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Title: Cavitation during the protein misfolding cyclic amplification (PMCA) method – The trigger for de novo prion generation?

The protein misfolding cyclic amplification (PMCA) technique has become a widely-adopted method for amplifying minute amounts of the infectious conformer of the prion protein (PrP). PMCA involves repeated cycles of 20 kHz sonication and incubation, during which the infectious conformer seeds the conversion of normally folded protein by a templating interaction. Recently, it has proved possible to create an infectious PrP conformer without the need for an infectious seed, by including RNA and the phospholipid POPG as essential cofactors during PMCA. The mechanism underpinning this de novo prion formation remains unknown. In this study, we first establish by spin trapping methods that cavitation bubbles formed during PMCA provide a radical-rich environment. Using a substrate preparation comparable to that employed in studies of de novo prion formation, we demonstrate by immuno-spin trapping that PrP- and RNA-centered radicals are generated during sonication, in addition to PrP-RNA cross-links. We further show that serial PMCA produces protease-resistant PrP that is oxidatively modified. We suggest a unique confluence of structural (membrane-mimetic hydrophobic/hydrophilic bubble interface) and chemical (ROS) effects underlie the phenomenon of de novo prion formation by PMCA, and that these effects have meaningful biological counterparts of possible relevance to spontaneous prion formation in vivo. - Highlights:more » • Sonication during PMCA generates free radicals at the surface of cavitation bubbles. • PrP-centered and RNA-centered radicals are formed in addition to PrP-RNA adducts. • De novo prions may result from ROS and structural constraints during cavitation.« less
Authors:
 [1] ;  [2]
  1. Department of Pathology, The University of Melbourne, Victoria 3010 (Australia)
  2. Florey Department of Neuroscience and Mental Health, The University of Melbourne, Victoria 3010 (Australia)
Publication Date:
OSTI Identifier:
22462068
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 461; Journal Issue: 3; Other Information: Copyright (c) 2015 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ADDUCTS; AMPLIFICATION; BUBBLES; CAVITATION; COMPARATIVE EVALUATIONS; ELECTRON SPIN RESONANCE; IN VIVO; INCUBATION; LIMITING VALUES; MEMBRANES; OXIDATION; OXIDES; PHOSPHOLIPIDS; PROTEINS; RADICALS; RNA; SPIN; SURFACES