The normal cellular prion protein (PrPC) is an essential substrate in all forms of prion diseases and a receptor for Aβ oligomers in Alzheimer’s disease. However, it is not fully understood how cells regulate PrPC levels. Recently, we identified glycogen synthase kinase-3β (GSK-3β) as a potential regulator of PrPC levels in a whole genome knockout screen. Here, we show that both cell surface and total PrPC levels can be reduced either by siRNA-mediated Gsk3b (but not Gsk3a) knockdown or by CRISPR-mediated Gs3b knockout. Whole cell mass spectrometric analysis showed that PrPC was the 60th most significantly reduced protein (out of 7227 total proteins detected) in Gsk3b knockout cells, compared to wild-type cells. Two different GSK-3 inhibitors, laduviglusib (CHIR-99021) and AZD-1080, reduced PrPC levels in mouse CAD5 and human BE(2)-C cells, both in undifferentiated and differentiated states. PrPC levels were similarly reduced by cycloheximide treatment in both Gsk3b knockout and WT cells, indicating that GSK-3β regulates PrPC levels through a post-translational mechanism. Finally, treatment with either laduviglusib or AZD-1080 reduced PrPSc levels in CAD5 cells infected with three different rodent prion strains. Overall, the results reveal that GSK-3β activity controls PrPC levels in living cells, revealing a novel regulatory mechanism and promising therapeutic target.
Kathryn S. Beauchemin, Anna M. Schmoker, J. Watts et al.· bioRxiv· 0 citations
The pathogenic conversion of the cellular prion protein (PrPC) into the β-sheet-rich isoform PrPSc is the pivotal pathogenic event in prion disease, yet the molecular steps that govern this structural transition remain elusive. In this study, we introduce a new approach to monitor site-specific conformational transitions that occur during infectious prion formation. The method relies on genetically encoded substitution of a fluorescent, environmentally sensitive noncanonical amino acid, l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA), into recombinant PrP substrate molecules, allowing real-time monitoring of structural changes in high-efficiency in vitro PrPSc conversion reactions. As proof of principle, we show that the W99 7-HCAA recPrP substrate efficiently propagates two different PrPSc conformers (infectious cofactor PrPSc and noninfectious protein-only PrPSc). Bioassays in knock-in mice expressing bank vole PrP confirm that W99 7-HCAA cofactor PrPSc produced by serial propagation is infectious, causing scrapie with an incubation period and neuropathological profile like those induced by wild-type cofactor PrPSc. Marked differences in fluorescence intensity were observed between native, misfolded, and denatured states of W99 7-HCAA PrP, confirming that 7-HCAA reports on local changes in PrP conformation. Together, these findings establish 7-HCAA as a site-specific and sensitive probe of local PrP conformation. Moreover, the results suggest a new strategy for studying conformational dynamics in amyloid-forming proteins.
Jessica de Alcantara Ferreira, Daniel J. Walsh, Evelyn M. Turnbaugh et al.· Biochemistry· 0 citations
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