Krystyna Surewicz

Mammalian Prions Generated from Bacterially Expressed Prion Protein in the Absence of Any Mammalian Cofactors

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases that are associated with the conformational conversion of a normal prion protein, PrPC, to a misfolded aggregated form, PrPSc. The protein-only hypothesis asserts that PrPSc itself represents the infectious TSE agent. Although this model is supported by rapidly growing experimental data, unequivocal proof has been elusive. The protein misfolding cyclic amplification reactions have been recently shown to propagate prions using brain-derived or recombinant prion protein, but only in the presence of additional cofactors such as nucleic acids and lipids. Here, using a protein misfolding cyclic amplification variation, we show that prions causing transmissible spongiform encephalopathy in wild-type hamsters can be generated solely from highly purified, bacterially expressed recombinant hamster prion protein without any mammalian or synthetic cofactors (other than buffer salts and detergent). These findings provide strong support for the protein-only hypothesis of TSE diseases, as well as argue that cofactors such as nucleic acids, other polyanions, or lipids are non-obligatory for prion protein conversion to the infectious form.

Molecular conformation and dynamics of the Y145Stop variant of human prion protein in amyloid fibrils

A C-terminally truncated Y145Stop variant of the human prion protein (huPrP23–144) is associated with a hereditary amyloid disease known as PrP cerebral amyloid angiopathy. Previous studies have shown that recombinant huPrP23–144 can be efficiently converted in vitro to the fibrillar amyloid state, and that residues 138 and 139 play a critical role in the amyloidogenic properties of this protein. Here, we have used magic-angle spinning solid-state NMR spectroscopy to provide high-resolution insight into the protein backbone conformation and dynamics in fibrils formed by 13C,15N-labeled huPrP23–144. Surprisingly, we find that signals from ≈100 residues (i.e., ≈80% of the sequence) are not detected above approximately −20°C in conventional solid-state NMR spectra. Sequential resonance assignments revealed that signals, which are observed, arise exclusively from residues in the region 112–141. These resonances are remarkably narrow, exhibiting average 13C and 15N linewidths of ≈0.6 and 1 ppm, respectively. Altogether, the present findings indicate the existence of a compact, highly ordered core of huPrP23–144 amyloid encompassing residues 112–141. Analysis of 13C secondary chemical shifts identified likely β-strand segments within this core region, including β-strand 130–139 containing critical residues 138 and 139. In contrast to this relatively rigid, β-sheet-rich amyloid core, the remaining residues in huPrP23–144 amyloid fibrils under physiologically relevant conditions are largely unordered, displaying significant conformational dynamics.

Molecular basis of barriers for interspecies transmissibility of mammalian prions

Spongiform encephalopathies are believed to be transmitted by a unique mechanism involving self-propagating conformational conversion of prion protein into a misfolded form. Here we demonstrate that fundamental aspects of mammalian prion propagation, including the species barrier and strain diversity, can be reproduced in vitro in a seeded fibrillization of the recombinant prion protein variant Y145Stop. Our data show that species-specific substitution of a single amino acid in a critical region completely changes the seeding specificity of prion protein fibrils. Furthermore, we demonstrate that sequence-based barriers that prevent cross-seeding between proteins from different species can be bypassed, and new barriers established, by a template-induced adaptation process that leads to the emergence of new strains of prion fibrils. Although the seeding barriers observed in this study do not fully match those seen in animals, the present findings provide fundamental insight into mechanistic principles of these barriers at a molecular level.

Conformational flexibility of Y145stop human prion protein amyloid fibrils probed by solid-state nuclear magnetic resonance spectroscopy

