Nobel Lecture: Prions

Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm.

Prions are composed largely, if not entirely, of prion protein (PrPSc in the case of scrapie). Although the formation of PrPSc from the cellular prion protein (PrPC) is a post-translational process, no candidate chemical modification was identified, suggesting that a conformational change features in PrPSc synthesis. To assess this possibility, we purified both PrPC and PrPSc by using nondenaturing procedures and determined the secondary structure of each. Fourier-transform infrared (FTIR) spectroscopy demonstrated that PrPC has a high alpha-helix content (42%) and no beta-sheet (3%), findings that were confirmed by circular dichroism measurements. In contrast, the beta-sheet content of PrPSc was 43% and the alpha-helix 30% as measured by FTIR. As determined in earlier studies, N-terminally truncated PrPSc derived by limited proteolysis, designated PrP 27-30, has an even higher beta-sheet content (54%) and a lower alpha-helix content (21%). Neither PrPC nor PrPSc formed aggregates detectable by electron microscopy, while PrP 27-30 polymerized into rod-shaped amyloids. While the foregoing findings argue that the conversion of alpha-helices into beta-sheets underlies the formation of PrPSc, we cannot eliminate the possibility that an undetected chemical modification of a small fraction of PrPSc initiates this process. Since PrPSc seems to be the only component of the "infectious" prion particle, it is likely that this conformational transition is a fundamental event in the propagation of prions.

https://www.pnas.org/content/pnas/90/23/10962.full.pdf

Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins.

We present a structural model for amyloid fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta(1-40)), based on a set of experimental constraints from solid state NMR spectroscopy. The model additionally incorporates the cross-beta structural motif established by x-ray fiber diffraction and satisfies constraints on Abeta(1-40) fibril dimensions and mass-per-length determined from electron microscopy. Approximately the first 10 residues of Abeta(1-40) are structurally disordered in the fibrils. Residues 12-24 and 30-40 adopt beta-strand conformations and form parallel beta-sheets through intermolecular hydrogen bonding. Residues 25-29 contain a bend of the peptide backbone that brings the two beta-sheets in contact through sidechain-sidechain interactions. A single cross-beta unit is then a double-layered beta-sheet structure with a hydrophobic core and one hydrophobic face. The only charged sidechains in the core are those of D23 and K28, which form salt bridges. Fibrils with minimum mass-per-length and diameter consist of two cross-beta units with their hydrophobic faces juxtaposed.

https://www.pnas.org/content/pnas/99/26/16742.full.pdf

A structural model for Alzheimer's β-amyloid fibrils based on experimental constraints from solid state NMR

Strains of transmissible spongiform encephalopathies are distinguished by differing physicochemical properties of PrPSc, the disease-related isoform of prion protein, which can be maintained on transmission to transgenic mice. 'New variant' Creutzfeldt-Jakob disease (CJD) has strain characteristics distinct from other types of CJD and which resemble those of BSE transmitted to mice, domestic cat and macaque, consistent with BSE being the source of this new disease. Strain characteristics revealed here suggest that the prion protein may itself encode disease phenotype.

Molecular analysis of prion strain variation and the aetiology of 'new variant' CJD

Mice, homozygous for prion protein (PrP) gene ablation (Prn-p0/0), develop normally and remain well > 500 days after inoculation with murine scrapie prions. In contrast, wild-type mice developed scrapie < 165 days after inoculation and most Prn-p0/+ mice, heterozygous for disruption of the PrP gene, exhibited signs of central nervous system dysfunction between 400 and 465 days after inoculation. In situ immunoblots showed widespread deposition of scrapie PrP (PrPSc) in the brains of both wild-type Prn-p+/+ and Prn-p0/+ mice, while neither cellular PrP (PrPC) nor PrPSc was detected in the brains of Prn-p0/0 mice. In contrast to Prn-p+/+ and Prn-p0/+ mice, Prn-p0/0 mice failed to propagate prion infectivity as measured by bioassays. Syrian hamster (SHa) PrP transgenes rendered Prn-p0/0 mice susceptible to prions containing SHaPrPSc. Immunization of Prn-p0/0 mice with purified, infectious mouse or SHa prions dispersed in Freund's adjuvant produced antisera that bound mouse, SHa, and human PrP on Western blots. Presumably, the lack of PrPC expression in Prn-p0/0 mice prevents them from becoming tolerant to the immunogen. The resistance of Prn-p0/0 mice to developing scrapie after inoculation with murine prions supports the hypothesis that PrPSc is essential for both transmission and pathogenesis of the prion diseases.

