Familial Mutations and the Thermodynamic Stability of the Recombinant Human Prion Protein

Wieslaw Swietnicki,Robert B Petersen,Pierluigi Gambetti,Witold K Surewicz

doi:10.1074/jbc.273.47.31048

Wieslaw Swietnicki, Robert B Petersen + Show 2 more

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https://doi.org/10.1074/jbc.273.47.31048

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Abstract

Hereditary forms of human prion disease are linked to specific mutations in the PRNP gene. It has been postulated that these mutations may facilitate the pathogenic process by reducing the stability of the prion protein (PrP). To test this hypothesis, we characterized the recombinant variants of human PrP(90-231) containing point mutations corresponding to Gerstmann-Straussler-Scheinker disease (P102L), Creutzfeld-Jakob disease (E200K), and fatal familial insomnia (M129/D178N). The first two of these mutants could be recovered form from the periplasmic space of Escherichia coli in a soluble form, whereas the D178N variant aggregated into inclusion bodies. The secondary structure of the two soluble variants was essentially identical to that of the wild-type protein. The thermodynamic stability of these mutants was assessed by unfolding in guanidine hydrochloride and thermal denaturation. The stability properties of the P102L variant were indistinguishable from those of wild-type PrP, whereas the E200K mutation resulted in a very small destabilization of the protein. These data, together with the predictive analysis of other familial mutations, indicate that some hereditary forms of prion disease cannot be rationalized using the concept of mutation-induced thermodynamic destabilization of the cellular prion protein.

Highlights

Prion diseases, known as spongiform encephalopathies, are disorders of the central nervous system
Our data show that these mutations do not exert a uniform effect on the thermodynamic stability of the protein, indicating that not all hereditary forms of prion disease can be rationalized using the concept of mutation-induced thermodynamic destabilization of PrPC
Within the framework of the protein only hypothesis, the central molecular event in the pathogenesis of prion diseases is the conversion of PrPC into an abnormal, conformationally altered isoform, PrPres [1,2,3,4, 19]

Summary

Introduction

Known as spongiform encephalopathies, are disorders of the central nervous system. According to the “protein only” hypothesis [7, 8], the key factor in the disease is an abnormal protein, designated PrPres (or PrPSc).1 This protein is an altered isoform of a normal cellular glycoprotein, PrPC, that is host-encoded by a chromosomal gene (PRNP) and expressed in mammalian cells. PrPC is highly soluble and degraded by proteinase K, PrPres exists as an insoluble aggregate that is resistant to proteinase K digestion and often has the characteristics of an amyloid [10, 11] These differences in physical properties most likely reflect different conformations of the two isoforms: PrPC is highly ␣-helical, whereas PrPres appears to contain a large proportion of ␤-sheet structure [12,13,14,15,16,17,18]. Our data show that these mutations do not exert a uniform effect on the thermodynamic stability of the protein, indicating that not all hereditary forms of prion disease can be rationalized using the concept of mutation-induced thermodynamic destabilization of PrPC

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