New Principal Investigators 12

The infectious isoform of the prion protein, PrP^Sc, is responsible for diseases such as bovine spongiform encephalopathy (BSE, also known as “Mad Cow Disease”), chronic wasting disease (CWD) in deer and elk, and Creutzfeldt-Jakob disease (CJD) in humans. The identification of the first home-grown case of BSE in Canada in May 2003 led to devastating economic losses, with estimates of the damage surpassing $7 billion. The proven ability of BSE prions to infect other species such as sheep, cats, and humans gives BSE-related research relevance in agriculture, veterinary medicine, and human healthcare. Furthermore, in recent years it has become apparent that protein-misfolding diseases such as Alzheimer’s disease, Parkinson’s disease, and Lou Gehrig’s disease propagate throughout the tissue of afflicted individuals with a prion-like mechanism, emphasizing similarities in pathology and structural biology.

The research in my laboratory focuses on the structure of amyloids and other disease-related, misfolded proteins. In particular, we are interested in PrP^Sc (and its truncated variants) and the structure-function relationship underlying its infectious nature. The scope of our experimental approaches is centred on electron microscopy and helical reconstruction techniques, supplemented by other biochemical and biophysical methods.

So far, the structure of PrP^Sc has eluded experimental determination due to its insolubility and propensity to aggregate. Molecular modelling has been used to predict its structure, but the various modelling approaches produced conflicting results indicating the limitations of this method. In absence of a three-dimensional structure, a variety of experimental techniques have been used to gain insights into the fold of this isoform. Negative stain electron microscopy, X-ray fiber diffraction, and molecular modelling indicated that the beta-sheets of PrP^Sc form a beta-helix or beta-solenoid structure with a height of four beta-strands (rungs) per molecule (= 19.2 Å).

In our recent efforts to analyse the structure of PrP^Sc we have employed a hybrid approach. In particular, the helical periodicity that is inherent to most amyloid fibrils can be used to generate a three-dimensional structure from two-dimensional electron micrographs and electron tomograms. Higher-resolution structures require electron micrographs to be recorded using cryo low-dose imaging. Preliminary, helical reconstructions of negatively stained fibrils showed repeating densities along the fibril axis spaced at ~40 Å (2x ~19.2 Å), in good agreement with our earlier X-ray fiber diffraction results. Cryo low-dose electron micrographs of PrP^Sc amyloid fibrils routinely exhibit a 4.8 Å spacing, confirming the presence of beta-strands in a cross-beta configuration. Current efforts are targeted towards visualizing the 4.8 Å beta-strands in individual helical reconstructions, with the ultimate aim to decipher the structure of the PrP^Sc to at least 4.8 Å. Such an outcome would provide us with unique, structure-based insights into the molecular replication mechanism and also enable structure-based drug design approaches.