Assemblages of prion fragments: novel model systems for understanding amyloid toxicity.
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 alpha-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 beta-sheet structure with a significant percentage of helical conformation and beta-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 Abeta 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.
Bioorganic and Neurochemistry Laboratory, Central Leather Research Institute, Adyar, Chennai 600 020, India.
K S
KS
Satheeshkumar
J
J
Murali
R
R
Jayakumar
D000682:Amyloid / Q000737:chemistry* / Q000633:toxicity; D016229:Amyloid beta-Peptides / Q000737:chemistry; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004195:Disease Models, Animal; D008055:Lipids / Q000737:chemistry; D021962:Membrane Microdomains; D046529:Microscopy, Electron, Transmission; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011328:Prions / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011721:Pyrenes / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013574:Synaptosomes / Q000378:metabolism; D013696:Temperature; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology
D000682:Amyloid; D016229:Amyloid beta-Peptides; D008055:Lipids; D010446:Peptide Fragments; D010455:Peptides; D011328:Prions; D011721:Pyrenes; D013778:Tetrazolium Salts; D013844:Thiazoles; D012967:Sodium Dodecyl Sulfate; C022616:thiazolyl blue