Different structural stability and toxicity of PrP(ARR) and PrP(ARQ) sheep prion protein variant.

Different structural stability and toxicity of PrPARR and PrPARQ sheep prion protein variants Domenico Paludi,* Stefano Thellung,! Katia Chiovitti," Alessandro Corsaro,! Valentina Villa,! Claudio Russo,§ Adriana Ianieri,¶ Uwe Bertsch,** Hans A. Kretzschmar,** Antonio Aceto" and Tullio Florio! *Department of Scienze degli Alimenti, Veterinary School, University of Teramo, Teramo, Italy !Department of Oncology, Biology and Genetics, Section of Pharmacology, University of Genova, Genova, Italy "Section of Biochemistry, Department of Biomedical Sciences, University G. D’Annunzio of Chieti, Chieti, Italy §Department of Health Sciences, University of Molise, Campobasso, Italy ¶Department of Produzioni animali, biotecnologie veterinarie, qualita` e sicurezza degli alimenti, University of Parma, Parma, Italy **Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Munich, Germany Abstract The polymorphisms at amino acid residues 136, 154, and 171 in ovine prion protein (PrP) have been associated with different susceptibility to scrapie: animals expressing PrPARQ [PrP(Ala136/Arg154/Gln171)] show vulnerability, whereas those that express PrPARR [PrP(Ala136/Arg154/Arg171)] are resistant to scrapie. The aim of this study was to evaluate the in vitro toxic effects of PrPARR and PrPARQ variants in relation with their structural characteristics. We show that both peptides cause cell death inducing apoptosis but, unexpectedly, the scrapie resistant PrPARR form was more toxic than the scrapie susceptible PrPARQ variant. Moreover, the a-helical conformation of PrPARR was less stable than that of PrPARQ and the structural determinants responsible of these different conformational stabilities were characterized by spectroscopic analysis. We observed that PrP toxicity was inversely related to protein structural stability, being the unfolded conformation more toxic than the native one. However, the PrPARQ variant displays a higher propensity to form large aggregates than PrPARR. Interestingly, in the presence of small amounts of PrPARR, PrPARQ aggregability was reduced to levels similar to that of PrPARR. Thus, in contrast to PrPARR toxicity, scrapie transmissibility seems to reside in the more stable conformation of PrPARQ that allows the formation of large amyloid fibrils. Keywords: cell death, prion protein genetic polymorphism, sheep prion protein. J. Neurochem. (2007) 103, 2291–2300. Transmissible spongiform encephalopathies (TSE) represent a group of fatal neurodegenerative disorders that affect both animals and humans, occurring through infective, genetic, or sporadic mechanisms. TSE share a common histopathology with a pathognomonic triad: spongiform vacuolation of the gray matter, neuronal death, and glial proliferation, with amyloid deposition less constantly observed. In contrast to other transmissible diseases in which the infectious particles are viruses or bacteria, the only agent currently associated with TSE is a structural isoform of the cellular prion protein (PrPC) known as the scrapie conformation (PrPSc) (Prusiner 2001). Several hypotheses have been proposed concerning the nature of the infectious agents, but the most widely accepted, the ‘protein only’ hypothesis, designates prion protein as the infectious agent. According to this hypothesis, the key event in the pathogenesis is the conversion of the host PrPC into the pathogenic isoform (PrPSc) characterized by insolubility, high content in b-sheet, and partial protease resistance. The difference between PrPSc and PrPC is only conformational, as no covalent modifications differentiate PrPSc from PrPC. Scrapie in sheep is the archetypal prion disease. Polymorphisms in ovine PrP at amino acid residues 136, 154, and 171 are associated with variation in susceptibility to natural scrapie (Baylis and Goldmann 2004). The homozygous genotype Ala136-Arg154-Arg171 (ARR) induces a resistant phenotype, while Val136-Arg154-Gln171 (VRQ) confers a high scrapie susceptibility phenotype. Between these two extreme variants, two other isoforms have also been described, Ala136-Arg154-Gln171 (ARQ) and Ala136-His154-Gln171 (AHQ), which are associated in homozygous animals with medium and low susceptibility to scrapie, respectively (Caplazi et al. 2004). Similarly, the experimental transmission of other TSE, and in particular the Received May 22, 2007; accepted August 7, 2007. Address correspondence and reprint requests to Tullio Florio, Department of Oncology Biology and Genetics, Section of Pharmacology, University of Genova, Viale Benedetto XV, 2, 16132 Genova, Italy. E-mail: tullio.florio@unige.it Abbreviations used: ARQ, Ala136-Arg154-Gln171; ARR, Ala136- Arg154-Arg171; CD, circular dichroism; MES, 2-(N-morpholino)-ethanesulfonic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PrP, prion protein; ThT, thioflavin T; TNS, 2-paratoluidinylnaphthalene- 6-sulfonate; TSE, transmissible spongiform encephalopathies. Journal of Neurochemistry, 2007, 103, 2291–2300 doi:10.1111/j.1471-4159.2007.04934.x ! 2007 The Authors Journal Compilation ! 2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 2291–2300 2291 Chronic Wasting Disease, was much more difficult in sheep with PrPARR genotype (Hamir et al. 2006), although the inoculation of the bovine spongiform encephalopathy agent may lead to PrPSc accumulation (Andreoletti et al. 2006) generating silent carriers of disease (Ronzon et al. 2006). Several PrP polymorphisms are also known to influence either the susceptibility of humans and mice to TSE or the incubation period of the disease. For example, in humans, the polymorphism methionine/valine, at the position 129, dictates the susceptibility to Kuru and to the new variant of Creutzfeldt-Jakob disease (Cervenakova et al. 1998; Ironside 2000). However, among mammals, sheep represent the species in which the strongest correlation between genotype and susceptibility phenotype can be demonstrated. According to the ‘protein only’ theory, the effect of PrP polymorphisms on the pathogenic transformation can be understood, if these polymorphisms affect the unfolding pathways or the interactions of the protein with still ill-defined factors involved in the PrPC–PrPSc transition (Prusiner 1998). Previous study of heat and chemical unfolding of sheep VRQ, ARQ, and ARR PrP variants has demonstrated significant differences in their free energies of denaturation and in their tertiary structure (Rezaei et al. 2000). Thus, it was proposed that the variants associated with susceptibility (VRQ and ARQ) possess higher stability than the variants associated with scrapie resistance (ARR). Interestingly, in heterozygous animals PrPARR is dominant, as the PrPARR/ARQ heterozygosis confers resistance to the infection (Eghiaian et al. 2004), involving a mechanism other than preferential allele usage (Caplazi et al. 2004). In this paper, we used two recombinant ovine PrP fragments [ovPrP (25–234)], corresponding to genotype ARR and ARQ, respectively, to investigate the pattern of in vitro toxicity of these variants and to relate the obtained results with their structural conformation. Moreover, we evaluated whether the co-treatment with both proteins may affect the biological activity of the two PrP variants and their structural features. In particular, we assessed PrPARR and PrPARQ cellular effects by exogenous treatment of the SH-SY5Y neuroblastoma cells, that were reported to represent a valuable model to study the PrP-dependent cell death in vitro (O’Donovan et al. 2001; Corsaro et al. 2003, 2006).