Interaction Het-S Prion / Membrane to get a Better Insight of Amyloid Toxicity

Abstract

Many neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, spongiform encephalopathies or amyloidopathies, correlate with amyloid formation. Several hypotheses have been proposed to explain the toxicity of amyloids: the existence of intermediates, from oligomers to protofilaments and/or the ability of amyloids to form pores which would destabilize the membranes but also change the cellular homeostasis.Our work aims to characterize the interaction amyloid/membrane in relation with the amyloid toxicity. We have chosen the non toxic Prion Forming Domain 218-289 of Het-s from the filamentous fungi Podospora anserina, as model. By random PCR mutagenesis we generated a toxic mutant in yeast (M8) that interacts in vivo with membrane vesicles. We have demonstrated that in vitro, M8 forms very unusual short amyloid fibers contrary to WT, which polymerizes as long fibers. Furthermore, ATR spectra have shown that M8 toxic mutant is essentially assembled into mixed parallel and anti-parallel β-sheets whereas WT displays a predominant parallel organization. Monolayers of phospholipids at the air-water interface were used as membrane model to investigate the interaction with non toxic amyloid (WT) and toxic amyloid (M8). Characterization of the molecular interactions between amyloid (WT and M8) and different lipid monolayers (DOPG, DOPC, DOPS DOPE and DOPI), at the air-water interface were performed by polarization-modulated infrared reflection absorption spectroscopy, Brewster angle microscopy and ellipsometry. BAM images reveal a high selectivity of the M8 toxic mutant for the anionic phospholipid monolayers (DOPG, DOPI and DOPS). In comparison, WT presents less evidences of interaction with the tested phospholipid monolayers. The secondary structure of amyloids (M8 or WT) interacting with a phospholipid monolayer is mainly made of anti-parallel β-sheets (observed by PMIRRAS) but the orientation of the β-sheet differs between toxic and non toxic mutant.