Reactive Oxygen Species-mediated β-Cleavage of the Prion Protein in the Cellular Response to Oxidative Stress*
- Nicole T. Watt,
- David R. Taylor,
- Andrew Gillott,
- Daniel A. Thomas1,
- W. Sumudhu S. Perera2 and
- Nigel M. Hooper3
- Proteolysis Research Group, School of Biochemistry and Microbiology, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT, United Kingdom
- 3 To whom correspondence should be addressed. Tel.: 44-113-343-3163; Fax: 44-113-343-3167; E-mail: n.m.hooper{at}leeds.ac.uk.
Abstract
The cellular prion protein (PrPC) is critical for the development of prion diseases. However, the physiological role of PrPC is less clear, although a role in the cellular resistance to oxidative stress has been proposed. PrPC is cleaved at the end of the copper-binding octapeptide repeats through the action of reactive oxygen species (ROS), a process termed β-cleavage. Here we show that ROS-mediated β-cleavage of cell surface PrPC occurs within minutes and was inhibited by the hydroxyl radical quencher dimethyl sulfoxide and by an antibody against the octapeptide repeats. A construct of PrP lacking the octapeptide repeats, PrPΔoct, failed to undergo ROS-mediated β-cleavage, as did two mutant forms of PrP, PG14 and A116V, associated with human prion diseases. As compared with cells expressing wild type PrP, when challenged with H2O2 and Cu2+, cells expressing PrPΔoct, PG14, or A116V had reduced viability and glutathione peroxidase activity and increased intracellular free radicals. Thus, lack of ROS-mediated β-cleavage of PrP correlated with the sensitivity of the cells to oxidative stress. These data indicate that the β-cleavage of PrPC is an early and critical event in the mechanism by which PrP protects cells against oxidative stress.
Footnotes
-
↵4 The abbreviations used are: PrPC, cellular form of the prion protein; PrPSc, infectious, protease resistant form of PrP; ALLM, N-acetyl-Leu-Leu-Met-aldehyde; FCS, fetal calf serum; PBS, phosphate-buffered saline; PNGase F, Peptide:N-glycosidase F; ROS, reactive oxygen species; wtPrP, wild type PrP; GPI, glycosylphosphatidylinositol.
-
↵* This work was supported by the Medical Research Council of Great Britain, European Union Grant QLG3-CT-2001-02353, and the Wellcome Trust (Bioimaging Facility, University of Leeds). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 Recipient of a Biotechnology and Biological Sciences Research Council studentship.
-
↵2 Recipient of an Emma and Leslie Reid studentship from the University of Leeds. Present address: Institut für Zoologie, Abteilung Molekulare Zellbiologie, Universität Mainz, D-55128 Mainz, Germany.
-
- Received July 6, 2005.
- Revision received August 22, 2005.
- The American Society for Biochemistry and Molecular Biology, Inc.
