No description available.

No description available.

No description available.

No description available.

No description available.

The cell biology of Prion formation and transfer is not well understood. In order to further elucidate the dynamics of PrPc and PrPsc in a cellular context, fusions between Green Fluorescent Protein (GFP) and PrP were constructed and infected/uninfected cell line pairs expressing these constructs were created. Biochemical analysis indicated that the C-terminal PrPc portion of the fusion protein successfully converted to PrPsc. However, further studies demonstrated that proteolysis occurs between GFP and PrP and therefore the fusion protein cannot be employed as a direct reporter for PrPsc. A Time-Lapse microscopy system was set up and studies were undertaken with infected and uninfected cell lines expressing the fusion construct or cytoplasmic markers to observe events that may be related to transfer of infectivity. Although no exchange of fusion protein is observed, cytoplasmic material is released from both infected and uninfected cell lines. Fluorescence recovery after photobleaching (FRAP) was carried out to establish a system for further investigation of PrP dynamics in the plane of the membrane. Early experiments indicate the possibility of a difference in the diffusion of PrP between infected and uninfected contexts. It is not currently known how Prion glycoform profile is transmitted and maintained following a new infection. The glycoform profile of PrPscwas perturbed in order to investigate the causal role of PrPsc glycotypes in transmission and maintanence of Prion glycoform profile. The results indicate that perturbation of PrPsc glycoform profile in an infectious source does not lead to a correlated perturbation of glycoform profile in the newly established infection. Therefore the glycosylation of PrPsc in an infectious source is not a required source of information for establishing the glycoform profile of a Prion infection.

Analysis of how prions are propagated and transmitted in the yeast Saccharomyces cerevisiae has begun to reveal how an amyloid-forming protein can act as an epigenetic determinant of cell phenotype. Through the ability of prions to self propagate, genetic traits encoded by prions are inherently dominant, yet the underlying mechanism is only just beginning to emerge. One approach to elucidating the mechanism of prion propagation is to establish why certain mutations can impact negatively on inheritance of a prion-based trait. This thesis reports on a combined in vivo and structural analysis of one such class of mutant - PNM2-1 - a dominant negative mutation that inhibits propagation of [PS/+], the prion form of the translation termination factor Sup35p. The original PNM2-1 allele, a G58D mutation lies in one of a series of five oligopeptide repeats in the amino-terminal prion domain of the Sup35p (eRF3) and cells expressing this allele cannot efficiently propagate the [PS/+] prion. To establish the mechanism by which the PNM2-1 allele mediates this effect, a series of PNM2-1G58/G59/Y60 mutants of Sup35p was constructed. By combining genetic crossing, phenotypic analysis and solution NMR structural studies, a clear correlation between the conformational changes in the oligopeptide repeat caused by these mutations and the relative impact of these mutations on the in vivo propagation of [PS/+] was demonstrated. The conformational constraints associated with these mutations were also shown to the affect the ability of the protein to form and/or incorporate different of prion variants. These findings provide a molecular explanation of the dominant negative effects of PNM2-1 mutation on the maintenance of the [PS/+] prion and provide important new insights into the importance of conformation in prion propagation.

