Glycosylation is the most abundant post-translational modification of proteins and has the ability to change the physical properties of the protein and its cell biology. The cellular prion protein (PrPC) is a membrane bound host glycoprotein present in a number of isoforms in vivo due to variable occupancy of the two N-linked glycosylation sites. The function of PrPC is still unclear but it is essential for disease in transmissible spongiform encephalopathies (TSEs). The significance of the PrP glycoforms in the physiological function is unknown. Gene targeted mice have been created with point mutations that selectively abolish the glycosylation sites of PrPC. These GlycoD mutants have been used to study the effect of glycosylation at the different sites on the cell biology of PrPC. This study showed that both glycosylation sites played a role in the cell biology of PrPC. Removal of a single or both glycosylation sites significantly reduced total PrPC protein. The relative amount of the truncated protein produced through proteolytic cleaving was slightly reduced in the GlycoD mutants; however the proportion of truncated to full length PrP was increased, further reducing full length protein. The maintenance of truncated protein levels indicates a potential importance of the fragment in PrPC function. Wild type PrP is predominantly diglycosylated and localised to the cell surface. In this study it was shown that all GlycoD mutants had reduced amounts of cell surface PrPC and an increased proportion of PrPC associated with the secretory pathway. Removal of either the first or the second glycosylation site produced changes in cell biology that were almost indistinguishable from each other whilst disruption of both glycosylation sites produces a more extreme phenotype than removal of a single site. Previous studies have shown an altered susceptibility for TSE disease GlycoD mice. An in vitro conversion assay was used to investigate the ability of the glycoforms to initiate conversion from PrPC to the disease associated PrPSc. Mice which had only the second site abolished were much more efficient at seeding conversion than all other glycoforms. This may reflect the difference in susceptibility between the two monoglycosylated PrPs but does not explain the increased resistance compared to wild type mice. All other GlycoD mutants had similar seeding times to wild type mice despite having increased TSE resistance. The differences observed in the cell biology of PrPC of the GlycoD mutants may go some way to explaining the differences in TSE susceptibilities seen with these mice.

No description available.

The Prion protein (PrP) is a cell surface glycoprotein that has been directly implicated in the pathogenesis of a range of neurological disorders referred to as the transmissible spongiform encephalopathies (TSE’s). The protein has been shown to bind copper within its unstructured N-terminus but the affinity and stoichiometry of the association is a matter of some debate. In addition, the functional significance of this copper binding has yet to be elucidated. This study aimed to determine accurate metal binding parameters for PrP through the use of calorimetry and to provide insight into the potential redox implications of metal once bound. A method of analysis for complex binding to proteins is thoroughly assessed and found to be suitable. The study also aimed to qualify the involvement of metals in the proteins remarkable ability to survive in the environment. This study confirms that PrP binds copper with an affinity relative to the amount of copper available to the protein. A high nanomolar affinity is reported within two regions on the protein, the octarepeat and the 5th site. Binding within the octarepeat region is found to be highest at low copper concentrations, reducing to micromolar affinity when copper levels exceed equivalents of 1. There is also strong evidence of a complex and cooperative binding mechanism. The 5th site also displays high nanomolar affinity for a single atom of copper. These two regions on the protein also interact in the coordination of copper (II). The copper bound protein is highly redox active and is capable of fully reversible cycling of electrons that are dependent mainly on the octarepeat. The protein does bind other divalent cations but none appear to be physiologically relevant considering the amount of these free metal ions in the body. When adsorbed to model clays, PrP is able to survive for long periods at room temperature. This longevity is increased significantly by the presence of metals in the soil, especially manganese. These data provide confirmation of the precise parameters of divalent cation binding to PrP. It also confirms that the copper bound protein is capable of a physiological redox role.

Universiẗat, Diss., 2006--Würzburg.

Thesis (M. A.)--Florida State University, 2006. / Advisor: Yun-Hwa Peggy Hsieh, Florida State University, College of Human Sciences , Dept. of Nutrition, Food, and Exercise Science. Title and description from dissertation home page (viewed Jan. 2, 2007). Document formatted into pages; contains xi, 115 pages. Includes bibliographical references.

