Abstract
Some mutant forms of the cellular prion protein (PrPC) carrying artificial deletions or point mutations associated with familial human prion diseases are capable of inducing spontaneous ionic currents across the cell membrane, conferring hypersensitivity to certain antibiotics to a wide range of cultured cells and primary cerebellar granular neurons (CGNs). These effects are abrogated when the wild type (WT) form is co-expressed, suggesting that they might be related to a physiological activity of PrPC. Interestingly, the prion protein family member Shadoo (Sho) makes cells hypersensitive to the same antibiotics as mutant PrP-s, an effect that is diminished by the co-expression of WT-PrP. Here, we report that Sho engages in another mutant PrP-like activity: it spontaneously induces large ionic currents in cultured SH-SY5Y cells, as detected by whole-cell patch clamping. These currents are also decreased by the co-expression of WT-PrP. Furthermore, deletion of the N-terminal (RXXX)8 motif of Sho, mutation of the eight arginine residues of this motif to glutamines, or replacement of the hydrophobic domain by that of PrP, also diminish Sho-induced ionic currents. Our results suggest that the channel activity that is also characteristic to some pathogenic PrP mutants may be linked to a physiological function of Sho.
Highlights
A great deal of evidence indicates that the prion protein (PrPC), encoded by the prnp gene, is involved in several neurodegenerative disorders, including Creutzfeldt-Jacob disease, Gerstmann-Sträussler-Scheinker syndrome, fatal familial insomnia, kuru, and possibly Alzheimer’s disease
Using an expression system employed previously for the expression of wild type (WT)-Sho in SH-SY5Y cells, which harbour no detectable amount of endogenous Sho[26], we developed cells stably expressing this deletion-mutant
The mobility shift caused by the deletion of the (RXXX)[8] motif is detectable when the protein is deglycosylated (Fig. 2b) as a result of peptide N-glycosidase F (PNGaseF) treatment
Summary
A great deal of evidence indicates that the prion protein (PrPC), encoded by the prnp gene, is involved in several neurodegenerative disorders, including Creutzfeldt-Jacob disease, Gerstmann-Sträussler-Scheinker syndrome, fatal familial insomnia, kuru, and possibly Alzheimer’s disease These pathological conditions are characterized by protein aggregation and formation of β-sheet rich amyloid deposits, which might use PrPC to deliver toxic signals[1,2,3,4,5,6,7,8]. Further insights came from studies investigating two other members of the prion protein family[12]: doppel, a neurotoxic protein sharing structural similarities with the C-terminal globular fold of PrPC 23, and Sho, a natively disordered protein displaying structural features similar to the N-terminal, flexible tail of PrPC 24 The latter has been shown to possess WT-PrP-like protective properties, as its expression suppresses the toxicity of doppel and PrPΔ32-121, proteins that display ΔCR-PrP-like toxic effects[25]. We report that, to ΔCR-PrP, Sho spontaneously induces ionic currents across the plasma membrane, as detected by patch clamping experiments
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