofactor binding (7), PrP conversion (11), and amyloidogenicity (12).…”

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Prion Protein Prolines 102 and 105 and the Surrounding Lysine Cluster Impede Amyloid Formation

Kraus

Anson

Raymond

et al. 2015

Journal of Biological Chemistry

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Background:We investigated how pathogenic prion protein mutations P102L and P105L lead to misfolding. Results: Mutations of these proline and adjacent lysine residues accelerated in vitro formation of amyloid with properties reminiscent of PrP Sc . Conclusion: Specific proline and lysine residues might delay spontaneous prion disease by hindering PrP conversion into amyloid. Significance: These findings suggest mechanisms for genetic prion diseases.

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Charge Neutralization of the Central Lysine Cluster in Prion Protein (PrP) Promotes PrPSc-like Folding of Recombinant PrP Amyloids

Cited by 43 publications

References 58 publications

Detection of Atypical H-Type Bovine Spongiform Encephalopathy and Discrimination of Bovine Prion Strains by Real-Time Quaking-Induced Conversion

Detection of Atypical H-Type Bovine Spongiform Encephalopathy and Discrimination of Bovine Prion Strains by Real-Time Quaking-Induced Conversion

PrP^Sc-Specific Antibody Reveals C-Terminal Conformational Differences between Prion Strains

Prion Protein Prolines 102 and 105 and the Surrounding Lysine Cluster Impede Amyloid Formation

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Charge Neutralization of the Central Lysine Cluster in Prion Protein (PrP) Promotes PrPSc-like Folding of Recombinant PrP Amyloids

Cited by 43 publications

References 58 publications

Detection of Atypical H-Type Bovine Spongiform Encephalopathy and Discrimination of Bovine Prion Strains by Real-Time Quaking-Induced Conversion

Detection of Atypical H-Type Bovine Spongiform Encephalopathy and Discrimination of Bovine Prion Strains by Real-Time Quaking-Induced Conversion

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

Prion Protein Prolines 102 and 105 and the Surrounding Lysine Cluster Impede Amyloid Formation

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PrP^Sc-Specific Antibody Reveals C-Terminal Conformational Differences between Prion Strains