Prion diseases are a group of fatal neurodegenerative protein-misfolding diseases. Microglia, the resident myeloid cells found within the brain, have been shown to demonstrate a reactive morphology during the disease process with conflicting evidence for both a neurotoxic and neuroprotective role. The studies presented here aimed to investigate the role of microglia activation using transcriptomic and morphological analysis of prion disease in mice. Initially, the host immune response to prion disease was explored using a publically available mouse prion disease dataset. Re-analysis of this dataset was performed using BioLayout Express3D; a novel software tool that supports the visualisation and clustering of correlation networks. Disease-associated genes up-regulated during the later stages of infection were present in two main clusters. The cellular origin of these genes was explored by examining their expression in a dataset comprised of pure populations of cells. This demonstrated that the primary cluster of up-regulated transcripts encompassed genes expressed mainly by microglia and to a lesser extent astrocytes and neurons. The secondary cluster comprised almost exclusively of interferon response genes. The conclusions of these analyses were different from those of the original study that suggested disease-associated genes were primarily neuronal in origin. Mouse models of prion disease were established by infecting a novel line of BALB/cJ inbred mice, expressing EGFP under control of a myeloid specific Csf1r promoter, with the 79A prion strain. Quantification of the morphological changes of EGFP expressing microglia suggested the cells accumulated in the medulla at sites of early misfolded protein deposition with minimal change in their overall appearance. An activated microglia morphology was not observed until protein deposition was extensive. Isolation of EGFP expressing microglia was performed for transcriptome analysis. The vast majority of disease associated genes demonstrated increased expression at the onset of clinical symptoms. The gene list was found to be highly enriched for genes associated with an innate immune response regulated by the NFκB signalling cascade. Also highly enriched were processes associated with protein translation, energy production and stress response. These data suggest a high metabolic load is burdened by proliferating microglia; and as part of a response which is strikingly more pro-inflammatory in nature than has previously been attributed to the microglia phenotype within prion disease. As an active contributor to normal homeostasis, microglia are more than just innate immune surveillance and are now considered an integral component in both the healthy and diseased brain. The ramifications of activation toward the microglia phenotype shown here will have direct and potentially cytotoxic influence on neighbouring microglia and other brain cell types implying microglia as major contributors to the neurotoxic environment found within the CNS during prion disease. Furthermore the identification of genes associated with metabolism offer many intriguing possibilities for manipulating the activity of microglia in pre-clinical therapeutic intervention.

Pathogenesis of prion diseases in animals is associated with the misfolding of the cellular prion protein PrPC to the infectious form, PrPSc. We hypothesized that an animal’s susceptibility to prions is correlated with the propensity of an animal’s PrPC to adopt a β-sheet, PrPSc-like, conformation. We have developed a method which uses circular dichroism (CD) to directly calculate the relative population of PrP molecules that adopt a β-sheet conformation or the ‘β-state’, as a function of denaturant concentration and pH. We find that the PrP from animals that are more susceptible to prion diseases, like hamsters and mice, adopt the β-state more readily than the PrP from rabbits. The X-ray crystal structure of rabbit PrP reveals a helix-capping motif that may lower the propensity to form the β-state. PrP in the β-state contains both monomeric and octameric β-structured species, and possesses cytotoxic properties.

Pathogenesis of prion diseases in animals is associated with the misfolding of the cellular prion protein PrPC to the infectious form, PrPSc. We hypothesized that an animal’s susceptibility to prions is correlated with the propensity of an animal’s PrPC to adopt a β-sheet, PrPSc-like, conformation. We have developed a method which uses circular dichroism (CD) to directly calculate the relative population of PrP molecules that adopt a β-sheet conformation or the ‘β-state’, as a function of denaturant concentration and pH. We find that the PrP from animals that are more susceptible to prion diseases, like hamsters and mice, adopt the β-state more readily than the PrP from rabbits. The X-ray crystal structure of rabbit PrP reveals a helix-capping motif that may lower the propensity to form the β-state. PrP in the β-state contains both monomeric and octameric β-structured species, and possesses cytotoxic properties.

No description available.

No description available.