The protease-sensitive N-terminal polybasic region of prion protein modulates its conversion to the pathogenic prion conformer

Xiangyi Zhang,Yi-Hsuan Pan,Ying Chen,Chenhua Pan,Ji Ma,Chonggang Yuan,Guohua Yu,Jiyan Ma

doi:10.1016/j.jbc.2021.101344

Abstract

Conversion of normal prion protein (PrPC) to the pathogenic PrPSc conformer is central to prion diseases such as Creutzfeldt–Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrPC-to-PrPSc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrPSc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrPSc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrPSc mutants were significantly different from each other and from that of WT recPrPSc. Together, our results support that the NPR greatly influences PrPC-to-PrPSc conversion, but it is not essential for the generation of PrPSc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.

Highlights

Prion disease, known as transmissible spongiform encephalopathy, is a large group of fatal neurodegenerative disorders affecting both humans and animals [1,2]
10 μl of the product was used to seed round PMCA and 30 μl of the products was subjected to proteinase K (PK) digestion and Western blotting
RecPrP in the substrate was replaced with water and no PK-resistant PrP was detected in the products of control serial protein misfolding cyclic amplification (sPMCA) reactions (Fig. 2A, seed only), supporting that all PK-resistant PrP signals resulted from the conversion of recPrP in the substrate

Summary

Introduction

Known as transmissible spongiform encephalopathy, is a large group of fatal neurodegenerative disorders affecting both humans and animals [1,2]. The pathogenic PrPSc conformer, consists solely of -sheets [15,16] and has a large PK-resistant C-terminal fragment [8,17,18] This PK-resistant fragment of PrPSc is sufficient to seed PrPC-to-PrPSc conversion and cause prion disease [19,20]. Journal Pre-proof produced in this system recapitulates all the hallmarks of naturally occurring prions and causes prion disease in wild-type mice via various routes [34,35] Using this sPMCA system, we studied the influence of NPR on PrPC-to-PrPSc conversion with two NPR mutants ∆N6 and Met (Fig. 1A). Clear differences in convertibility and POPG-interaction were observed between ∆N6 and Met, indicating that charges, but the identity of amino acids in NPR affects PrP conversion. The disease phenotypes were significantly different among mice inoculated with wild-type or either of the mutant recPrPSc, indicating that there are conformational differences among these three types of recPrPSc aggregates

Results

Discussion

Experimental Procedures