A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization

Katharina Annick Wüsten,Andrej Smiyakin,Gültekin Tamgüney,Maria Eugenia Bernis,Pasham Parameshwar Reddy

doi:10.1038/s41598-018-32581-1

Katharina Annick Wüsten, Andrej Smiyakin + Show 3 more

Open Access

https://doi.org/10.1038/s41598-018-32581-1

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Abstract

The prion protein (PrP) is a cell surface protein that in disease misfolds and becomes infectious causing Creutzfeldt-Jakob disease in humans, scrapie in sheep, and chronic wasting disease in deer and elk. Little is known regarding the dimerization of PrP and its role in disease. We developed a bioluminescent prion assay (BPA) to quantify PrP dimerization by bimolecular complementation of split Gaussia luciferase (GLuc) halves that are each fused to PrP. Fusion constructs between PrP and N- and C-terminal GLuc halves were expressed on the surface of RK13 cells (RK13-DC cells) and dimerized to yield a bioluminescent signal that was decreased in the presence of eight different antibodies to PrP. Dimerization of PrP was independent of divalent cations and was induced under stress. Challenge of RK13-DC cells with seven different prion strains did not lead to detectable infection but was measurable by bioluminescence. Finally, we used BPA to screen a compound library for compounds inhibiting PrP dimerization. One of the most potent compounds to inhibit PrP dimerization was JTC-801, which also inhibited prion replication in RML-infected ScN2a and SMB cells with an EC50 of 370 nM and 220 nM, respectively. We show here that BPA is a versatile tool to study prion biology and to identify anti-prion compounds.

Highlights

The prion protein (PrPC) is a natural protein that is predominantly expressed on the outer cell membrane of neurons[1]
To study dimerization of the prion protein (PrP) by bioluminescence in cells, we cloned fusion constructs between the PrP (Fig. 1a) and the N- and C-terminal halves of split Gaussia luciferase (GLuc) into a bicistronic expression vector that allows expression of two proteins from a single mRNA using an internal ribosomal entry site (IRES)
Our data show that fusion constructs between PrPC and N- and C-terminal GLuc halves readily dimerize under physiological conditions on the surface of cells implying that dimerization of PrPC is important for its function

Summary

Introduction

The prion protein (PrPC) is a natural protein that is predominantly expressed on the outer cell membrane of neurons[1]. To transmission experiments to animals many sophisticated tools have been developed over the years to detect and quantify prions and the effect of anti-prion drugs in vitro These include methods as simple as proteolytic treatment of prion-infected cell lysates with proteinase K (PK) to distinguish between PK-sensitive PrPC and PK-resistant PrPSc after immunodetection with antibodies to PrP. Based on the concept of protein-fragment complementation[22] and the fact that PrPC can dimerize, constructs between mouse PrP and split Gaussia luciferase halves were expressed in RK13 cells, which were bioluminescent and showed that GPI-anchored fusion constructs of PrPC dimerize on the cell surface under physiological conditions. Our data shows that the bioluminescent prion assay is a versatile tool to study the biology of prion proteins, and that it can be used to identify compounds inhibiting PrPC dimerization that inhibit prion replication

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