PrPC Substrate Research Articles

Human Prion Protein (PrP) 219K Is Converted to PrPSc but Shows Heterozygous Inhibition in Variant Creutzfeldt-Jakob Disease Infection

Prion protein gene (PRNP) E219K is a human polymorphism commonly occurring in Asian populations but is rarely found in patients with sporadic Creutzfeldt-Jakob disease (CJD). Thus the polymorphism E219K has been considered protective against sporadic CJD. The corresponding mouse prion protein (PrP) polymorphism variant (mouse PrP 218K) is not converted to the abnormal isoform (PrP(Sc)) and shows a dominant negative effect on wild-type PrP conversion. To define the conversion activity of this human molecule, we herein established knock-in mice with human PrP 219K and performed a series of transmission experiments with human prions. Surprisingly, the human PrP 219K molecule was converted to PrP(Sc) in variant CJD infection, and the conversion occurred more efficiently than PrP 219E molecule. Notably the knock-in mice with PRNP codon 219E/K showed the least efficient conversion compared with their hemizygotes with PRNP codon 219E/0 or codon 219K/0, or homozygotes with PRNP codon 219E/E or codon 219K/K. This phenomenon indicated heterozygous inhibition. This heterozygous inhibition was observed also in knock-in mice with PRNP codon 129M/V genotype. In addition to variant CJD infection, the human PrP 219K molecule is conversion-competent in transmission experiments with sporadic CJD prions. Therefore, the protective effect of PRNP E219K against sporadic CJD might be due to heterozygous inhibition.

Identifying Key Components of the PrPC-PrPSc Replicative Interface

In prion disease, direct interaction between the cellular prion protein (PrP(C)) and its misfolded disease-associated conformer PrP(Sc) is a crucial, although poorly understood step promoting the formation of nascent PrP(Sc) and prion infectivity. Recently, we hypothesized that three regions of PrP (corresponding to amino acid residues 23-33, 98-110, and 136-158) interacting specifically and robustly with PrP(Sc), likely represent peptidic components of one flank of the prion replicative interface. In this study, we created epitope-tagged mouse PrP(C) molecules in which the PrP sequences 23-33, 98-110, and 136-158 were modified. These novel PrP molecules were individually expressed in the prion-infected neuroblastoma cell line (ScN2a) and the conversion of each mutated mouse PrP(C) substrate to PrP(Sc) compared with that of the epitope-tagged wild-type mouse PrP(C). Mutations within PrP 98-110, substituting all 4 wild-type lysine residues with alanine residues, prevented conversion to PrP(Sc). Furthermore, when residues within PrP 136-140 were collectively scrambled, changed to alanines, or amino acids at positions 136, 137, and 139 individually replaced by alanine, conversion to PrP(Sc) was similarly halted. However, other PrP molecules containing mutations within regions 23-33 and 101-104 were able to readily convert to PrP(Sc). These results suggest that PrP sequence comprising residues 98-110 and 136-140 not only participates in the specific binding interaction between PrP(C) and PrP(Sc), but also in the process leading to conversion of PrP(Sc)-sequestered PrP(C) into its disease-associated form.

Selective Incorporation of Polyanionic Molecules into Hamster Prions

The central pathogenic event of prion disease is the conformational conversion of a host protein, PrPC, into a pathogenic isoform, PrPSc. We previously showed that the protein misfolding cyclic amplification (PMCA) technique can be used to form infectious prion molecules de novo from purified native PrPC molecules in an autocatalytic process requiring accessory polyanions (Deleault, N. R., Harris, B. T., Rees, J. R., and Supattapone, S. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 9741-9746). Here we investigated the molecular mechanism by which polyanionic molecules facilitate infectious prion formation in vitro. Ina PMCA reaction lacking PrPSc template seed, synthetic polyA RNA molecules induce hamster HaPrPC to adopt a protease-sensitive, detergent-insoluble conformation reactive against antibodies specific for PrPSc. During PMCA, labeled nucleic acids form nuclease-resistant complexes with HaPrP molecules. Strikingly, purified HaPrPC molecules subjected to PMCA selectively incorporate an approximately 1-2.5-kb subset of [32P]polyA RNA molecules from a heterogeneous mixture ranging in size from approximately 0.1 to >6 kb. Neuropathological analysis of scrapie-infected hamsters using the fluorescent dye acridine orange revealed that RNA molecules co-localize with large extracellular HaPrP aggregates. These findings suggest that polyanionic molecules such as RNA may become selectively incorporated into stable complexes with PrP molecules during the formation of native hamster prions.

SI08 Protein Misfolding Cyclic Amplification of Disease‐Associated Prion Protein Using Human Autopsy Tissues

The protein misfolding cyclic amplification (PMCA) technique allows for the in vitro production of protease‐resistant prion protein (PrPres) using a seed of disease‐associated prion protein (PrPsc) to convert normal cellular prion protein (PrPc). This system has been developed using brain tissue from scrapie infected hamsters to provide the seed and normal uninfected hamster brain to provide PrPc substrate and the required conversion factors. These are mixed and subjected to repeated rounds of sonication and incubation. Extremely low concentrations of PrPsc, such as those present in blood, have been detectable. In order to adapt this method for use in CJD research and in the development of diagnostic tests for human prion diseases it will be necessary to identify a source of human PrPc and co‐factors that support amplification of human PrPsc. We have tested whether human autopsy tissues held at the National CJD Surveillance Unit support PMCA. Using a seed extract from variant CJD brain mixed with homogenates of nonCJD neurological control brain we have been able to effect a >600‐fold increase in the detection limit of PrPres, as determined by Western Blotting. Further development of this technique in concert with high sensitivity PrPsc detection methods, such as conformation dependent immunoassay, may result in a method capable of acting as a confirmatory assay for disease‐associated prion protein in human tissues and biological fluids such as blood.