Contribution of two conserved glycine residues to fibrillogenesis of the 106–126 prion protein fragment. Evidence that a soluble variant of the 106–126 peptide is neurotoxic

Abstract

The fibrillogenic peptide corresponding to the residues 106-126 of the prion protein sequence (PrP 106-126) is largely used to explore the neurotoxic mechanisms underlying the prion disease. However, whether the neuronal toxicity of PrP 106-126 is caused by a soluble or fibrillar form of this peptide is still unknown. The aim of this study was to correlate the structural state of this peptide with its neurotoxicity. Here we show that the two conserved Gly114 and Gly119 residues, in force of their intrinsic flexibility, prevent the peptide assuming a structured conformation, favouring its aggregation in amyloid fibrils. The substitution of both Gly114 and Gly119 with alanine residues (PrP 106-126 AA mutated peptide) reduces the flexibility of this prion fragment and results in a soluble, b-structured peptide. Moreover, PrP 106-126 AA fragment was highly toxic when incubated with neuroblastoma cells, likely behaving as a neurotoxic protofibrillar intermediate of the wild-type PrP 106-126. These data further confirm that the fibrillar aggregation is not necessary for the induction of the toxic effects of PrP 106-126.

Computational approaches to shed light on molecular mechanisms in biological processes

“…Preliminary results concerning the analysis of the simulations carried out on 113-120 and 106-126 peptides evidenced a reduced relative mobility of Gly114 and Gly119 in both the peptides, and similar behaviour appears associated with the two terminal glycines in the 106-126 sequence. From secondary structure data and principal components analysis, it seems that Gly113 and Gly119 act as a sort of structural pivot, as also emerged from experiments [117]. The 113-120 sequence seems to present specific flexibility, preserved also in the larger 106-126 segment, in accordance with the experimental observation that the palindrome sequence modulates not only the neurotoxic activity of the 106-126 peptide but also its secondary structure properties [118].…”

Overstimulation of PrPC Signaling Pathways by Prion Peptide 106-126 Causes Oxidative Injury of Bioaminergic Neuronal Cells

“…Here, however, the time schedule of the deleterious events observed in 1C11-derived neuronal cells contrasts with the long delay between PrP-(106 -126) exposure and cell response as recorded with other experimental systems (42)(43)(44). Such a discrepancy may relate to the conditions of preparation of PrP-(106 -126), which influence the aggregation state of the peptide (45,46). Indeed, regarding entire PrP Sc , oligomers containing only [14][15][16] Sc monomers were recently shown to behave as the most infectious prion material, as compared with larger aggregates or amyloids (47), considered as inert dumping grounds for misfolded proteins (48 -50).…”

Accelerated Prion Disease Pathogenesis in Toll-Like Receptor 4 Signaling-Mutant Mice

“…with 1 ml of 4% thioglycolate; 4 days later M⌽s were collected by peritoneal lavage at greater than 98% purity, with those from each mouse strain pooled separately, and cultured as previously described (34). After cells were incubated overnight, the medium was replaced with medium containing either a peptide consisting of human PrP residues 106 to 126 (PrP 106-126 ; 40 M), PrP 106-126 scrambled (scrPrP 106-126 ; 40 M), or PrP 118-135 (80 M) (Bachem, King of Prussia, PA); LPS (Escherichia coli strain 055:B5; 1 ng/ml); zymosan (10 g/ml), or no added reagents and incubated for an additional 24 h. Prior to addition to M⌽ cultures, all PrP peptides (at 1.0 mM) had been aged at ambient temperature for 72 h in RPMI base medium to generate fibrils (PrP 106-126 and PrP 118-135 only), according to previously described methods (23,47). Endotoxin was not detected in any of the above reagents (except for LPS) using a Limulus amebocyte lysate assay (Sigma).…”