When prions are transmitted between species, there can be a delay in pathogenesis due to a phenomenon referred to as the transmission barrier. Some species also show very low susceptibility to prion disease. In this study I hypothesized that the susceptibility of species to prion disease is proportional to the tendency of their endogenous prion protein, PrP, to adopt the β-state, an oligomeric form of misfolded recombinant PrP that is rich in β sheet.
Using a novel method of two-wavelength CD analysis, it could be shown that recombinant PrP from prion-susceptible species have a higher propensity to refold to the β-state than resistant species. The crystal structure of rabbit PrPC 121-230 revealed a helix-cap motif at the N-terminus of helix-2 that contributes to the reduced β-state propensity of rabbit PrP.
Single amino acid changes in the sequence of PrP can lead to a transmission barrier and/or resistance in species. Mutating single residues in rabbit PrPC to those found in corresponding positions in hamster PrPC, ablated the helix-cap observed in the wild-type and caused an increase in the β-state propensity of rabbit PrP. Conversely, a decrease in β-state propensity was observed when rabbit mutations were introduced into PrP of hamster, a susceptible species.
A dimeric association is hypothesized to be involved in the function of PrP and/or the conversion mechanism to infectious prion. In the structures of the wild-type and mutant rabbit PrPCs a dimeric arrangement was observed in the asymmetric unit of the crystals. Using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), a dimer of rabbit and hamster PrPC was crosslinked in solution. The dimer crosslink was specific and dependent on the tertiary structure of PrPC. Crosslinking of the β-state octamer with EDC showed that similar contacts may be present in this oligomeric form.
Together these data provide strong evidence that species susceptibility is linked to β-state propensity.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/43717 |
| Date | 14 January 2014 |
| Creators | Sweeting, Braden |
| Contributors | Pai, Emil F. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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