1518 Research Article Introduction Prion diseases are transmissible neurodegenerative disorders causally linked to abnormal conformers (termed PrP Sc ) of the cellular prion protein (PrP C ). The role of PrP C within the cell has proved difficult to resolve, with suggested functions including copper homeostasis and trafficking, signal transduction, cellular adhesion and attenuation of oxidative stress (Brown and Besinger, 1998; Brown et al., 1999; Schmitt-Ulms et al., 2001; Spielhaupter and Schätzl, 2001; Stuermer et al., 2004). Protection against oxidative stress or reactive oxygen species (ROS) has been proposed to be enzymatic, wherein PrP C itself would have superoxide dismutase-like activity (Brown et al., 2001), or alternatively, mediated by signal transduction cascades, whereby the resultant reaction protects the cell (Watt et al., 2005). The regions of PrP C involved in such protection have been investigated and both the C- and N-termini have been determined to be involved (Rambold et al., 2008). The C-terminal amyloidogenic region has been more widely studied than the N-terminal region due to its propensity to misfold and so have a more prominent association with disease. However, mutations within the N-terminus are found in hereditary prion diseases, consisting of insertions or deletions within the copper- binding octameric repeat domain (Kovács et al., 2005). The N- terminal region, although not constituting part of the amyloid core of PrP Sc , is thought to be biologically active, associated with clathrin-mediated internalisation and intracellular trafficking of PrP C (Nunziante et al., 2003; Shyng et al., 1995; Sunyach et al., 2003), and with PrP C movement at the cell surface (Taylor et al., 2005). In particular, the most N-terminal amino acids of PrP C are highly conserved across mammalian species (Wopfner et al., 1999) and contain a polybasic domain (residues 23-28) shown to function as a glycosaminoglycan (GAG)-binding site (Pan et al., 2002). PrP C binding to cellular receptors, including low density lipoprotein receptor-related protein 1 (Parkyn et al., 2008) and the 37 kDa/ 67 kDa laminin receptor (Gauczynski et al., 2001), involves the N- terminal region. Further, the latter of these PrP receptors requires heparan sulphate to mediate binding and has additionally been shown to be involved in the internalisation of PrP Sc (Morel et al., 2005; Gauczynski et al., 2006). PrP cleavage fragments, corresponding to two internal cleavage sites, can be detected in both cell culture systems and brain tissue. In non-disease states the α-cleavage fragments (N1/C1) usually have a higher prevalence than the β-cleavage fragments (N2/C2). The latter cleavage fragments are increased in the brains of Creutzfeldt- Jakob Disease (CJD) patients and mice generated as models of prion disease (Chen et al., 1995; Yadavalli et al., 2004). This increase has mainly been considered a pro-pathogenic event, but equally might represent a neuronal protective response, attempting to compensate for increased stress during disease progression. Consistent with this hypothesis, cell lines expressing mutant PrP species that do not undergo N2/C2 cleavage are rendered unable Beta-cleavage of the neurodegenerative disease-associated prion protein (PrP) protects cells from death induced by oxidative insults. The beta-cleavage event produces two fragments, designated N2 and C2. We investigated the role of the N2 fragment (residues 23-89) in cellular stress response, determining mechanisms involved and regions important for this reaction. The N2 fragment differentially modulated the reactive oxygen species (ROS) response induced by serum deprivation, with amelioration when copper bound. Amino acid residues 23-50 alone mediated a ROS reduction response. PrP23-50 ROS reduction was not due to copper binding or direct antioxidant activity, but was instead mediated through proteoglycan binding partners localised in or interacting with cholesterol-rich membrane domains. Furthermore, mutational analyses of both PrP23-50 and N2 showed that their protective capacity requires the sterically constraining double proline motif within the N-terminal polybasic region. Our findings show that N2 is a biologically active fragment that is able to modulate stress-induced intracellular ROS through interaction of its structurally defined N-terminal polybasic region with cell- surface proteoglycans. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/122/10/1518/DC1 Key words: Prion, N-terminus, Oxidative stress, Beta-cleavage, GAG, Copper Summary Dominant roles of the polybasic proline motif and copper in the PrP23-89-mediated stress protection response Cathryn L. Haigh 1,2 , Simon C. Drew 1,2,3,4 , Martin P. Boland 1,2 , Colin L. Masters 2,5 , Kevin J. Barnham 1,2,3 , Victoria A. Lawson 1,2 and Steven J. Collins 1,2, * 1 Department of Pathology, The University of Melbourne, 3010, Australia 2 Mental Health Research Institute, The University of Melbourne, 3010, Australia 3 Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 3010, Australia 4 School of Physics, Monash University, Clayton, 3800, Australia 5 Centre for Neuroscience, The University of Melbourne, 3010, Australia *Author for correspondence (e-mail: stevenjc@unimelb.edu.au) Accepted 10 January 2009 Journal of Cell Science 122, 1518-1528 Published by The Company of Biologists 2009 doi:10.1242/jcs.043604 Journal of Cell Science JCS ePress online publication date 21 April 2009