The structure of the cellular prion protein (PrPc), conformational change into the scrapie-isoform (PrPSc or PrPres) and functional consequences thereof are at the heart of understanding prion diseases (transmissible spongi for mencephalopathies). The C-terminal half of PrPc is an intrinsically stable domain that folds autonomously and reversibly and possesses a hitherto unknown protein fold consisting of three alfa-helices and a two-stranded antiparallelbeta-sheet (Riek et al., 1996; Hornemann and Glockshuber, 1996). The centraldomain of PrP contains a neurotoxic segment (Forloni et al., 1993; Brown etal., 1994; Brown et al., 1996) as well as a cellular cleavage site. The N-terminal moiety may be essential for endocytosis of PrPc in clathrin-coatedpits (Shyng et al., 1995) and contains a well-preserved octa-repeat whose function is unknown. In contrast to the C-terminal half, the N-terminal and central domains seem to be largely unstructured under normal conditions (Glockshuber and Wuthrich, submitted for publication). This collaboration will concentrate on structural and functional features of all three domains of PrP, on possible interactions of these domains as well as on the conditions of conformational change from PrPc into PrPSc or PrPres.
To achieve this aim full-length recombinant PrPc and mutated PrPs as well as peptides from the three domains will be purified from procaryotic systems (Glockshuber, submitted for publication), synthesized in vitro and expressed in eucaryotic cell culture systems and transgenic mice. Biophysical technologies including NMR spectroscopy will be used to study structural features of the N-terminal and central domains and possible interactions with the C-terminal half under various physico-chemicai conditions. Efforts will be made to identify smaller fragments in PrP121-231 which still fold and adopt a defined three-dimensional structure in order to identify regions which are essential for stability or most unstable and are therefore most likely to form nucleation points for conversion of PrPc to PrPSc/res.
All biophysically characterized peptides and PrP mutants will be tested for phenotypic cell biological consequences and convertibility to PrPSc/res in a number of systems. These include primary cell cultures from wild-type and transgenic mice for the study of neurotoxic and neuroprotective effects of peptides and PrP-variants. Point mutations and deletion mutants of the three domains will be expressed and tested for phenotypical changes and convertibility to PrPres in a neuroblastome cell iine (N2a and ScN2a) in which PrP genes are expressed in a binary tetracycline (Tc)-inducible expression system consisting of a Tc controlled transactivator (tTA) and the PrP gene under the control of a transactivator-responsive promoter. Transgenic animals expressing various deletion mutants on a PrPÀ'À background (mostly from the laboratory of Charles Weissmann) will be phenotypically investigated and additional transgenic lines will be generated with the details of the genetic design depending on the results of ongoing biophysical studies. Tests for infectivity will be performed in wild-type and transgenic animals.
Funding SchemeCSC - Cost-sharing contracts
CB2 2PY Cambridge
NE4 6BE Newcastle Upon Tyne