Abstract

Prion diseases are neurodegenerative disorders that affect many mammalian species. Mammalian prion proteins (PrPs) can misfold into many different aggregates. However, only a small subpopulation of these structures is infectious. One of the major unresolved questions in prion research is identifying which specific structural features of these misfolded protein aggregates are important for prion infectivity in vivo Previously, two types of proteinase K-resistant, self-propagating aggregates were generated from the recombinant mouse prion protein in the presence of identical cofactors. Although these two aggregates appear biochemically very similar, they have dramatically different biological properties, with one of them being highly infectious and the other one lacking any infectivity. Here, we used several MS-based structural methods, including hydrogen-deuterium exchange and hydroxyl radical footprinting, to gain insight into the nature of structural differences between these two PrP aggregate types. Our experiments revealed a number of specific differences in the structure of infectious and noninfectious aggregates, both at the level of the polypeptide backbone and quaternary packing arrangement. In particular, we observed that a high degree of order and stability of β-sheet structure within the entire region between residues ∼89 and 227 is a primary attribute of infectious PrP aggregates examined in this study. By contrast, noninfectious PrP aggregates are characterized by markedly less ordered structure up to residue ∼167. The structural constraints reported here should facilitate development of experimentally based high-resolution structural models of infectiosus mammalian prions.

Highlights

  • Prion diseases are neurodegenerative disorders that affect many mammalian species

  • 3 The abbreviations used are: PrPC, cellular prion protein, PrPSc, scrapie prion protein; prion proteins (PrPs), prion protein; PK, proteinase K; rPrP, recombinant prion protein; PMCA, protein misfolding cyclic amplification; HXMS, hydrogen– deuterium exchange coupled with MS; UPLC, ultra-HPLC; HRF, hydroxyl radical-mediated footprinting; GdnHCl, guanidinium hydrochloride; TRF, time-resolved fluorescence

  • It was previously shown that highly infectious mammalian prions, rPrP-resRNA, can be generated de novo from the recombinant mouse prion protein in the presence of two cofactors, mouse liver RNA and a synthetic phospholipid, phosphatidylglycerol [24]

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Summary

The abbreviations used are

PrPC, cellular prion protein, PrPSc, scrapie prion protein; PrP, prion protein; PK, proteinase K; rPrP, recombinant prion protein; PMCA, protein misfolding cyclic amplification; HXMS, hydrogen– deuterium exchange coupled with MS; UPLC, ultra-HPLC; HRF, hydroxyl radical-mediated footprinting; GdnHCl, guanidinium hydrochloride; TRF, time-resolved fluorescence. It was shown that, in addition to these highly infectious rPrP-resRNA prions, using the same PMCA protocol and identical cofactors one can generate de novo another type of self-propagating rPrP-res aggregate, denoted rPrP-resRNA-low, that biochemically appears very similar to rPrP-resRNA but lacks any infectivity [26, 27]. The availability of these two selfpropagating aggregate types with dramatically different biological properties opened up new avenues for exploring the structural basis of prion infectivity. Our data reveal major structural differences between rPrPresRNA and rPrP-resRNA-low, both at the level of the polypeptide backbone and quaternary packing arrangement, providing new insight into the structural basis of prion infectivity

Results
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