Proteolytic processing of the cellular prion protein ‐ its importance in health and as a modulator of TSE disease susceptibility in sheep
Campbell, Lauren Smith
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Expression of the cellular prion protein (PrPC) from the PRNP gene is crucial for the development of a group of fatal neurodegenerative disorders called prion diseases. During prion infection a misfolded protein homologue of PrPC, PrPSc causes further misfolding on interaction with native PrPC molecules. PrPSc is highly resistant to proteinase K and aggregation of this protein is considered a hallmark of infection. Sheep are considered a model of natural infection and susceptibility to scrapie in sheep is defined by polymorphisms in the PRNP gene. It is still not fully understood how these polymorphisms regulate the conversion process or which other co-‐factors are involved. One such factor may be the truncation of PrPC via proteolytic processing in the form of two main cleavage events, known as α-‐ and β-‐cleavage. In sheep α-‐cleavage cuts at amino acid 115, creating two truncated proteins C1 and N1 and represents the main cleavage event in healthy brain. β-‐Cleavage creates a longer C-‐terminal fragment, C2 and corresponding N-‐terminal fragment N2, cutting around amino acid 92 in sheep. Truncated forms of PrPC have been shown to represent around 50 % of total residual PrP in brain and may be an important determinant of disease through both decreasing the amount of full length PrPC available for conversion and through functions associated with the truncated fragments. The research presented has shown that increased production of an α-‐cleavage fragment C1 in brain is associated with TSE resistant genotype ARR/ARR, while the presence of C2 fragment is affiliated with scrapie susceptible PRNP genotypes in brain. There was no difference in the levels of full length PrPC in these genotypes suggesting that PrP expression does not directly correlate to susceptibility in this model. To assess if PrPC fragments could affect the conversion during disease in-‐vitro fibrillisation assays were performed using novel truncated recombinant proteins. These truncated proteins, although not thought to convert to PK resistant PrPSc during disease, can form amyloid fibrils. However, these fibrils appear to be less neurotoxic when compared to fibrils produced by full length PrPC. Only the truncated fragments derived from the ARR allele inhibit in-‐vitro fibrillisation of other allelic PrPC variants. Furthermore, treatment of infected cells in culture with recombinant C1ARR led to a decrease in the formation of disease associated PrPSc. In conclusion, genetic variations in levels of PrP truncated fragments may add to the complexity of genetic determinants of prion disease. In parallel with polymorphism-‐dependant conversion abilities, varying α-‐cleavage of ovine PrPC may help to explain genetic resistance in sheep. The inhibitory effects of C1, illustrated in-‐ vitro may represent a therapeutic avenue in the treatment of prion disease.