Biochemistry and Pharmacology - Research Publications

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    Conservation of a Glycine-rich Region in the Prion Protein Is Required for Uptake of Prion Infectivity
    Harrison, CF ; Lawson, VA ; Coleman, BM ; Kim, Y-S ; Masters, CL ; Cappai, R ; Barnham, KJ ; Hill, AF (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010-06-25)
    Prion diseases are associated with the misfolding of the endogenously expressed prion protein (designated PrP(C)) into an abnormal isoform (PrP(Sc)) that has infectious properties. The hydrophobic domain of PrP(C) is highly conserved and contains a series of glycine residues that show perfect conservation among all species, strongly suggesting it has functional and evolutionary significance. These glycine residues appear to form repeats of the GXXXG protein-protein interaction motif (two glycines separated by any three residues); the retention of these residues is significant and presumably relates to the functionality of PrP(C). Mutagenesis studies demonstrate that minor alterations to this highly conserved region of PrP(C) drastically affect the ability of cells to uptake and replicate prion infection in both cell and animal bioassay. The localization and processing of mutant PrP(C) are not affected, although in vitro and in vivo studies demonstrate that this region is not essential for interaction with PrP(Sc), suggesting these residues provide conformational flexibility. These data suggest that this region of PrP(C) is critical in the misfolding process and could serve as a novel, species-independent target for prion disease therapeutics.
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    Anionic Phospholipid Interactions of the Prion Protein N Terminus Are Minimally Perturbing and Not Driven Solely by the Octapeptide Repeat Domain
    Boland, MP ; Hatty, CR ; Separovic, F ; Hill, AF ; Tew, DJ ; Barnham, KJ ; Haigh, CL ; James, M ; Masters, CL ; Collins, SJ (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010-10-15)
    Although the N terminus of the prion protein (PrP(C)) has been shown to directly associate with lipid membranes, the precise determinants, biophysical basis, and functional implications of such binding, particularly in relation to endogenously occurring fragments, are unresolved. To better understand these issues, we studied a range of synthetic peptides: specifically those equating to the N1 (residues 23-110) and N2 (23-89) fragments derived from constitutive processing of PrP(C) and including those representing arbitrarily defined component domains of the N terminus of mouse prion protein. Utilizing more physiologically relevant large unilamellar vesicles, fluorescence studies at synaptosomal pH (7.4) showed absent binding of all peptides to lipids containing the zwitterionic headgroup phosphatidylcholine and mixtures containing the anionic headgroups phosphatidylglycerol or phosphatidylserine. At pH 5, typical of early endosomes, quartz crystal microbalance with dissipation showed the highest affinity binding occurred with N1 and N2, selective for anionic lipid species. Of particular note, the absence of binding by individual peptides representing component domains underscored the importance of the combination of the octapeptide repeat and the N-terminal polybasic regions for effective membrane interaction. In addition, using quartz crystal microbalance with dissipation and solid-state NMR, we characterized for the first time that both N1 and N2 deeply insert into the lipid bilayer with minimal disruption. Potential functional implications related to cellular stress responses are discussed.
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    A rigorous method to enrich for exosomes from brain tissue
    Vella, LJ ; Scicluna, BJ ; Cheng, L ; Bawden, EG ; Masters, CL ; Ang, C-S ; Willamson, N ; McLean, C ; Barnham, KJ ; Hill, AF (TAYLOR & FRANCIS LTD, 2017-07-26)
    Extracellular vesicles, including exosomes, are released by all cells, including those of the nervous system. Capable of delivering lipid, protein and nucleic acids to both nearby and distal cells, exosomes have been hypothesized to play a role in progression of many diseases of the nervous system. To date, most analyses on the role of these vesicles in the healthy and diseased state have relied on studying vesicles from in vitro sources, such as conditioned cell culture media, or body fluids. Here we have taken a critical approach to the enrichment and characterization of exosomes from human frontal cortex. This method maintains the integrity of the vesicles and their cargo, and comprehensive proteomic and genomic characterization confirms the legitimacy of the resulting extracellular vesicles as endosome-derived exosomes. This method will enable neuroscientists to acquire more detailed information about exosomes in the brain and explore the role(s) this form of intercellular communication and unique source of lipid, protein and RNA has in healthy brain function and pathogenic conditions. Furthermore, this method may have important utility in the isolation of exosomes from other tissues.
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    The hypoxia imaging agent CuII(atsm) is neuroprotective and improves motor and cognitive functions in multiple animal models of Parkinson's disease
    Hung, LW ; Villemagne, VL ; Cheng, L ; Sherratt, NA ; Ayton, S ; White, AR ; Crouch, PJ ; Lim, S ; Leong, SL ; Wilkins, S ; George, J ; Roberts, BR ; Pham, CLL ; Liu, X ; Chiu, FCK ; Shackleford, DM ; Powell, AK ; Masters, CL ; Bush, AI ; O'Keefe, G ; Culvenor, JG ; Cappai, R ; Cherny, RA ; Donnelly, PS ; Hill, AF ; Finkelstein, DI ; Barnham, KJ (ROCKEFELLER UNIV PRESS, 2012-04-09)
    Parkinson's disease (PD) is a progressive, chronic disease characterized by dyskinesia, rigidity, instability, and tremors. The disease is defined by the presence of Lewy bodies, which primarily consist of aggregated α-synuclein protein, and is accompanied by the loss of monoaminergic neurons. Current therapeutic strategies only give symptomatic relief of motor impairment and do not address the underlying neurodegeneration. Hence, we have identified Cu(II)(atsm) as a potential therapeutic for PD. Drug administration to four different animal models of PD resulted in improved motor and cognition function, rescued nigral cell loss, and improved dopamine metabolism. In vitro, this compound is able to inhibit the effects of peroxynitrite-driven toxicity, including the formation of nitrated α-synuclein oligomers. Our results show that Cu(II)(atsm) is effective in reversing parkinsonian defects in animal models and has the potential to be a successful treatment of PD.
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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.