https://www.pnas.org/content/pnas/90/22/10608.full.pdf

Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies

Because the insolubility of the scrapie prion protein (PrP(Sc)) has frustrated structural studies by x-ray crystallography or NMR spectroscopy, we used electron crystallography to characterize the structure of two infectious variants of the prion protein. Isomorphous two-dimensional crystals of the N-terminally truncated PrP(Sc) (PrP 27-30) and a miniprion (PrP(Sc)106) were identified by negative stain electron microscopy. Image processing allowed the extraction of limited structural information to 7 A resolution. By comparing projection maps of PrP 27-30 and PrP(Sc)106, we visualized the 36-residue internal deletion of the miniprion and localized the N-linked sugars. The dimensions of the monomer and the locations of the deleted segment and sugars were used as constraints in the construction of models for PrP(Sc). Only models featuring parallel beta-helices as the key element could satisfy the constraints. These low-resolution projection maps and models have implications for understanding prion propagation and the pathogenesis of neurodegeneration.

https://www.pnas.org/content/pnas/99/6/3563.full.pdf

Structural studies of the scrapie prion protein by electron crystallography

With the discovery of the prion protein (PrP), immunodiagnostic procedures were applied to diagnose Creutzfeldt–Jakob disease (CJD). Before development of the conformation-dependent immunoassay (CDI), all immunoassays for the disease-causing PrP isoform (PrPSc) used limited proteolysis to digest the precursor cellular PrP (PrPC). Because the CDI is the only immunoassay that measures both the protease-resistant and protease-sensitive forms of PrPSc, we used the CDI to diagnose human prion disease. The CDI gave a positive signal for PrPSc in all 10–24 brain regions (100%) examined from 28 CJD patients. A subset of 18 brain regions from 8 patients with sporadic CJD (sCJD) was examined by histology, immunohistochemistry (IHC), and the CDI. Three of the 18 regions (17%) were consistently positive by histology and 4 of 18 (22%) by IHC for the 8 sCJD patients. In contrast, the CDI was positive in all 18 regions (100%) for all 8 sCJD patients. In both gray and white matter, ≈90% of the total PrPSc was protease-sensitive and, thus, would have been degraded by procedures using proteases to eliminate PrPC. Our findings argue that the CDI should be used to establish or rule out the diagnosis of prion disease when a small number of samples is available as is the case with brain biopsy. Moreover, IHC should not be used as the standard against which all other immunodiagnostic techniques are compared because an immunoassay, such as the CDI, is substantially more sensitive.

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Diagnosis of human prion disease

Scrapie prion infectivity can be enriched from hamster brain homogenates by using limited proteolysis and detergent extraction. Purified fractions contain both scrapie infectivity and the protein PrP 27-30, which is aggregated in the form of prion rods. During purification, PrP 27-30 is produced from a larger membrane protein, PrPSc, by limited proteolysis with proteinase K. Brain homogenates from scrapie-infected hamsters do not contain prion rods prior to exposure to detergents and proteases. To determine whether both detergent extraction and limited proteolysis are required for the formation of prion rods, microsomal membranes were prepared from infected brains in the presence of protease inhibitors. The isolated membranes were then detergent extracted as well as protease digested to evaluate the effects of these treatments on the formation of prion rods. Neither detergent (2% Sarkosyl) extraction nor limited proteinase K digestion of scrapie microsomes produced recognizable prion amyloid rods. Only after combining detergent extraction with limited proteolysis were numerous prion rods observed. Rod formation was influenced by the protease concentration, the specificity of the protease, and the duration of digestion. Rod formation also depended upon the detergent; some combinations of protease and detergent did not produce prion amyloid rods. Similar results were obtained with purified PrPSc fractions prepared by repeated detergent extractions in the presence of protease inhibitors. These fractions contained amorphous structures but not rods; however, prion rods were produced upon conversion of PrPSc to PrP 27-30 by limited proteolysis. We conclude that the formation of prion amyloid rods in vitro requires both detergent extraction and limited proteolysis. In vivo, amyloid filaments found in the brains of animals with scrapie resemble prion rods in their width and their labeling with prion protein (PrP) antisera; however, filaments are typically longer than rods. Whether limited proteolysis and some process equivalent to detergent extraction are required for amyloid filament formation in vivo remains to be established.

Ana Serban

Papers

Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins.

Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies

Structural studies of the scrapie prion protein by electron crystallography

Diagnosis of human prion disease

Scrapie prion rod formation in vitro requires both detergent extraction and limited proteolysis.