Predicted alpha-helical regions of the prion protein when synthesized as peptides form amyloid.

Abstract

By comparing the amino acid sequences of 11 mammalian and 1 avian prion proteins (PrP), structural analyses predicted four a-helical regions. Peptides corresponding to these regions of Syrian hamster PrP were synthesized, and, contrary to predictions, three ofthe four spontaneously formed amyloids as shown by electron microscopy and Congo red staining. By IR spectroscopy, these amyloid peptides exhibited secondary structures composed largely of 13-sheets. The first of the predicted helices is the 14-amino acid peptide corresponding to residues 109-122; this peptide and the overlapping 15-residue sequence 113-127 both form amyloid. The most highly amyloidogenic peptide is AGAAAAGA, which corresponds to Syrian hamster PrP residues 113-120 and is conserved across all species for which the PrP sequence has been determined. Two other predicted a-helices corresponding to residues 178-191 and 202-218 form amyloids and exhibit considerable 13-sheet structure when synthesized as peptides. These findings suggest the possibility that the conversion of the cellular isoform of PrP to the scrapie isoform of PrP Involves the transition of one or more putative PrP a-helices into 13-sheets and that prion diseases are disorders of protein conformation. 27-30 (19, 20).In the study reported here, we synthesized peptides homologous to portions of the Syrian hamster (SHa) PrP sequence and examined their physical characteristics by attenuated total reflection (ATR) Fourier transform IR spectroscopy (FTIR) (21). The particular peptides were chosen based on structural predictions where the PrP amino acid sequences from 11 mammalian and 1 avian sources were compared (22). Those analyses suggested the possibility that PrP might fold into a monomeric molecule with four a-helices (ref. 23; J.-M.G., F.C., R.F., and S.B.P., unpublished results). Unexpectedly, synthetic peptides corresponding to three of the four putative a-helices exhibit extensive 13-sheet structure as well as the ultrastructural and tinctorial properties of amyloid.MATERIALS AND METHODS All PrP peptides (Table 1) were assembled by using the 9-fluorenylmethoxycarbonyl variant of the Merrifield solidphase method on an Applied Biosystems model 432A Synergy peptide synthesizer. Peptides with side-chain protection were cleaved from the resins by using thioanisole/ethanedithiol/ trifluoroacetic acid (TFA) and precipitated with tert-butyl methyl ether. They were pelleted by centrifugation, suspended in ether, washed three times, repelleted, dissolved in water/ isopropanol, and filtered. Peptides without side-chain protection were cleaved with 90%o (vol/vol) TFA. The solutions were lyophilized, and the peptides were analyzed by reversed-phase HPLC and plasma desorption mass spectrometry. The majority ofpeptides were >80%o pure and were used without further purification. The initial product for peptide H4 was =20%6 pure, and it was purified by preparative HPLC and reanalyzed. The synthesis of SHaPrP-(113-120) gave 40%o Ala6Gly2 (the desired product) and 60% Ala5Gly2, which ...

CD and NMR Studies of Prion Protein (PrP) Helix 1

“…The nature of this initiation site, however, remains unclear. For both regions proposed to be involved in the early stages of transformation, the stretch of apolar residues in the region 110 -120 (12,33) and the short anti-parallel ␤-sheet of the prion protein, no indication for the existence of stable extended conformers could be found. This might be because of the higher flexibility of the used model peptides compared with the fulllength prion protein in which where tertiary interactions stabilize the two ␤-strands of PrP C .…”

Metal‐dependent α‐helix formation promoted by the glycine‐rich octapeptide region of prion protein

“…The aggregated PrP sc acquires resistance to proteolytic degradation and causes fatal neurodegenerative diseases, including scrapie and bovine spongiform encephalopathy in animals and Creutzfeld-Jakob disease in humans [2]. Although the c~-helix-to-[3-sheet transition in the C-terminal half region may be related to the PrP c ~ PrP sc transformation [8,11,12], the molecular mechanism of the conformational transition has not yet been clarified.…”

Genetic analysis of the SPRN gene in ruminants reveals polymorphisms in the alanine-rich segment of shadoo protein

“…Lampo et al (2007) , that it modulates the formation of transmembrane PrP isoforms (Hegde et al, 1998) and that it is important for basolateral sorting of PrP in a cell (Uelhoff et al, 2005). Furthermore, the AGAAAAGA palindrome may play a part in the diseasespecific PrP Sc -PrP C interaction (Norstrom & Mastrianni, 2005): peptides containing this sequence are neurotoxic (Forloni et al, 1993) and amyloidogenic (Gasset et al, 1992), and the variant AGAAVAGA is linked to Gerstmann-Sträussler-Scheinker syndrome, a genetic form of human prion disease (Mallucci et al, 1999). The homology in this region between PrP and Sho implies that there is similar cellular sorting and ligands and that they use similar mechanisms to promote their neuroprotective properties .…”