Selective incorporation of polyanionic molecules into hamster prions.

The Journal of biological chemistry (2007-10-18)
James C Geoghegan, Pablo A Valdes, Nicholas R Orem, Nathan R Deleault, R Anthony Williamson, Brent T Harris, Surachai Supattapone
RESUMEN

The central pathogenic event of prion disease is the conformational conversion of a host protein, PrPC, into a pathogenic isoform, PrPSc. We previously showed that the protein misfolding cyclic amplification (PMCA) technique can be used to form infectious prion molecules de novo from purified native PrPC molecules in an autocatalytic process requiring accessory polyanions (Deleault, N. R., Harris, B. T., Rees, J. R., and Supattapone, S. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 9741-9746). Here we investigated the molecular mechanism by which polyanionic molecules facilitate infectious prion formation in vitro. Ina PMCA reaction lacking PrPSc template seed, synthetic polyA RNA molecules induce hamster HaPrPC to adopt a protease-sensitive, detergent-insoluble conformation reactive against antibodies specific for PrPSc. During PMCA, labeled nucleic acids form nuclease-resistant complexes with HaPrP molecules. Strikingly, purified HaPrPC molecules subjected to PMCA selectively incorporate an approximately 1-2.5-kb subset of [32P]polyA RNA molecules from a heterogeneous mixture ranging in size from approximately 0.1 to >6 kb. Neuropathological analysis of scrapie-infected hamsters using the fluorescent dye acridine orange revealed that RNA molecules co-localize with large extracellular HaPrP aggregates. These findings suggest that polyanionic molecules such as RNA may become selectively incorporated into stable complexes with PrP molecules during the formation of native hamster prions.

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Sodium deoxycholate, ≥97% (titration)
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Anti-Actin (20-33) antibody produced in rabbit, IgG fraction of antiserum, buffered aqueous solution
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SP Sepharose®, Fast Flow, aqueous ethanol suspension, 45-165 μm (wet), exclusion limit ~4,000,000 Da

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