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Intra- and interspecies interactions between prion proteins and effects of mutations and polymorphisms
Christoph Hundt1, Sabine Gauczynski, Christoph Leucht
1Laboratorium für Molekulare Biologie-Genzentrum, Institut für Biochemie der Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, D-81377 München, Germany.
Abstract:
Recently, crystallization of the prion protein in a dimeric form was reported. Here we show that native soluble homogeneous FLAG-tagged prion proteins from hamster, man and cattle expressed in the baculovirus system are predominantly dimeric. The PrP/PrP interaction was confirmed in Semliki Forest virus-RNA transfected BHK cells co-expressing FLAG- and oligohistidine-tagged human PrP. The yeast two-hybrid system identified the octarepeat region and the C-terminal structured domain (aa90-aa230) of PrP as PrP/PrP interaction domains. Additional octarepeats identified in patients suffering from fCJD reduced (wtPrP versus PrP + 9OR) and completely abolished (PrP + 9OR versus PrP + 9OR) the PrP/PrP interaction in the yeast two-hybrid system. In contrast, the Met/Val polymorphism (aa129), the GSS mutation Pro102Leu and the FFI mutation Asp178Asn did not affect PrP/PrP interactions. Proof of interactions between human or sheep and bovine PrP, and sheep and human PrP, as well as lack of interactions between human or bovine PrP and hamster PrP suggest that interspecies PrP interaction studies in the yeast two-hybrid system may serve as a rapid pre-assay to investigate species barriers in prion diseases.
Insights
Prion protein (PrP) predominantly forms dimers. Specific regions, including octarepeats, mediate PrP/PrP interactions, influencing prion disease transmission barriers.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Prion diseases are linked to the misfolding and aggregation of prion proteins (PrP).
- Recent studies suggest prion protein can crystallize as a dimer, hinting at its functional relevance.
Purpose of the Study:
- To investigate the predominant form of soluble prion protein expressed in a baculovirus system.
- To identify the domains responsible for prion protein self-interaction.
- To assess the impact of specific genetic variations and interspecies interactions on prion protein binding.
Main Methods:
- Expression and purification of FLAG-tagged prion proteins from hamster, human, and cattle using the baculovirus system.
- Confirmation of PrP/PrP interaction in mammalian cells (BHK) co-expressing differentially tagged human PrP.
- Utilizing the yeast two-hybrid system to map PrP interaction domains and test the effects of mutations and polymorphisms.
Main Results:
- Soluble prion proteins from various species expressed in baculovirus are predominantly dimeric.
- The octarepeat region and the C-terminal structured domain (aa90-aa230) were identified as key PrP/PrP interaction domains.
- Additional octarepeats associated with familial Creutzfeldt-Jakob disease (fCJD) significantly reduced or abolished PrP/PrP interaction.
- Common polymorphisms (Met/Val at aa129) and disease-associated mutations (Pro102Leu, Asp178Asn) did not affect PrP/PrP interactions.
- Interspecies PrP interactions varied, with evidence of human-bovine and sheep-human binding, but not human-hamster binding.
Conclusions:
- Prion protein exists predominantly as a dimer, with specific domains mediating self-interaction.
- The octarepeat region plays a crucial role in regulating PrP/PrP interactions, and variations within it can impair binding.
- Yeast two-hybrid system analysis of interspecies PrP interactions can serve as a preliminary method for predicting prion disease species barriers.