Cavitation during the protein misfolding cyclic amplification (PMCA) method – The trigger for de novo prion generation?
Introduction
Prions are proteinaceous infectious agents that cause transmissible neurodegenerative diseases such as Creutzfeldt Jakob Disease (CJD) and Bovine Spongiform Encephalopathy (BSE). Prion diseases are associated with the conversion of the normal structure of the prion protein (PrPC) into an infectious conformer (PrPSc). PrPSc accumulates in disease and provides a template for further PrPC misfolding and conversion.
The protein misfolding cyclic amplification (PMCA) technique is a cell-free method developed to amplify very small amounts of PrPSc. The process of PMCA involves “seeding” natively-folded PrPC (from whole brain, cell culture, or recombinant expression) with a minute quantity of infectious PrPSc, followed by repeated cycles of incubation and sonication [1]. A templating interaction with PrPSc leads to the conversion of α-helical PrP to a β-sheet-rich protease-resistant PrPres, whose properties are purported to be identical to those of brain-derived PrPSc. In this way, PMCA is viewed as a means to accelerate the conversion process that occurs in vivo, such that the presence of PrPSc in the original titre can then be inferred by immunodetection of the amplified PrPres.
Recently, the de novo generation of infectious PrP (ie. without addition of a PrPSc seed) was reported by PMCA using only recombinant PrP (rPrP) expressed from E. coli, with RNA and the lipid POPG as essential cofactors [1], [2], [3], [4]. Although it has been possible to propagate PrPSc seeds using rPrP in the absence of RNA and POPG, both by PMCA [6], [7] and vigorous shaking [8], the de novo generation of a recombinant PrP conformer that induces prion disease in wild type mice has only been demonstrated using PMCA in the presence of these cofactors [2], [3], [4]. The molecular mechanism underlying this phenomenon remains unknown.
Sonication of water at frequencies between 20 and 800 kHz causes a repeated expansion and collapse of microbubbles (“acoustic cavitation”). The extremely high local temperature and pressure during cavitation leads to the splitting (sonolysis) of entrapped gaseous water molecules to form hydroxyl () and hydrogen () radicals, in addition to downstream formation of hydrogen peroxide (H2O2) and superoxide radical anions () (Fig. 1A) [9], [10].
In this study, we sought to determine whether bacterially-expressed PrP and any of its essential cofactors for de novo prion formation underwent observable free radical damage during PMCA. Using a combination of spin trapping, immuno-spin trapping and immunodetection of RNA oxidation, we identified the production of hydrogen, hydroxyl and superoxide radicals during PMCA, which caused protein-centred radicals, RNA-centred radicals and polypropylene-centred radicals, together with cross-linking of protease-resistant oxidised PrP-RNA adducts. This extensive free radical damage, in conjunction with cofactor interactions at the hydrophilic/hydrophobic interface of cavitation bubbles, may provide the necessary conditions for producing an oxidatively-modified conformer that subsequently acquires infectious properties.
Section snippets
Reagents
Bovine serum albumin (BSA), 2-Oleoyl-1-palmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (POPG) and phosphate-buffered saline (PBS; 10 mM phosphate, 2.7 mM KCl, 137 mM NaCl, pH 7.4) were purchased from Sigma–Aldrich. Thin-walled, RNAase-free 0.2 mL PCR tubes were obtained from Scientific Specialties Inc. Bacterially-expressed α-folded recombinant murine PrP(23–231) was obtained from Prionatis. Mouse Xpress Ref universal total RNA (whole body) was purchased from Qiagen.
PMCA provides a radical rich environment
The standard instrument for PMCA is the Misonix Sonicator 4000 with cup-horn accessory [1]. The frequency (20 kHz) and power (∼1–3 Watts/cm3) of sonication used for PMCA should be sufficient to trigger the free radical production (Fig. 1A). We first established that free radicals are produced under these conditions. To enable detection of these short-lived radicals, we used the spin trapping technique to form longer-lived radical adducts and then identified these adducts using electron
Discussion
Using a substrate preparation comparable to that employed in previous studies of de novo prion formation [2], [3], [4], the present data demonstrate that sonication during PMCA provides a radical-rich environment that causes oxidation of protein and RNA, together with covalently cross-linked PrP-RNA adducts. Although the relevance to de novo prion formation remains unclear, we also observed radical damage to the polypropylene tubes in which the substrates were contained, which may also undergo
Conflict of interest
None.
Acknowledgments
This work was supported by a Future Fellowship (FT110100199) awarded to S.C.D. and administered by the Australian Research Council.
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