Amyloid aggregates of a C-truncated Y145Stop mutant of human prion protein, huPrP23-144, associated with a heritable amyloid angiopathy, have previously been shown to contain a compact, relatively rigid, and beta-sheet-rich approximately 30-residue amyloid core near the C-terminus under physiologically relevant conditions. In contrast, the remaining huPrP23-144 residues display considerable conformational dynamics, as evidenced by the absence of corresponding signals in cross-polarization (CP)-based solid-state NMR (SSNMR) spectra under ambient conditions and their emergence in analogous spectra recorded at low temperature on frozen fibril samples. Here, we present the direct observation of residues comprising the flexible N-terminal domain of huPrP23-144 amyloid by using 2D J-coupling-based magic-angle spinning (MAS) SSNMR techniques. Chemical shifts for these residues indicate that the N-terminal domain is effectively an ensemble of protein chains with random-coil-like conformations. Interestingly, a detailed analysis of signal intensities in CP-based 3D SSNMR spectra suggests that non-negligible molecular motions may also be occurring on the NMR time scale within the relatively rigid core of huPrP23-144 amyloid. To further investigate this hypothesis, quantitative measurements of backbone dipolar order parameters and transverse spin relaxation rates were performed for the core residues. The observed order parameters indicate that, on the submicrosecond time scale, these residues are effectively rigid and experience only highly restricted and relatively uniform motions similar to those characteristic for well-structured regions of microcrystalline proteins. On the other hand, significant variations in magnitude of transverse spin relaxation rates were noted for residues present at different locations within the core region and correlated with observed differences in spectral intensities. While interpreted only qualitatively at the present time, the extent of the observed variations in transverse relaxation rates is consistent with the presence of relatively slow, microsecond-millisecond time scale chemical exchange type phenomena within the huPrP23-144 amyloid core.

Conformational diversity in prion protein variants influences intermolecular β-sheet formation

A conformational transition of normal cellular prion protein (PrPC) to its pathogenic form (PrPSc) is believed to be a central event in the transmission of the devastating neurological diseases known as spongiform encephalopathies. The common methionine/valine polymorphism at residue 129 in the PrP influences disease susceptibility and phenotype. We report here seven crystal structures of human PrP variants: three of wild‐type (WT) PrP containing V129, and four of the familial variants D178N and F198S, containing either M129 or V129. Comparison of these structures with each other and with previously published WT PrP structures containing M129 revealed that only WT PrPs were found to crystallize as domain‐swapped dimers or closed monomers; the four mutant PrPs crystallized as non‐swapped dimers. Three of the four mutant PrPs aligned to form intermolecular β‐sheets. Several regions of structural variability were identified, and analysis of their conformations provides an explanation for the structural features, which can influence the formation and conformation of intermolecular β‐sheets involving the M/V129 polymorphic residue.

Nucleation-dependent conformational conversion of the Y145Stop variant of human prion protein: Structural clues for prion propagation

One of the most intriguing disease-related mutations in human prion protein (PrP) is the Tyr to Stop codon substitution at position 145. This mutation results in a Gerstmann–Straussler–Scheinker-like disease with extensive PrP amyloid deposits in the brain. Here, we provide evidence for a spontaneous conversion of the recombinant polypeptide corresponding to the Y145Stop variant (huPrP23–144) from a monomeric unordered state to a fibrillar form. This conversion is characterized by a protein concentration-dependent lag phase and has characteristics of a nucleation-dependent polymerization. Atomic force microscopy shows that huPrP23–144 fibrils are characterized by an apparent periodicity along the long axis, with an average period of 20 nm. Fourier-transform infrared spectra indicate that the conversion is associated with formation of β-sheet structure. However, the infrared bands for huPrP23–144 are quite different from those for a synthetic peptide PrP106–126, suggesting conformational non-equivalence of β-structures in the disease-associated Y145Stop variant and a frequently used short model peptide. To identify the region that is critical for the self-seeded assembly of huPrP23–144 amyloid, experiments were performed by using the recombinant polypeptides corresponding to prion protein fragments 23–114, 23–124, 23–134, 23–137, 23–139, and 23–141. Importantly, none of the fragments ending before residue 139 showed a propensity for conformational conversion to amyloid fibrils, indicating that residues within the 138–141 region are essential for this conversion.

Rapidly progressive Alzheimer's disease features distinct structures of amyloid-β.

