Assemblages of prion fragments: novel model systems for understanding amyloid toxicity K.S. Satheeshkumar, J. Murali, and R. Jayakumar * Bioorganic and Neurochemistry Laboratory, Central Leather Research Institute, Adyar, Chennai 600 020, India Received 2 April 2004, and in revised form 10 May 2004 Available online 2 July 2004 Abstract We report the conformational and toxic properties of two novel fibril-forming prion amyloid sequences, GAVVGGLG (PrP(119–126)) and VVGGLGG (PrP(121–127)). The conformational preferences of these fragments were studied in differing microenvironments of TFE/water mixtures and SDS solution. Interestingly, with an increase in TFE concentration, PrP(119–126) showed a helical conformational propensity, whereas PrP(121–127) adopted a more random coil structure. In 5% SDS, PrP(119–126) showed more a-helical content than in TFE solution, and PrP(121–127) exhibited a predominantly random coil conformation. However, both peptides took a random coil conformation in water, and over time the random coil transformed into a b-sheet structure with a significant percentage of helical conformation and b-turn structure in PrP(119–126) and PrP(121– 127), respectively, as observed with CD spectroscopy. The aged fibrils of PrP(119–126) were insoluble in SDS, and PrP(121–127) was extractable with SDS solution. These fibrils were characterized by transmission electron microscopy. Both PrP(119–126) and PrP(121–127) formed stable monolayer’s consisting of multimeric assemblages at the air–water interface. Monomeric PrP(119– 126) was more toxic to astrocytes than the control Ab peptide; however, the fibrillar form of PrP(119–126) was less toxic to astrocytes. PrP(121–127) elicited moderate toxicity in both soluble and fibrillar forms on astrocytes. Furthermore, quenching experiments using acroyl-labeled PrP(119–126) and PrP(121–127) with eosin-labeled synaptosomal membrane revealed that these prion fragments bind to anion-exchange protein. The binding of PrP(119–126) and PrP(121–127) with a membrane microdomain (lipid raft) was also analyzed using pyrenated derivatives. We conclude that the formation of PrP(119–126) and PrP(121–127) fibrils is a concentration-dependent process that involves coil to sheet conversion with aging. PrP(119–126), the sequence with intrinsic helical propensity, is more toxic in monomer form, and the fibril formation in this case seems to be protective to cells. For PrP(121–127), the SDS-soluble fibrils are more cytotoxic, indicating that a higher order assemblage structure is required for cytotoxic activity of this peptide. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Conformational transition; Peptide aggregation; Helix-sheet transition; Prion; Mad cow disease; Lipid rafts 1. Introduction Misfolded isoforms of the naturally occurring prion protein (PrP) have been shown to be the causative agents in many mammalian neurodegenerative disor- ders, including Cruetzfeldt–Jakob disease (CJD) in hu- man, scrapie in sheep, and bovine spongiform encephalopathy in cows (Prusiner, 1997). Prion infec- tivity is unique in that the pathogenic prion form (PrP Sc ) is involved in the conversion of the endogenous con- formation (PrP C ) into transformed PrP Sc . The ‘‘protein- only’’ hypothesis (Prusiner, 1998) asserts further that no extraneous agents are necessary to explain the unusual behavior of prions. Several groups have analyzed the secondary structure and fibrillogenic properties of syn- thetic peptides of PrP (Baldwin et al., 1995; Pan et al., 1993). Extensive studies of these peptides have estab- lished that the consecutive segment of prion protein spanning residues 106–147 is important for the fibrillo- genic properties of the protein (DeGioia et al., 1994; Tagliavini et al., 1991, 1993, 1994). In particular, the * Corresponding author. Fax: +91-4424911589. E-mail address: karkuvi77@yahoo.co.uk (R. Jayakumar). 1047-8477/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2004.05.006 Journal of Structural Biology 148 (2004) 176–193 Journal of Structural Biology www.elsevier.com/locate/yjsbi