Abstract
The critical step in the pathogenesis of transmissible spongiform encephalopathies (prion diseases) is the conversion of a cellular prion protein (PrP(c)) into a protease-resistant, beta-sheet rich form (PrP(Sc)). Although the disease transmission normally requires direct interaction between exogenous PrP(Sc) and endogenous PrP(C), the pathogenic process in hereditary prion diseases appears to develop spontaneously (i.e. not requiring infection with exogenous PrP(Sc)). To gain insight into the molecular basis of hereditary spongiform encephalopathies, we have characterized the biophysical properties of the recombinant human prion protein variant containing the mutation (Phe(198) --> Ser) associated with familial Gerstmann-Straussler-Scheinker disease. Compared with the wild-type protein, the F198S variant shows a dramatically increased propensity to self-associate into beta-sheet-rich oligomers. In a guanidine HCl-containing buffer, the transition of the F198S variant from a normal alpha-helical conformation into an oligomeric beta-sheet structure is about 50 times faster than that of the wild-type protein. Importantly, in contrast to the wild-type PrP, the mutant protein undergoes a spontaneous conversion to oligomeric beta-sheet structure even in the absence of guanidine HCl or any other denaturants. In addition to beta-sheet structure, the oligomeric form of the protein is characterized by partial resistance to proteinase K digestion, affinity for amyloid-specific dye, thioflavine T, and fibrillar morphology. The increased propensity of the F198S variant to undergo a conversion to a PrP(Sc)-like form correlates with a markedly decreased thermodynamic stability of the native alpha-helical conformer of the mutant protein. This correlation supports the notion that partially unfolded intermediates may be involved in conformational conversion of the prion protein.
Highlights
Transmissible spongiform encephalopathies, or prion diseases, comprise a group of fatal neurodegenerative disorders that affect both animals and humans
Over 20 mutations in the human PrP gene have been identified to date that segregate with familial Creuzfeldt-Jakob disease, GSS, or fatal familial insomnia [1,2,3, 15]
Because the PrPC3 PrPSc conversion in hereditary prion diseases appears to occur spontaneously, studies with prion protein variants containing familial mutations could provide important clues regarding the molecular mechanism of the pathogenic process in spongiform encephalopathies
Summary
Plasmid Construction and Protein Purification—The plasmid encoding huPrP90 –231 with a N-terminal linker containing a His tail and a thrombin cleavage site was described previously [17]. In a typical equilibrium unfolding experiment, two stock solutions of huPrP90 –231 at identical protein concentrations (1.2 M) were prepared: one in buffer alone (native protein) and one in an appropriate buffer containing 9 M urea (unfolded protein). Dynamic Light Scattering—The light scattering experiments were performed at room temperature on a DynoPro-801 dynamic light scattering instrument (Protein Solution, Inc.). Small aliquots of each sample (5 or 10 l for 120 or 60 M protein solutions, respectively) were withdrawn at different time points and transferred to a quartz cell containing 10 M ThT in 50 mM potassium phosphate, pH 6.0. Electron Microscopy—Samples for electron microscopy were prepared by incubating the wild-type huPrP90 –231 or F198S huPrP90 – 231 (120 M) at 37 °C in a 50 mM sodium acetate buffer, pH 5.5. Following incubation for 3–5 days, a drop of each sample was placed on a carbon-coated 600-mesh copper grid (Electron Microscope Sciences)
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