Oxidation of Helix-3 Methionines Precedes the Formation of PK Resistant PrPSc

María Gasset,Assaf Friedler,Kati Frid,Tamar Canello,Jackob Moskovitz,Silvia Lisa,Ruth Gabizon,Ronen Gabizon

doi:10.1371/journal.ppat.1000977

Abstract

While elucidating the peculiar epitope of the α-PrP mAb IPC2, we found that PrPSc exhibits the sulfoxidation of residue M213 as a covalent signature. Subsequent computational analysis predicted that the presence of sulfoxide groups at both Met residues 206 and 213 destabilize the α-fold, suggesting oxidation may facilitate the conversion of PrPC into PrPSc. To further study the effect of oxidation on prion formation, we generated pAbs to linear PrP peptides encompassing the Helix-3 region, as opposed to the non-linear complexed epitope of IPC2. We now show that pAbs, whose epitopes comprise Met residues, readily detected PrPC, but could not recognize most PrPSc bands unless they were vigorously reduced. Next, we showed that the α-Met pAbs did not recognize newly formed PrPSc, as is the case for the PK resistant PrP present in lines of prion infected cells. In addition, these reagents did not detect intermediate forms such as PK sensitive and partially aggregated PrPs present in infected brains. Finally, we show that PrP molecules harboring the pathogenic mutation E200K, which is linked to the most common form of familial CJD, may be spontaneously oxidized. We conclude that the oxidation of methionine residues in Helix-3 represents an early and important event in the conversion of PrPC to PrPSc. We believe that further investigation into the mechanism and role of PrP oxidation will be central in finally elucidating the mechanism by which a normal cell protein converts into a pathogenic entity that causes fatal brain degeneration.

Highlights

Prions are infectious agents that cause neurodegenerative diseases, such as scrapie, bovine spongiform encephalopathy (BSE) and Creutzfeld Jacob disease (CJD)
We show that before PrPC initiates its conversion from proteinase K sensitive to resistant and from soluble to aggregated in the pathway to becoming PrPSc, it first undergoes oxidation of the most hidden Met residues located in a protein region exhibiting sequence identity for all species
While the cellular events promoting such oxidation in this transmissible disease remain unclear, we present evidence that PrP molecules carrying a mutation ascribed to the most common familial prion disease spontaneously oxidizes at these same Met residues

Summary

Introduction

Prions are infectious agents that cause neurodegenerative diseases, such as scrapie, bovine spongiform encephalopathy (BSE) and CJD They are believed to be composed mainly of PrPSc, a misfolded form of the GPI-anchored glycoprotein termed PrPC[1]. The finding that M213 as well as the other conserved Helix-3 Met residue, M206, were oxidized in PrPSc was first reported in the seminal work of Stahl et al following sequencing of the PrP27-30 endoLysC peptides [9] The fact that these specific Met residues are oxidized in PrPSc is intriguing since they are the most buried residues among methionines in the 3D PrP a-fold and are less accessible to reactive oxygen species (ROS) [10]. It was shown that while mice overexpressing superoxide dismutase (SOD), which inhibits oxidation, presented prolonged incubation periods upon RML infection, ablation of the MsrA system did not reduce the time from infection to disease outbreak [14]

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