In vitro conversion and seeded fibrillization of posttranslationally modified prion protein
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Jan Stöhr
Abstract
The conversion of the cellular isoform of the prion protein (PrPC) into the pathologic isoform (PrPSc) is the key event in prion diseases. To study the conversion process, an in vitro system based on varying the concentration of low amounts of sodium dodecyl sulfate (SDS) has been employed. In the present study, the conversion of full-length PrPC isolated from Chinese hamster ovary cells (CHO-PrPC) was examined. CHO-PrPC harbors native, posttranslational modifications, including the GPI anchor and two N-linked glyco-sylation sites. The properties of CHO-PrPC were compared with those of full-length and N-terminally truncated recombinant PrP. As shown earlier with recombinant PrP (recPrP90-231), transition from a soluble α-helical state as known for native PrPC into an aggregated, β-sheet-rich PrPSc-like state could be induced by dilution of SDS. The aggregated state is partially proteinase K (PK)-resistant, exhibiting a cleavage site similar to that found with PrPSc. Compared to recPrP (90-231), fibril formation with CHO-PrPC requires lower SDS concentrations (0.0075%), and can be drastically accelerated by seeding with PrPSc purified from brain homogenates of terminally sick hamsters. Our results show that recPrP 90-231 and CHO-PrPC behave qualitatively similar but quantitatively different. The in vivo situation can be simulated closer with CHO-PrPC because the specific PK cleave site could be shown and the seed-assisted fibrillization was much more efficient.
©2011 by Walter de Gruyter Berlin New York
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- Recent insights into regulation of transcription by RNA polymerase III and the cellular functions of its transcripts
- Genes and Nucleic Acids
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- Protein Structure and Function
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