Prions are aggregates of misfolded proteins that have acquired an amyloid-like structure and ability to propagate through recruitment of new proteins. [PSI+], a prion form of eukaryotic release factor Sup35 (eRF1) is widely used as a model for research on prion formation and propagation and in this study [PSI+] is used to explore an effect of three previously identified proteins on de novo prion formation. One mechanism proposed to affect prion formation is direct interaction of Sup35p with its binding partners and search for proteins that interact with Sup35p identified Ppq 1 p, a putative Ser/Thr protein phosphatase (M.F. Tuite and T. von der Haar). Another approach was to identify proteins that function to protect translational apparatus from environmental and . endogenous oxidative damage. and this approach identified two ribosome associated peroxiredoxins, Tsa1 p and Tsa2p (T. Sideri and C.M. Grant). The data presented here shows that the deletion of PPQ1 gene greatly increases the rate of de novo formation of [PSI+] but the mechanism by which loss of Ppq1 p affects [PSI+] formation is not known. Analysis of the distribution of fluorescently-tagged Ppq 1 P showed that the protein co-localises with mitochondria. A further line of evidence linking Ppq 1 P to mitochondria was an observed reduction in respiratory capacity of a ppq1 Δ strain. That exposure to environmental sources of oxidative stress promotes [PSI+] prion formation was previously reported (Tyedmers et al., 2008). Results presented here show that an endogenous source of oxidative stress, brought about by deleting the ribosomally- associated peroxiredoxins (Prx) encoded by genes TSA 1/2 (Trotter et al., 2008; Sideri et al., 2010), also increases the rate of de novo [PSI+]formation. This result provides a direct link between oxidative stress and the eukaryotic release factor Sup35p.

No description available.

The prion protein (PrPC) is a cell surface glycoprotein that binds Cu2+ ions. The misfolding and oligomerisation of PrPC is responsible for a range of transmissible spongiform encephalopathies (TSEs) in mammals. As changes in PrPC conformation are intimately linked with disease pathogenesis, the effect of Cu2+ ions on the structure and stability of PrPC has been investigated. In chapter 3, urea unfolding studies indicate that Cu2+ ions destabilise the native fold of PrPC. The mid-point of the unfolding transition is reduced by 0.73 ± 0.05 M urea in the presence of Cu2+ ions equating to an appreciable difference in free energy of unfolding (∆∆GU[D]50%), 2.02 ± 0.05 kJmol-1. This suggests that in Prion diseases, Cu2+ ions could destabilise the native fold of PrPC and make the transition to a misfolded state more favourable. Furthermore, Cu2+ induced changes in secondary structure observed for small fragments of the protein are related to the full-length prion protein. An increase in -sheet like character is observed when Cu2+ ions are present, this is due to local Cu2+ ion coordination to the individual binding sites of the amyloidgenic region. Cu2+ induced changes in the secondary structure of the N-terminal domain, PrP(23-126) and full-length PrP(23-231), are attributed to Cu2+ ions binding within the octarepeat region of PrPC. Oxidative stress is also a well-recognised feature of Prion diseases. In chapter 4, the effects of Cu2+ catalysed oxidation of PrP on the structure and stability are discussed. 2D 1H-15N HSQC NMR studies of PrPC indicate that specific key residues are perturbed upon methionine (Met) oxidation by H2O2. These residues are involved in the hydrophobic packing of the structured core of the protein, stabilising its ternary structure. Urea unfolding studies indicate that the oxidation of PrPC by H2O2 and to a greater extent Cu2+ ions with peroxide significantly reduce the thermodynamic stability of PrPC. Cu2+ catalysed oxidation of PrP causes much more significant alteration of the structure. 2D 1H-15N HSQC NMR spectroscopy indicates that the structured C-terminal portion of PrP becomes a large molten-globule, made of monomeric species. FT-IR and far-UV-CD spectroscopy indicate that this molten-globule is rich in β-sheet. These observations supports a hypothesis that oxidation of PrP destabilises the native fold of PrPC, making the transition to PrPSc more energetically favourable. This study gives a structural and thermodynamic explanation for the high levels of oxidised Met residues in scrapie isolates. Finally, in chapter 5 the interaction of PrPC with Aβ peptide, responsible for Alzheimer’s disease (AD), is investigated. In particular, the influence of full length PrP and fragments on the kinetics of Aβ fibril growth is investigated. The complete inhibition Aβ fibril formation is observed when as little as one-twentieth of the molar ratio of PrPC is used. The unstructured N-terminus of PrPC, residues 23 to115, is thought to be crucial for this inhibition, while, residues 116 to 231 have no influence on the fibril formation. Gel filtration chromatography indicates that the complex formed by PrPC with an Aβ oligomer is 12 to 24 monomers in size.