Biochemistry and Pharmacology - Research Publications

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Author: Barnham, Kevin × Author: Hill, Andrew × End date: 2014 × Start date: 2014 × Type: Journal Article ×

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    C-terminal peptides modelling constitutive PrPC processing demonstrate ameliorated toxicity predisposition consequent to α-cleavage
    Johanssen, VA ; Johanssen, T ; Masters, CL ; Hill, AF ; Barnham, KJ ; Collins, SJ (PORTLAND PRESS LTD, 2014-04-01)
    Misfolding of PrPC (cellular prion protein) to β-strand-rich conformations constitutes a key event in prion disease pathogenesis. PrPC can undergo either of two constitutive endoproteolytic events known as α- and β-cleavage, yielding C-terminal fragments known as C1 and C2 respectively. It is unclear whether C-terminal fragments generated through α- and β-cleavage, especially C2, influence pathogenesis directly. Consequently, we compared the biophysical properties and neurotoxicity of recombinant human PrP fragments recapitulating α- and β-cleavage, namely huPrP-(112-231) (equating to C1) and huPrP-(90-231) (equating to C2). Under conditions we employed, huPrP-(112-231) could not be induced to fold into a β-stranded isoform and neurotoxicity was not a feature for monomeric or multimeric assemblies. In contrast, huPrP-(90-231) easily adopted a β-strand conformation, demonstrated considerable thermostability and was toxic to neurons. Synthetic PrP peptides modelled on α- and β-cleavage of the unique Y145STOP (Tyr145→stop) mutant prion protein corroborated the differential toxicity observed for recombinant huPrP-(112-231) and huPrP-(90-231) and suggested that the persistence of soluble oligomeric β-strand-rich conformers was required for significant neurotoxicity. Our results additionally indicate that α- and β-cleavage of PrPC generate biophysically and biologically non-equivalent C-terminal fragments and that C1 generated through α-cleavage appears to be pathogenesis-averse.
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    Pathogenic Mutations within the Hydrophobic Domain of the Prion Protein Lead to the Formation of Protease-Sensitive Prion Species with Increased Lethality
    Coleman, BM ; Harrison, CF ; Guo, B ; Masters, CL ; Barnham, KJ ; Lawson, VA ; Hill, AF ; Caughey, BW (AMER SOC MICROBIOLOGY, 2014-03)
    UNLABELLED: Prion diseases are a group of fatal and incurable neurodegenerative diseases affecting both humans and animals. The principal mechanism of these diseases involves the misfolding the host-encoded cellular prion protein, PrP(C), into the disease-associated isoform, PrP(Sc). Familial forms of human prion disease include those associated with the mutations G114V and A117V, which lie in the hydrophobic domain of PrP. Here we have studied the murine homologues (G113V and A116V) of these mutations using cell-based and animal models of prion infection. Under normal circumstances, the mutant forms of PrP(C) share similar processing, cellular localization, and physicochemical properties with wild-type mouse PrP (MoPrP). However, upon exposure of susceptible cell lines expressing these mutants to infectious prions, very low levels of protease-resistant aggregated PrP(Sc) are formed. Subsequent mouse bioassay revealed high levels of infectivity present in these cells. Thus, these mutations appear to limit the formation of aggregated PrP(Sc), giving rise to the accumulation of a relatively soluble, protease sensitive, prion species that is highly neurotoxic. Given that these mutations lie next to the glycine-rich region of PrP that can abrogate prion infection, these findings provide further support for small, protease-sensitive prion species having a significant role in the progression of prion disease and that the hydrophobic domain is an important determinant of PrP conversion. IMPORTANCE: Prion diseases are transmissible neurodegenerative diseases associated with an infectious agent called a prion. Prions are comprised of an abnormally folded form of the prion protein (PrP) that is normally resistant to enzymes called proteases. In humans, prion disease can occur in individuals who inherited mutations in the prion protein gene. Here we have studied the effects of two of these mutations and show that they influence the properties of the prions that can be formed. We show that the mutants make highly infectious prions that are more sensitive to protease treatment. This study highlights a certain region of the prion protein as being involved in this effect and demonstrates that prions are not always resistant to protease treatment.