A fundamental event in the pathogenesis of
transmissible spongiform encephalopathies (TSE) is the conversion of a normal,
proteinase K-sensitive, host-encoded
protein,
PrP-sen, into its
protease-resistant
isoform,
PrP-res. During the formation of
PrP-res,
PrP-sen undergoes conformational changes that involve an increase of beta-sheet secondary structure. While previous studies in which
PrP-sen deletion mutants were expressed in transgenic mice or
scrapie-infected cell cultures have identified regions in
PrP-sen that are important in the formation of
PrP-res, the exact role of
PrP-sen secondary structures in the conformational transition of
PrP-sen to
PrP-res has not yet been defined. We constructed
PrP-sen mutants with deletions of the first beta-strand, the second beta-strand, or the first alpha-helix and tested whether these mutants could be converted to
PrP-res in both
scrapie-infected
neuroblastoma cells (Sc(+)-MNB cells) and a cell-free conversion assay. Removal of the second beta-strand or the first alpha-helix significantly altered both processing and the cellular localization of
PrP-sen, while deletion of the first beta-strand had no effect on these events. However, all of the mutants significantly inhibited the formation of
PrP-res in Sc(+)-MNB cells and had a greatly reduced ability to form
protease-resistant PrP in a cell-free assay system. Thus, our results demonstrate that deletion of the beta-strands and the first alpha-helix of
PrP-sen can fundamentally affect
PrP-res formation and/or
PrP-sen processing.