Temperature-Dependent Structural Variability of Prion Protein Amyloid Fibrils.
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ABSTRACT:
SUBMITTER: Ziaunys M
PROVIDER: S-EPMC8151363 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Protein aggregation into amyloid fibrils is linked to multiple neurodegenerative disorders, such as Alzheimer's, Parkinson's or Creutzfeldt-Jakob disease. A better understanding of the way these aggregates form is vital for the development of drugs. A large detriment to amyloid research is the ability of amyloidogenic proteins to spontaneously aggregate into multiple structurally distinct fibrils (strains) with different stability and seeding properties. In this work we show that prion proteins are capable of forming more than one type of fibril under the exact same conditions by assessing their Thioflavin T (ThT) binding ability, morphology, secondary structure, stability and seeding potential.
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Project description:Mass per length (mpl) measurements on single amyloid fibrils that specifically propagate the [VH], [VK], and [VL] strains of the yeast prion [PSI] reveal unanticipated differences in their structures. Many fibrils have approximately 1.0 prion molecule per 4.7-A cross-beta repeat period, which is consistent with a self-replicating model built by parallel beta-sheet hydrogen-bonding of like prion peptide segments, but other fibrils are definitely heavier. The predominantly straight fibrils of the dominant [VH] strain have a bimodal mpl distribution, corresponding to components with approximately 1.0 and 1.2 prions per repeat. Fibrils of the weaker [VK] strain, which are almost all wavy, have a monodisperse mpl distribution with a mean of 1.15 prions per repeat. The recessive [VL] strain sample has approximately 1.05 prions per repeat in single fibrils and includes approximately 10% double fibrils, which are rare in the duplicate [VH] and [VK] samples. All of these samples were assembled from purified recombinant Sup35 prion protein by seeded growth on nuclei extracted from yeast bearing the three [PSI] strains. Infectious and noninfectious spontaneously assembled fibrils of the recombinant prion protein also display different heterogeneous morphologies. The strain-specific morphological differences we have observed directly confirm the structural prediction of the protein-only prion theory but do not have an obvious molecular explanation.
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Project description:Propagation and infectivity of prions in human prionopathies are likely associated with conversion of the mainly alpha-helical human prion protein, HuPrP, into an aggregated form with amyloid-like properties. Previous reports on efficient conversion of recombinant HuPrP have used mild to harsh denaturing conditions to generate amyloid fibrils in vitro. Herein we report on the in vitro conversion of four forms of truncated HuPrP (sequences 90-231 and 121-231 with and without an N-terminal hexa histidine tag) into amyloid-like fibrils within a few hours by using a protocol (phosphate buffered saline solutions at neutral pH with intense agitation) close to physiological conditions. The conversion process monitored by thioflavin T, ThT, revealed a three stage process with lag, growth and equilibrium phases. Seeding with preformed fibrils shortened the lag phase demonstrating the classic nucleated polymerization mechanism for the reaction. Interestingly, comparing thioflavin T kinetics with solubility and turbidity kinetics it was found that the protein initially formed nonthioflavionophilic, morphologically disordered aggregates that over time matured into amyloid fibrils. By transmission electron microscopy and by fluorescence microscopy of aggregates stained with luminescent conjugated polythiophenes (LCPs); we demonstrated that HuPrP undergoes a conformational conversion where spun and woven fibrils protruded from morphologically disordered aggregates. The initial aggregation functioned as a kinetic trap that decelerated nucleation into a fibrillation competent nucleus, but at the same time without aggregation there was no onset of amyloid fibril formation. The agitation, which was necessary for fibril formation to be induced, transiently exposes the protein to the air-water interface suggests a hitherto largely unexplored denaturing environment for prion conversion.
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Project description:One of the most puzzling aspects of the prion diseases is the intricate relationship between prion strains and interspecies transmissibility barriers. Previously we have shown that certain fundamental aspects of mammalian prion propagation, including the strain phenomenon and species barriers, can be reproduced in vitro in seeded fibrillization of the Y145Stop prion protein variant. Here, we use solid-state nuclear magnetic resonance spectroscopy to gain atomic level insight into the structural differences between Y145Stop prion protein amyloids from three species: human, mouse, and Syrian hamster. Remarkably, we find that these structural differences are largely controlled by only two amino acids at positions 112 and 139, and that the same residues appear to be key to the emergence of structurally distinct amyloid strains within the same protein sequence. The role of these residues as conformational switches can be rationalized based on a model for human Y145Stop prion protein amyloid, providing a foundation for understanding cross-seeding specificity.Prion diseases can be transmitted across species. Here the authors use solid-state NMR to study prion protein (PrP) amyloids from human, mouse and Syrian hamster and show that their structural differences are mainly governed by two residues, which helps to understand interspecies PrP propagation on a molecular level.
