The ultrastructure of infectious L-type bovine spongiform encephalopathy prions constrains molecular models

Abstract

Bovine spongiform encephalopathy (BSE) is a prion disease of cattle that is caused by the misfolding of the cellular prion protein (PrPC) into an infectious conformation (PrPSc). PrPC is a predominantly α-helical membrane protein that misfolds into a β-sheet rich, infectious state, which has a high propensity to self-assemble into amyloid fibrils. Three strains of BSE prions can cause prion disease in cattle, including classical BSE (C-type) and two atypical strains, named L-type and H-type BSE. To date, there is no detailed information available about the structure of any of the infectious BSE prion strains. In this study, we purified L-type BSE prions from transgenic mouse brains and investigated their biochemical and ultrastructural characteristics using electron microscopy, image processing, and immunogold labeling techniques. By using phosphotungstate anions (PTA) to precipitate PrPSc combined with sucrose gradient centrifugation, a high yield of proteinase K-resistant BSE amyloid fibrils was obtained. A morphological examination using electron microscopy, two-dimensional class averages, and three-dimensional reconstructions revealed two structural classes of L-type BSE amyloid fibrils; fibrils that consisted of two protofilaments with a central gap and an average width of 22.5 nm and one-protofilament fibrils that were 10.6 nm wide. The one-protofilament fibrils were found to be more abundant compared to the thicker two-protofilament fibrils. Both fibrillar assemblies were successfully decorated with monoclonal antibodies against N- and C-terminal epitopes of PrP using immunogold-labeling techniques, confirming the presence of polypeptides that span residues 100–110 to 227–237. The fact that the one-protofilament fibrils contain both N- and C-terminal PrP epitopes constrains molecular models for the structure of the infectious conformer in favour of a compact four-rung β-solenoid fold.

2.7 Å cryo-EM structure of ex vivo RML prion fibrils

“…Within such architectures the glycans themselves may also interact with one another and contribute to the overall stability of the assembly and to the ability of prions to evade host defences. Our observation of paired protofilaments in the purified RML prion samples may suggest such a mechanism and paired protofilaments have also recently been reported in purified samples from L-type BSE-prion-infected transgenic mouse brain 56 . Importantly, while we have established that protofilament pairing per se is not simply a PTA-induced artefact, PTA may contribute heterogeneity to protofilament pairing, or conversely, PTA may disrupt the interface of paired assemblies leading to the generation of single protofilaments.…”

Conformation-dependent influences of hydrophobic amino acids in two in-register parallel β-sheet amyloids, an α-synuclein amyloid and a local structural model of PrPSc

“…Detailed structures of PrP Sc are needed for such investigation, but even whether PrP Sc is an in-register parallel β -sheet amyloid or a β -solenoid has been controversial due to its incompatibility with conventional high-resolution structural analyses. 11–19 In 2021, while our study was under revision, a high-resolution structure of a fully infectious, brain-derived prion was solved using cryo-electron microscopy (cryo-EM) by Kraus et al 20 This PrP Sc (263K strain) has an in-register parallel β -sheet architecture without paired protofibrils. Then, cryo-EM structures of the glycosylphosphatidylinositol (GPI)-anchored and anchorless mouse-adapted RML scrapie strains proved that the conformations of PrP Sc differ depending on the strains.…”

Excess PrPCinhibits muscle cell differentiation via miRNA-enhanced liquid–liquid phase separation implicated in myopathy

“…PrP C contains an IDR in its N-terminal domain 7,48 −52,54−56,68 . We next employed an optogenetic tool that uses a blue light (488-nm laser) to activate IDR-mediated LLPS of proteins in living cells 71 −73 and immunogold electron microscopy 74 −76 to test this hypothesis. We used this ‘‘optoDroplet’’ system (Fig.…”