Genetic and environmental factors that increase the risk of late-onset Alzheimer disease are now well recognized but the cause of variable progression rates and phenotypes of sporadic Alzheimers disease is largely unknown. We aimed to investigate the relationship between diverse structural assemblies of amyloid-β and rates of clinical decline in Alzheimers disease. Using novel biophysical methods, we analysed levels, particle size, and conformational characteristics of amyloid-β in the posterior cingulate cortex, hippocampus and cerebellum of 48 cases of Alzheimers disease with distinctly different disease durations, and correlated the data with APOE gene polymorphism. In both hippocampus and posterior cingulate cortex we identified an extensive array of distinct amyloid-β42 particles that differ in size, display of N-terminal and C-terminal domains, and conformational stability. In contrast, amyloid-β40 present at low levels did not form a major particle with discernible size, and both N-terminal and C- terminal domains were largely exposed. Rapidly progressive Alzheimers disease that is associated with a low frequency of APOE e4 allele demonstrates considerably expanded conformational heterogeneity of amyloid-β42, with higher levels of distinctly structured amyloid-β42 particles composed of 30-100 monomers, and fewer particles composed of < 30 monomers. The link between rapid clinical decline and levels of amyloid-β42 with distinct structural characteristics suggests that different conformers may play an important role in the pathogenesis of distinct Alzheimers disease phenotypes. These findings indicate that Alzheimers disease exhibits a wide spectrum of amyloid-β42 structural states and imply the existence of prion-like conformational strains.

Soluble Prion Protein Inhibits Amyloid-β (Aβ) Fibrillization and Toxicity

Background: Prion protein was found to interact with Aβ, but the consequences of this interaction are largely unknown. Results: Prion protein and its N-terminal fragment inhibit Aβ1–42 amyloidogenesis and cytotoxicity. Conclusion: Soluble prion protein is a potent inhibitor of Aβ1–42 assembly into toxic oligomers. Significance: The results have important implications for understanding the pathogenesis of AD and for the development of novel therapeutic strategies. The pathogenesis of Alzheimer disease appears to be strongly linked to the aggregation of amyloid-β (Aβ) peptide and, especially, formation of soluble Aβ1–42 oligomers. It was recently demonstrated that the cellular prion protein, PrPC, binds with high affinity to these oligomers, acting as a putative receptor that mediates at least some of their neurotoxic effects. Here we show that the soluble (i.e. glycophosphatidylinositol anchor-free) prion protein and its N-terminal fragment have a strong effect on the aggregation pathway of Aβ1–42, inhibiting its assembly into amyloid fibrils. Furthermore, the prion protein prevents formation of spherical oligomers that normally occur during Aβ fibrillogenesis, acting as a potent inhibitor of Aβ1–42 toxicity as assessed in experiments with neuronal cell culture. These findings may provide a molecular level foundation to explain the reported protective action of the physiologically released N-terminal N1 fragment of PrPC against Aβ neurotoxicity. They also suggest a novel approach to pharmacological intervention in Alzheimer disease.

Intermolecular Alignment in Y145Stop Human Prion Protein Amyloid Fibrils Probed by Solid-State NMR Spectroscopy

The Y145Stop mutant of human prion protein, huPrP23-144, has been linked to PrP cerebral amyloid angiopathy, an inherited amyloid disease, and also serves as a valuable in vitro model for investigating the molecular basis of amyloid strains. Prior studies of huPrP23-144 amyloid by magic-angle-spinning (MAS) solid-state NMR spectroscopy revealed a compact β-rich amyloid core region near the C-terminus and an unstructured N-terminal domain. Here, with the focus on understanding the higher-order architecture of huPrP23-144 fibrils, we probed the intermolecular alignment of β-strands within the amyloid core using MAS NMR techniques and fibrils formed from equimolar mixtures of (15)N-labeled protein and (13)C-huPrP23-144 prepared with [1,3-(13)C(2)] or [2-(13)C]glycerol. Numerous intermolecular correlations involving backbone atoms observed in 2D (15)N-(13)C spectra unequivocally suggest an overall parallel in-register alignment of the β-sheet core. Additional experiments that report on intermolecular (15)N-(13)CO and (15)N-(13)Cα dipolar couplings yielded an estimated strand spacing that is within ∼10% of the distances of 4.7-4.8 Å typical for parallel β-sheets.

Small Protease Sensitive Oligomers of PrPSc in Distinct Human Prions Determine Conversion Rate of PrPC

The mammalian prions replicate by converting cellular prion protein (PrPC) into pathogenic conformational isoform (PrPSc). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrPSc on conversion of PrPC in vitro using PrPSc seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrPSc. The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrPSc. The tight correlation between conversion potency of small oligomers of human sPrPSc observed in vitro and duration of the disease suggests that sPrPSc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.