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Project description:Many peptides and proteins self-assemble into amyloid fibrils. Examples include mammalian and fungal prion proteins, polypeptides associated with human amyloid diseases, and proteins that may have biologically functional amyloid states. To understand the propensity for polypeptides to form amyloid fibrils and to facilitate rational design of amyloid inhibitors and imaging agents, it is necessary to elucidate the molecular structures of these fibrils. Although fibril structures were largely mysterious 15 years ago, a considerable body of reliable structural information about amyloid fibril structures now exists, with essential contributions from solid state nuclear magnetic resonance (NMR) measurements. This Account reviews results from our laboratories and discusses several structural issues that have been controversial. In many cases, the amino acid sequences of amyloid fibrils do not uniquely determine their molecular structures. Self-propagating, molecular-level polymorphism complicates the structure determination problem and can lead to apparent disagreements between results from different laboratories, particularly when different laboratories study different polymorphs. For 40-residue β-amyloid (Aβ₁₋₄₀) fibrils associated with Alzheimer's disease, we have developed detailed structural models from solid state NMR and electron microscopy data for two polymorphs. These polymorphs have similar peptide conformations, identical in-register parallel β-sheet organizations, but different overall symmetry. Other polymorphs have also been partially characterized by solid state NMR and appear to have similar structures. In contrast, cryo-electron microscopy studies that use significantly different fibril growth conditions have identified structures that appear (at low resolution) to be different from those examined by solid state NMR. Based on solid state NMR and electron paramagnetic resonance (EPR) measurements, the in-register parallel β-sheet organization found in β-amyloid fibrils also occurs in many other fibril-forming systems. We attribute this common structural motif to the stabilization of amyloid structures by intermolecular interactions among like amino acids, including hydrophobic interactions and polar zippers. Surprisingly, we have recently identified and characterized antiparallel β-sheets in certain fibrils that are formed by the D23N mutant of Aβ₁₋₄₀, a mutant that is associated with early-onset, familial neurodegenerative disease. Antiparallel D23N-Aβ₁₋₄₀ fibrils are metastable with respect to parallel structures and, therefore, represent an off-pathway intermediate in the amyloid fibril formation process. Other methods have recently produced additional evidence for antiparallel β-sheets in other amyloid-formation intermediates. As an alternative to simple parallel and antiparallel β-sheet structures, researchers have proposed β-helical structural models for some fibrils, especially those formed by mammalian and fungal prion proteins. Solid state NMR and EPR data show that fibrils formed in vitro by recombinant PrP have in-register parallel β-sheet structures. However, the structure of infectious PrP aggregates is not yet known. The fungal HET-s prion protein has been shown to contain a β-helical structure. However, all yeast prions studied by solid state NMR (Sup35p, Ure2p, and Rnq1p) have in-register parallel β-sheet structures, with their Gln- and Asn-rich N-terminal segments forming the fibril core.
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Project description: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 ?-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 ?-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 ?-sheet core. Additional experiments that report on intermolecular (15)N-(13)CO and (15)N-(13)C? dipolar couplings yielded an estimated strand spacing that is within ?10% of the distances of 4.7-4.8 Å typical for parallel ?-sheets.
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Project description: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 (13)C,(15)N-labeled huPrP23-144. Surprisingly, we find that signals from approximately 100 residues (i.e., approximately 80% of the sequence) are not detected above approximately -20 degrees 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 (13)C and (15)N linewidths of approximately 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 (13)C secondary chemical shifts identified likely beta-strand segments within this core region, including beta-strand 130-139 containing critical residues 138 and 139. In contrast to this relatively rigid, beta-sheet-rich amyloid core, the remaining residues in huPrP23-144 amyloid fibrils under physiologically relevant conditions are largely unordered, displaying significant conformational dynamics.
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