Koren Nishina

Prion Protein Glycosylation Is Not Required for Strain-Specific Neurotropism

ABSTRACT In this study, we tested the hypothesis that the glycosylation of the pathogenic isoform of the prion protein (PrPSc) might encode the selective neurotropism of prion strains. We prepared unglycosylated cellular prion protein (PrPC) substrate molecules from normal mouse brain by treatment with PNGase F and used reconstituted serial protein cyclic misfolding amplification reactions to produce RML and 301C mouse prions containing unglycosylated PrPSc molecules. Both RML- and 301C-derived prions containing unglycosylated PrPSc molecules were infectious to wild-type mice, and neuropathological analysis showed that mice inoculated with these samples maintained strain-specific patterns of PrPSc deposition and neuronal vacuolation. These results show that PrPSc glycosylation is not necessary for strain-dependent prion neurotropism.

SarA of Staphylococcus aureus Binds to the sarA Promoter To Regulate Gene Expression

The 375-bp sarA open reading frame is driven by three promoters, P1, P3, and P2. Using gel shift and DNase I footprinting assays, we found that SarA binds to two 26-bp sequences and one 31-bp sequence within the P1 and P3 promoters, respectively. Together with the results of transcription analyses, our data indicate that SarA binds to its own promoter to down-regulate sarA expression.

Post-transcriptional suppression of pathogenic prion protein expression in Drosophila neurons.

A wealth of evidence supports the view that conformational change of the prion protein, PrPC, into a pathogenic isoform, PrPSc, is the hallmark of sporadic, infectious, and inherited forms of prion disease. Although the central role played by PrPSc in the pathogenesis of prion disease is appreciated, the cellular mechanisms that recognize PrPSc and modulate its production, clearance, and neural toxicity have not been elucidated. To address these questions, we used a tissue‐specific expression system to express wild‐type and disease‐associated PrP molecules heterologously in Drosophila melanogaster. Our results indicate that Drosophila brain possesses a specific and saturable mechanism that suppresses the accumulation of PG14, a disease‐associated insertional PrP mutant. We also found that wild‐type PrP molecules are maintained in a detergent‐soluble conformation throughout life in Drosophila brain neurons, whereas they become detergent‐insoluble in retinal cells as flies age. PG14 protein expression in Drosophila eye did not cause retinal pathology. Our work reveals the presence of mechanisms in neurons that specifically counterbalance the production of misfolded PrP conformations, and provides an opportunity to study these processes in a model organism amenable to genetic analysis.

A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers

Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrPSc. A critical prediction of the protein-only hypothesis is that autocatalytic PrPSc molecules should be infectious. However, some autocatalytic recombinant PrPSc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >105-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrPC substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrPC. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrPC molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrPSc structure.