Characterization of Conformation-dependent Prion Protein Epitopes

Kang, Hyung-Lyun; Weng, Chu Chun; Saijo, Eri; Saylor, Vicki; Bian, Jifeng; Kim, Sehun; Ramos, Laylaa; Angers, Rachel; Langenfeld, Katie; Khaychuk, Vadim; Calvi, Carla; Bartz, Jason C.; Hunter, Nora; Telling, Glenn C.

doi:10.1074/jbc.m112.395921

Cited by 30 publications

(31 citation statements)

References 58 publications

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“…For serial PMCA, 6 μL of amplified material was diluted into 54 μL of fresh normal brain homogenate. Western blots were developed using anti-PrP mAbs 6H4 (Prionics AG) or PRC5 (32).…”

Section: Methodsmentioning

confidence: 99%

Structural effects of PrP polymorphisms on intra- and interspecies prion transmission

Angers

Christiansen

Nalls

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Understanding the molecular parameters governing prion propagation is crucial for controlling these lethal, proteinaceous, and infectious neurodegenerative diseases. To explore the effects of prion protein (PrP) sequence and structural variations on intra-and interspecies transmission, we integrated studies in deer, a species naturally susceptible to chronic wasting disease (CWD), a burgeoning, contagious epidemic of uncertain origin and zoonotic potential, with structural and transgenic (Tg) mouse modeling and cell-free prion amplification. CWD properties were faithfully maintained in deer following passage through Tg mice expressing cognate PrP, and the influences of naturally occurring PrP polymorphisms on CWD susceptibility were accurately reproduced in Tg mice or cellfree systems. Although Tg mice also recapitulated susceptibility of deer to sheep prions, polymorphisms that provided protection against CWD had distinct and varied influences. Whereas substitutions at residues 95 and 96 in the unstructured region affected CWD propagation, their protective effects were overridden during replication of sheep prions in Tg mice and, in the case of residue 96, deer. The inhibitory effects on sheep prions of glutamate at residue 226 in elk PrP, compared with glutamine in deer PrP, and the protective effects of the phenylalanine for serine substitution at the adjacent residue 225, coincided with structural rearrangements in the globular domain affecting interaction between α-helix 3 and the loop between β2 and α-helix 2. These structure-function analyses are consistent with previous structural investigations and confirm a role for plasticity of this tertiary structural epitope in the control of PrP conversion and strain propagation.protein structure | prion replication | protective polymorphisms | structural plasticity

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“…For serial PMCA, 6 μL of amplified material was diluted into 54 μL of fresh normal brain homogenate. Western blots were developed using anti-PrP mAbs 6H4 (Prionics AG) or PRC5 (32).…”

Section: Methodsmentioning

confidence: 99%

Structural effects of PrP polymorphisms on intra- and interspecies prion transmission

Angers

Christiansen

Nalls

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…PMCA reactions were performed as described previously (32) at a seed to substrate ratio of 1:4,000, digested with PK at a final concentration of 0.33 μg/μL, and analyzed by Western blotting using anti-PrP mAb 6H4 (Prionics AG) or PRC5 (61).…”

Section: Methodsmentioning

confidence: 99%

Quinacrine promotes replication and conformational mutation of chronic wasting disease prions

Bian

Kang

Telling

2014

Proc. Natl. Acad. Sci. U.S.A.

