A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization
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 b io...
Saved in:
Published in | Scientific reports Vol. 8; no. 1; pp. 14178 - 14 |
---|---|
Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
London
Nature Publishing Group UK
21.09.2018
Nature Publishing Group |
Subjects | |
Online Access | Get full text |
Cover
Loading…
Summary: | 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
b
ioluminescent
p
rion
a
ssay (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 EC
50
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. |
---|---|
Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 2045-2322 2045-2322 |
DOI: | 10.1038/s41598-018-32581-1 |