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Hundt, Christoph (2002): Interaction studies of the cellular prion protein. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Prion diseases are rare but fatal neurodegenerative diseases which occur both in humans and mammals caused by the prion protein (PrP) which is well conserved among the species. In this thesis the biochemical properties and the function of prion protein were investiagted using different methods. The oligomerisation state of the prion protein analysed by size exclusion chromatography revealed that the prion protein is dimeric under native conditions. This was proven in the yeast two-hybrid system followed by the identification of two interaction domains. The influence of mutations and polymorphisms within the prion gene was investigated in the yeast two-hybrid system. This method is also an useful tool for the investigation of the species barrier. To determine the role of dimeric prion proteins on the scrapie prion protein formation a covalently-linked PrP dimer was constructed and expressed in yeast Pichia pastoris. The protein was expressed as a glycosylated, proteinase K sensitive protein which is transported to the plasma membrane of yeast cells. Recently, the 37-kDa/67-kDa laminin receptor LRP/LR was identified as the receptor for cellular PrP. Besides the in vitro interaction of PrP and LRP the binding domains on PrP and LRP were mapped in the yeast two-hybrid system. In addition, cell binding assays revealed a second HSPG-dependent binding domain leading to a comprehensive model of the PrP/LRP complex on the cell surface. The binding of heparan sulfates to the prion protein was investigated and binding domains located on the N-terminus of the prion protein were characterized using biosensor and ELISA methodology. The prion-like protein Doppel (Dpl) shows neither an interaction with PrP nor with LRP, the receptor for the cellular prion protein, in the yeast two-hybrid system, but is well expressed in yeast cells. The tyrosine kinase Fyn, activated by PrP in neuronal cells, interact directly with PrP in the yeast two-hybrid system whereas LRP failed to interact with Fyn.