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Quinacrine's ability to reduce levels of pathogenic prion protein (PrP Sc ) in mouse cells infected with experimentally adapted prions led to several unsuccessful clinical studies in patients with prion diseases, a 10-y investment to understand its mechanism of action, and the production of related compounds with expectations of greater efficacy. We show here, in stark contrast to this reported inhibitory effect, that quinacrine enhances deer and elk PrP Sc accumulation and promotes propagation of prions causing chronic wasting disease (CWD), a fatal, transmissible, neurodegenerative disorder of cervids of uncertain zoonotic potential. Surprisingly, despite increased prion titers in quinacrine-treated cells, transmission of the resulting prions produced prolonged incubation times and altered PrP Sc deposition patterns in the brains of diseased transgenic mice. This unexpected outcome is consistent with quinacrine affecting the intrinsic properties of the CWD prion. Accordingly, quinacrine-treated CWD prions were comprised of an altered PrP Sc conformation. Our findings provide convincing evidence for drug-induced conformational mutation of prions without the prerequisite of generating drug-resistant variants of the original strain. More specifically, they show that a drug capable of restraining prions in one species/strain setting, and consequently used to treat human prion diseases, improves replicative ability in another and therefore force reconsideration of current strategies to screen antiprion compounds.prion therapeutics | prion enhancing drugs P rions are protein-based infectious agents that cause an increasing variety of fatal, infectious neurodegenerative disorders, frequently as epidemics. Variant Creutzfeldt-Jakob disease (CJD) resulting from bovine spongiform encephalopathy exemplifies prion zoonosis, and the continued, unpredictable emergence of additional epidemics in other species, particularly chronic wasting disease (CWD) in deer, elk, and moose, raises additional concerns. Its unprecedented contagious spread, widening host range, and conflicting evidence surrounding its zoonotic potential place CWD at the forefront of public health concerns. Although the notion, that prion replication occurs by corruption of host-encoded cellular prion protein (PrP C ) by abnormally conformed PrP Sc , is now widely accepted, the details of this mechanism remain enigmatic.Except under certain experimental conditions, treatments capable of arresting or of effectively modifying the course of disease do not exist for prion disorders. Compounds targeting prevention of PrP misfolding have been aggressively pursued as therapeutics. Cells chronically infected with rodent prions are used as a means either to screen compounds inhibiting PrP Sc accumulation (1-5) or to confirm the proposed antiprion effects of compounds discovered by other means (6, 7). Such strategies rest on the assumption that compounds with efficacy against experimentally adapted rodent prions will be effective against naturally occurring prions, in ...

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“…23 Likewise human, bank vole, or mouse PrP C expressing RK13 transfectants also failed to propagate certain prion isolates. 13,24 The ability of RK13 cells to propagate prions upon expression of exogenous PrP C from multiple species 12,13,16,17 clearly confirmed that RK13 cells have the required cellular machinery for efficient prion propagation. Although PRNP haplotype 3 and 4 caprine scrapie inocula were able to produce clinical scrapie in recipient goats 9 and Tg338 mice 21 which was very similar to PRNP haplotype 1 and 2 caprine scrapie inoculated animals, the lack of scrapie prion propagation in cpRK13cells when inoculated with the same PRNP haplotypes 3 and 4 scrapie inocula highlight the possible involvement of other unidentified factors to establish successful infection.…”

Section: Cprk13 Cells Support Propagation Of Classical Caprine Scrapimentioning

confidence: 82%

“…12,13,16,17 PCR amplified caprine PRNP haplotype 2 was cloned into a mammalian expression plasmid (pIRESpuro3-cp) (Fig. S1A).…”

Section: Resultsmentioning

confidence: 99%

A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

Dassanayake

Zhuang

Truscott

et al. 2016

Prion

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ABSTRACT. To assess scrapie infectivity associated with caprine-origin tissues, bioassay can be performed using kids, lambs or transgenic mice expressing caprine or ovine prion (PRNP) alleles, but the incubation periods are fairly long. Although several classical ovine scrapie prion permissive cell lines with the ability to detect brain-derived scrapie prion have been available, no classical caprine scrapie permissive cell line is currently available. Therefore, the aims of this study were to generate a rabbit kidney epithelial cell line (RK13) stably expressing caprine wild-type PRNP (cpRK13) and then to assess permissiveness of cpRK13 cells to classical caprine scrapie prion propagation. The cpRK13 and plasmid control RK13 (pcRK13) cells were incubated with brain-derived classical caprine scrapie inocula prepared from goats or ovinized transgenic mice (Tg338, express ovine VRQ allele) infected with caprine scrapie. Significant PrP Sc accumulation, which is indicative of scrapie prion propagation, was detected by TSE ELISA and immunohistochemistry in cpRK13 cells inoculated with classical caprine scrapie inocula. Western blot analysis revealed the typical proteinase K-resistant 3 PrP res isoforms in the caprine scrapie prion inoculated cpRK13 cell lysate. Importantly, PrP Sc accumulation was not detected in similarly inoculated pcRK13 cells, whether by TSE ELISA, immunohistochemistry, or western blot. These findings suggest that caprine scrapie prions can be propagated in cpRK13 cells, thus this cell line may be a useful tool for the assessment of classical caprine prions in the brain tissues of goats.

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Characterization of Conformation-dependent Prion Protein Epitopes

Cited by 30 publications

References 58 publications

Structural effects of PrP polymorphisms on intra- and interspecies prion transmission

Structural effects of PrP polymorphisms on intra- and interspecies prion transmission

Quinacrine promotes replication and conformational mutation of chronic wasting disease prions

A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

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