Chemical Induction of Misfolded Prion Protein Conformers in Cell Culture

Julie Ullman,Sina Ghaemmaghami,Susan St Martin,Misol Ahn,Stanley B Prusiner

doi:10.1074/jbc.m109.045112

Julie Ullman, Sina Ghaemmaghami + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m109.045112

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Abstract

Prion-infected cells accumulate a heterogeneous population of aberrantly folded PrP conformers, including the disease-causing isoform (PrP(Sc)). We found that specific chemicals can modulate the levels of various PrP conformers in cultured cells. Positively charged polyamidoamines (dendrimers) eliminated protease-resistant (r) PrP(Sc) from prion-infected cells and induced the formation of insoluble, protease-sensitive PrP aggregates (designated PrP(A)). Larger, positively charged polyamidoamines more efficaciously induced the formation of PrP(A) and cleared rPrP(Sc), whereas negatively charged polyamidoamines neither induced PrP(A) nor cleared rPrP(Sc). Although the biochemical properties of PrP(A) were shown to be similar to protease-sensitive (s) PrP(Sc), bioassays of PrP(A) indicated that it is not infectious. Our studies argue that PrP(A) represents an aggregated PrP species that is off-pathway relative to the formation of rPrP(Sc). It remains to be established whether the formation of PrP(A) inhibits the formation of rPrP(Sc) by sequestering PrP(C) in the form of benign, insoluble aggregates.

Highlights

The prion hypothesis proposes that proteins can act as infectious, self-replicating agents [1]
Prion-infected N2a-cl3 (ScN2a-cl3) cells form rPrPSc at levels that are within the same order of magnitude as those found in the brains of Rocky Mountain Laboratory (RML)-infected, wild-type mice
We found that in ScN2a-cl3, the amount of prion protein (PrP) that is phosphotungstic acid (PTA) insoluble is greater than the amount that is resistant to proteinase K (PK) digestion (Fig. 1A)

Summary

Introduction

The prion hypothesis proposes that proteins can act as infectious, self-replicating agents [1]. An aberrantly folded conformer of the prion protein (PrP) propagates by catalyzing a post-translational conversion reaction, utilizing cellular PrP (PrPC) as substrate [2]. This conversion reaction transforms endogenous, ␣-helix-rich PrPC to ␤-sheet-rich, disease-causing conformations (PrPSc) [3, 4]. In prion-infected cells, multiple conformers of PrPSc with distinct biochemical properties coexist at steady state [9]. It is not clear whether these conformers represent intermediates along a single misfolding pathway or distinct, thermodynamically stable, self-propagating, conformationally distinct strains. We report that positively charged polyamidoamines clear protease-resistant PrPSc and induce the formation of a novel, non-infectious PrP aggregate

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