In Vitro Approach To Identify Key Amino Acids in Low Susceptibility of Rabbit Prion Protein to Misfolding

Abstract

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a group of rare progressive neurodegenerative disorders caused by an abnormally folded prion protein (PrP). This is capable of transforming the normal cellular prion protein (PrP) into new infectious PrP 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 PrP propagation, protein misfolding cyclic amplification (PMCA), which mimics PrP-to-PrP 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 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. 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.

Dogs are resistant to prion infection, due to the presence of aspartic or glutamic acid at position 163 of their prion protein

“…Protein misfolding cyclic amplification treated samples were incubated with 85‐200 μg/mL of protease K (PK) for 1 hours at 42°C with shaking (450 rpm) as described previously . Digestion was stopped by adding electrophoresis Laemmli loading buffer and the samples were analyzed by Western blotting.…”

Evolutionary Divergent Suppressor Mutations in Conformational Diseases

“…It could be argued that the role of consensus amino acids in the different sensitivity of mammalian orthologues against disease might be a rare situation (let’s say, only for AGT and NQO1). However, it is well known that some mammalian species are particularly more resistant against disease-associated mutations than humans, which is likely associated to small divergences in the sequence and properties of the corresponding proteins [ 19 , 20 , 81 , 82 ]. Without a need to invoke inter-species differences in gene expression, regulation or protein homeostasis capacities, this situation has been investigated at the protein (sequence) level in this and other grounds, and referred to as compensated pathogenic deviation (CPD).…”

Insight From Animals Resistant to Prion Diseases: Deciphering the Genotype – Morphotype – Phenotype Code for the Prion Protein

“…The focus on rabbit PrP brought forth a number of studies to uncover the mechanisms mediating resistance to TSE. Though over a decade apart, two studies emerged as highly similar in goals ( Vorberg et al, 2003 ; Eraña et al, 2017 ). These studies identified 22 amino acid differences between full-length rabbit and mouse PrP, and set out to determine which residues impact prion transmissibility by complementary approaches.…”

“…The assay consisted on determining which substitutions inhibited the ability of recPrP from replicating prions. More recently, the Castilla group used recombinant rabbit PrP carrying mouse-specific changes ( Eraña et al, 2017 ). Their approach was to use the powerful cell-free PMCA (protein misfolding cyclic amplification) technique ( Saborio et al, 2001 ) to determine which mouse-specific substitutions enabled conversion of the naturally resistant rabbit PrP.…”