A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization

Wüsten, Katharina A.; Reddy, Pasham Parameshwar; Smiyakin, Andrej; Bernis, María Eugenia; Tamgüney, Gültekin

doi:10.1038/s41598-018-32581-1

Cited by 4 publications

(7 citation statements)

References 55 publications

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“…In support of this, a previous study using cross‐linkable unnatural amino acids in recombinant protein demonstrated that dimerization is facilitated by the CR 59 . Dimerization has also been studied in cell culture using bioluminescent complementation, 60 native PAGE and cysteine crosslinking 61 …”

Section: Resultsmentioning

confidence: 78%

“…It is possible that homodimerization orients the otherwise toxic effector N‐terminus in a way that prevents its misregulation. A previous study showed that C‐terminal antibodies block dimerization as measured by bioluminescent complementation 60 . If the N‐terminus is regulated by a PrP C homodimer, then, an antibody blocking dimerization would thus dislodge the N‐terminus to elicit toxicity.…”

Section: Discussionmentioning

confidence: 99%

“…In support of this, a previous study using cross-linkable unnatural amino acids in recombinant protein demonstrated that dimerization is facilitated by the CR. 59 Dimerization has also been studied in cell culture using bioluminescent complementation, 60 native PAGE and cysteine crosslinking. 61 It was previously reported that ∆HD (∆114-133) PrP C , a deletion similar to that in ∆CR PrP C , has a reduced dimerization propensity in N2a cells using native PAGE.…”

Section: Reintroduction Of α-Cleavage In G5 Prp C Generates Currentsmentioning

confidence: 99%

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Intrinsic toxicity of the cellular prion protein is regulated by its conserved central region

Roseman

Wadolkowski

et al. 2020

FASEB j.

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The conserved central region (CR) of PrPC has been hypothesized to serve as a passive linker connecting the protein's toxic N‐terminal and globular C‐terminal domains. Yet, deletion of the CR causes neonatal fatality in mice, implying the CR possesses a protective function. The CR encompasses the regulatory α‐cleavage locus, and additionally facilitates a regulatory metal ion‐promoted interaction between the PrPC N‐ and C‐terminal domains. To elucidate the role of the CR and determine why CR deletion generates toxicity, we designed PrPC constructs wherein either the cis‐interaction or α‐cleavage are selectively prevented. These constructs were interrogated using nuclear magnetic resonance, electrophysiology, and cell viability assays. Our results demonstrate the CR is not a passive linker and the native sequence is crucial for its protective role over the toxic N‐terminus, irrespective of α‐cleavage or the cis‐interaction. Additionally, we find that the CR facilitates homodimerization of PrPC, attenuating the toxicity of the N‐terminus.

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Section: Resultsmentioning

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Section: Reintroduction Of α-Cleavage In G5 Prp C Generates Currentsmentioning

confidence: 99%

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Intrinsic toxicity of the cellular prion protein is regulated by its conserved central region

Roseman

Wadolkowski

et al. 2020

FASEB j.

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“…When PrP dimerizes in these cells, a luminescent signal is produced. Dimerization of PrP was detected in these cells upon incubation with several different strains of scrapie, including RML, 22L, ME7, 79A, 87V, 22A, and 263K [ 75 ].…”

Section: Immortalized Cell Linesmentioning

confidence: 99%

From Cell Culture to Organoids-Model Systems for Investigating Prion Strain Characteristics

Pineau

Sim

2021

Biomolecules

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Prion diseases are the hallmark protein folding neurodegenerative disease. Their transmissible nature has allowed for the development of many different cellular models of disease where prion propagation and sometimes pathology can be induced. This review examines the range of simple cell cultures to more complex neurospheres, organoid, and organotypic slice cultures that have been used to study prion disease pathogenesis and to test therapeutics. We highlight the advantages and disadvantages of each system, giving special consideration to the importance of strains when choosing a model and when interpreting results, as not all systems propagate all strains, and in some cases, the technique used, or treatment applied, can alter the very strain properties being studied.

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“…An ELISA-based screen of approximately 44,500 compounds using two different human cell lines has also led to the identification of potential PrP C -lowering drugs (Silber et al, 2014). Very recently, a novel RK13-derived cell line has been established in which PrP dimerization can be assessed using a split luciferase system (Wusten, Reddy, Smiyakin, Bernis, & Tamguney, 2018). This bioluminescent prion assay, which should be readily adaptable for HTS, may be useful for identifying inhibitors of early events in prion propagation.…”

Section: Identific Ation and De Velopment Of Anti -Prion Drug Smentioning

confidence: 99%

Cellular models for discovering prion disease therapeutics: Progress and challenges

Krance

Luke

Shenouda

et al. 2020

Journal of Neurochemistry

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Prion diseases, or transmissible spongiform encephalopathies (TSEs), are neurodegenerative disorders that affect both humans and animals. The first TSE observed historically was scrapie, which affects both ovine (sheep) and caprine (goat) species. Subsequently, prion diseases have been observed in cattle (bovine spongiform encephalopathy; BSE or "mad cow disease"); cervids such as deer, elk and moose (chronic wasting disease; CWD); minks (transmissible mink encephalopathy), felines (feline spongiform encephalopathy) and, most recently, dromedary camels (camel prion disease) (Babelhadj et al., AbstractPrions, which cause fatal neurodegenerative disorders such as Creutzfeldt-Jakob disease, are misfolded and infectious protein aggregates. Currently, there are no treatments available to halt or even delay the progression of prion disease in the brain.The infectious nature of prions has resulted in animal paradigms that accurately recapitulate all aspects of prion disease, and these have proven to be instrumental for testing the efficacy of candidate therapeutics. Nonetheless, infection of cultured cells with prions provides a much more powerful system for identifying molecules capable of interfering with prion propagation. Certain lines of cultured cells can be chronically infected with various types of mouse prions, and these models have been used to unearth candidate anti-prion drugs that are at least partially efficacious when administered to prion-infected rodents. However, these studies have also revealed that not all types of prions are equal, and that drugs active against mouse prions are not necessarily effective against prions from other species. Despite some recent progress, the number of cellular models available for studying non-mouse prions remains limited. In particular, human prions have proven to be particularly challenging to propagate in cultured cells, which has severely hindered the discovery of drugs for Creutzfeldt-Jakob disease. In this review, we summarize the cellular models that are presently available for discovering and testing drugs capable of blocking the propagation of prions and highlight challenges that remain on the path towards developing therapies for prion disease.

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A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization

Cited by 4 publications

References 55 publications

Intrinsic toxicity of the cellular prion protein is regulated by its conserved central region

Intrinsic toxicity of the cellular prion protein is regulated by its conserved central region

From Cell Culture to Organoids-Model Systems for Investigating Prion Strain Characteristics

Cellular models for discovering prion disease therapeutics: Progress and challenges

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