Dataset Information

Requirements for mutant and wild-type prion protein misfolding in vitro.

ABSTRACT: Misfolding of the prion protein (PrP) plays a central role in the pathogenesis of infectious, sporadic, and inherited prion diseases. Here we use a chemically defined prion propagation system to study misfolding of the pathogenic PrP mutant D177N in vitro. This mutation causes PrP to misfold spontaneously in the absence of cofactor molecules in a process dependent on time, temperature, pH, and intermittent sonication. Spontaneously misfolded mutant PrP is able to template its unique conformation onto wild-type PrP substrate in a process that requires a phospholipid activity distinct from that required for the propagation of infectious prions. Similar results were obtained with a second pathogenic PrP mutant, E199K, but not with the polymorphic substitution M128V. Moreover, wild-type PrP inhibits mutant PrP misfolding in a dose-dependent manner, and cofactor molecules can antagonize this effect. These studies suggest that interactions between mutant PrP, wild-type PrP, and other cellular factors may control the rate of PrP misfolding in inherited prion diseases.

SUBMITTER: Noble GP

PROVIDER: S-EPMC4520438 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Requirements for mutant and wild-type prion protein misfolding in vitro.

Noble Geoffrey P GP Walsh Daniel J DJ Miller Michael B MB Jackson Walker S WS Supattapone Surachai S

Biochemistry 20150122 5

Misfolding of the prion protein (PrP) plays a central role in the pathogenesis of infectious, sporadic, and inherited prion diseases. Here we use a chemically defined prion propagation system to study misfolding of the pathogenic PrP mutant D177N in vitro. This mutation causes PrP to misfold spontaneously in the absence of cofactor molecules in a process dependent on time, temperature, pH, and intermittent sonication. Spontaneously misfolded mutant PrP is able to template its unique conformation ...[more]

PMID: 25584902

Similar Datasets

Project description:Prion diseases are fatal neurodegenerative diseases associated with the conversion of cellular prion protein (PrP(C)) in the central nervous system into the infectious isoform (PrP(Sc)). The mechanics of conversion are almost entirely unknown, with understanding stymied by the lack of an atomic-level structure for PrP(Sc). A number of pathogenic PrP(C) mutants exist that are characterized by an increased propensity for conversion into PrP(Sc) and that differ from wild-type by only a single amino-acid point mutation in their primary structure. These mutations are known to perturb the stability and conformational dynamics of the protein. Understanding of how this occurs may provide insight into the mechanism of PrP(C) conversion. In this work we sought to explore wild-type and pathogenic mutant prion protein structure and dynamics by analysis of the current fluctuations through an organic ?-hemolysin nanometer-scale pore (nanopore) in which a single prion protein has been captured electrophoretically. In doing this, we find that wild-type and D178N mutant PrP(C), (a PrP(C) mutant associated with both Fatal Familial Insomnia and Creutzfeldt-Jakob disease), exhibit easily distinguishable current signatures and kinetics inside the pore and we further demonstrate, with the use of Hidden Markov Model signal processing, accurate discrimination between these two proteins at the single molecule level based on the kinetics of a single PrP(C) capture event. Moreover, we present a four-state model to describe wild-type PrP(C) kinetics in the pore as a first step in our investigation on characterizing the differences in kinetics and conformational dynamics between wild-type and D178N mutant PrP(C). These results demonstrate the potential of nanopore analysis for highly sensitive, real-time protein and small molecule detection based on single molecule kinetics inside a nanopore, and show the utility of this technique as an assay to probe differences in stability between wild-type and mutant prion proteins at the single molecule level.

Project description:Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a group of rare progressive neurodegenerative disorders caused by an abnormally folded prion protein (PrPSc). This is capable of transforming the normal cellular prion protein (PrPC) into new infectious PrPSc Interspecies prion transmissibility studies performed by experimental challenge and the outbreak of bovine spongiform encephalopathy that occurred in the late 1980s and 1990s showed that while some species (sheep, mice, and cats) are readily susceptible to TSEs, others are apparently resistant (rabbits, dogs, and horses) to the same agent. To study the mechanisms of low susceptibility to TSEs of certain species, the mouse-rabbit transmission barrier was used as a model. To identify which specific amino acid residues determine high or low susceptibility to PrPSc propagation, protein misfolding cyclic amplification (PMCA), which mimics PrPC-to-PrPSc conversion with accelerated kinetics, was used. This allowed amino acid substitutions in rabbit PrP and accurate analysis of misfolding propensities. Wild-type rabbit recombinant PrP could not be misfolded into a protease-resistant self-propagating isoform in vitro despite seeding with at least 12 different infectious prions from diverse origins. Therefore, rabbit recombinant PrP mutants were designed to contain every single amino acid substitution that distinguishes rabbit recombinant PrP from mouse recombinant PrP. Key amino acid residue substitutions were identified that make rabbit recombinant PrP susceptible to misfolding, and using these, protease-resistant misfolded recombinant rabbit PrP was generated. Additional studies characterized the mechanisms by which these critical amino acid residue substitutions increased the misfolding susceptibility of rabbit PrP.IMPORTANCE Prion disorders are invariably fatal, untreatable diseases typically associated with long incubation periods and characteristic spongiform changes associated with neuronal loss in the brain. Development of any treatment or preventative measure is dependent upon a detailed understanding of the pathogenesis of these diseases, and understanding the mechanism by which certain species appear to be resistant to TSEs is critical. Rabbits are highly resistant to naturally acquired TSEs, and even under experimental conditions, induction of clinical disease is not easy. Using recombinant rabbit PrP as a model, this study describes critical molecular determinants that confer this high resistance to transmissible spongiform encephalopathies.

Dataset Information

Requirements for mutant and wild-type prion protein misfolding in vitro.

Publications

Requirements for mutant and wild-type prion protein misfolding in vitro.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets