In addition to its central role in fatal neurodegenerative syndromes like prion diseases (also known as transmissible spongiform encephalopathies), the prion protein (PrP) is now one of the most studied models for misfolding diseases. It is the first system where a protein has been shown to exist in, at least, two significantly different conformations, associated with radically different functions.

The cellular form of the prion protein (PrP^C) is a glysosyl-phosphatidylinositol (GPI) anchored polypeptide present on the outside leaf of the cellular membrane of most cell types in mammals. Upon conformational change it may convert into a pathological form denominated PrP^Sc, so far the only identified causative agent of prion diseases. The pre-pro-protein is composed of 253 amino acids in humans, which includes 22 amino acids of signal sequence at the N-terminus and 23 amino acids as GPI anchoring signal at the C-terminus. The N-terminal region of PrP^C encompasses five signature octarepeats that coordinate copper and, to a lesser extent, other metal ions.

The physiological function of PrP^C has not been established with certainty and one additional and relevant focus of the laboratory will be to follow-up recent findings indicating a possible role of the protein in neuronal differentiation and polarization (Kanaani et al., 2005).

Furthermore, we have been developing new expression systems for the characterization of the structure of PrP^C, the cellular form of the prion protein (Legname et al., 2002). Biophysical studies concerning PrP have largely been limited to investigations into structure/folding of recombinant material produced in Escherichia coli. This is due mainly because of the difficulties in isolating pure, natively folded wild type protein. This is a significant disadvantage, in fact lacking an assayable function, establishing the proper "folded" state upon in vitro refolding involves considerable guesswork. This uncertainty, coupled with the known N-terminal unstructured region of recombinant PrP, begs the question whether an in vivo production system, utilizing mammalian cells with their own protein folding machinery, would produce a more natively folded molecule. Such a system has been established in our lab using a vector which, when suitably transfected into mammalian cells, expresses a folded PrP fused with the Fc region of an immunoglobulin protein (denominated PrP-Fc). This protein shares many of the features of cellular PrP including glycosylation, yet lacks a GPI anchor signalling sequence on its C-terminus. This allows the protein to be secreted into the growth media of the cells, where it can be easily purified in quantity. This material should provide an exciting target for structural studies of the complete in vivo folded prion protein, while allowing for the simple inclusion of pathogenic variants (Legname et al., 2002).

Produced in mammalian cells, PrP-Fc is likely to possess a natively folded N-terminal (whether structured or not) and offers a unique opportunity to resolve the structure of this domain, and in particular the organization of the octarepeat region. Several crystallization screening approaches are carried in parallel. On one hand, the purified molecule is directly brought to a concentration of 10mg/ml and directly submitted to large scale automated nanodrop screening. So far this approach has not yielded protein crystals. In parallel, we are trying to crystallize PrP-Fc in combination with ligands, in particular copper and/or antibodies. We believe that antibodies recognizing native-like conformations of the N-terminus are likely to favor the formation of crystal lattice by stabilizing this dynamical domain. After initial trials of mixing Fab with PrP-Fc in the crystallization nanodrop, now we would like to co-purify the complex before crystallization in order to avoid problems due to incomplete complex formation.

All the above projects have provided the basis for a novel discovery in Prion Biology. Since prion amyloid deposition can be found in the brain of humans and animals affected by prion disease, we set to produce pure amyloid preparations using recombinant mouse prion protein. These preparations were used to inoculate mice intracranially to test the hypothesis that we have created infectious material in vitro. The accounts of this research have been reported in three recent publications, one in the journal Science and two in the Proceedings of the National Academy of Science, USA (Legname et al., 2004; Legname et al., 2005; Legname et al., 2006).