Prion Protein Fibrils Research Articles

Syntaxin-6 delays prion protein fibril formation and prolongs the presence of toxic aggregation intermediates.

Prions replicate via the autocatalytic conversion of cellular prion protein (PrPC) into fibrillar assemblies of misfolded PrP. While this process has been extensively studied in vivo and in vitro, non-physiological reaction conditions of fibril formation in vitro have precluded the identification and mechanistic analysis of cellular proteins, which may alter PrP self-assembly and prion replication. Here, we have developed a fibril formation assay for recombinant murine and human PrP (23-231) under near-native conditions (NAA) to study the effect of cellular proteins, which may be risk factors or potential therapeutic targets in prion disease. Genetic screening suggests that variants that increase syntaxin-6 expression in the brain (gene: STX6) are risk factors for sporadic Creutzfeldt-Jakob disease. Analysis of the protein in NAA revealed, counterintuitively, that syntaxin-6 is a potent inhibitor of PrP fibril formation. It significantly delayed the lag phase of fibril formation at highly sub-stoichiometric molar ratios. However, when assessing toxicity of different aggregation time points to primary neurons, syntaxin-6 prolonged the presence of neurotoxic PrP species. Electron microscopy and super-resolution fluorescence microscopy revealed that, instead of highly ordered fibrils, in the presence of syntaxin-6 PrP formed less-ordered aggregates containing syntaxin-6. These data strongly suggest that the protein can directly alter the initial phase of PrP self-assembly and, uniquely, can act as an 'anti-chaperone', which promotes toxic aggregation intermediates by inhibiting fibril formation.

Conformation-Specific Association of Prion Protein Amyloid Aggregates with Tau Protein Monomers.

Protein aggregation into amyloid fibrils is associated with several amyloidoses, including neurodegenerative Alzheimer's and Parkinson's diseases. Despite years of research and numerous studies, the process is still not fully understood, which significantly impedes the search for cures of amyloid-related disorders. Recently, there has been an increase in reports of amyloidogenic protein cross-interactions during the fibril formation process, which further complicates the already intricate process of amyloid aggregation. One of these reports displayed an interaction involving Tau and prion proteins, which prompted a need for further investigation into the matter. In this work, we generated five populations of conformationally distinct prion protein amyloid fibrils and examined their interaction with Tau proteins. We observed that there was a conformation-specific association between Tau monomers and prion protein fibrils, which increased the aggregate self-association and amyloidophilic dye binding capacity. We also determined that the interaction did not induce the formation of Tau protein amyloid aggregates, but rather caused their electrostatic adsorption to the prion protein fibril surface.

Inhibitory effects of sesquiterpene lactones on the aggregation and cytotoxicity of prion neuropeptide

The misfolding and conformational transformation of prion protein (PrP) are crucial to the progression of prion diseases. Screening for available natural inhibitors against prion proteins can contribute to the rational design and development of new anti-prion drugs and therapeutic strategies. The prion neuropeptide, PrP106-126 is commonly used as a model peptide of the abnormal PrPSc, and a number of potential inhibitors were explored against the amyloid fibril formation of PrP106-126. The well-known sesquiterpene lactone, artemisinin, shows diverse biological functions in anti-malarial, anti-cancer and lowering glucose. However, its inhibitory effect on PrP106-126 fibrillation is unclear. In this work, we selected two sesquiterpene lactones, artemisinin (1) and artesunate (2), to explore their roles in PrP106-126 aggregation by a series of physicochemical and biochemical methods. The results demonstrated that 1 and 2 could effectively impede the formation of amyloid fibrils and remodel the preformed fibrils. The binding of the small molecules to PrP106-126 was dominated by electrostatic, hydrophobic and hydrogen bonding interactions. In addition, both compounds exhibited neuroprotective effects by reducing peptide oligomerization. 2 showed better inhibition and regulation on peptide aggregation and cellular viability than 1 due to its specific succinate modification. Our study provides the information of sesquiterpene lactones to prevent PrP fibril formation and other related amyloidosis.

Direct Observation of Competing Prion Protein Fibril Populations with Distinct Structures and Kinetics.

In prion diseases, fibrillar assemblies of misfolded prion protein (PrP) self-propagate by incorporating PrP monomers. These assemblies can evolve to adapt to changing environments and hosts, but the mechanism of prion evolution is poorly understood. We show that PrP fibrils exist as a population of competing conformers, which are selectively amplified under different conditions and can "mutate" during elongation. Prion replication therefore possesses the steps necessary for molecular evolution analogous to the quasispecies concept of genetic organisms. We monitored structure and growth of single PrP fibrils by total internal reflection and transient amyloid binding super-resolution microscopy and detected at least two main fibril populations, which emerged from seemingly homogeneous PrP seeds. All PrP fibrils elongated in a preferred direction by an intermittent "stop-and-go" mechanism, but each population possessed distinct elongation mechanisms that incorporated either unfolded or partially folded monomers. Elongation of RML and ME7 prion rods likewise exhibited distinct kinetic features. The discovery of polymorphic fibril populations growing in competition, which were previously hidden in ensemble measurements, suggests that prions and other amyloid replicating by prion-like mechanisms may represent quasispecies of structural isomorphs that can evolve to adapt to new hosts and conceivably could evade therapeutic intervention.

Mapping the interactions between prion protein (PrPC) and prion protein fibrils (PrPSc).

Transmissible spongiform encephalopathies (TSEs), prion diseases, arise when normal prion protein (PrPC) misfolds and accumulates, forming prion fibrils (PrPSc) in the brain. Normal prion protein is GPI-anchored to the outer leaflet of the plasma membrane. Therefore, under pathological conditions, templated misfolding and aggregation occur on, or close to, the cell surface. However, this process of misfolding is distinct from neurotoxicity signaling pathways. Experimentally resolved three-dimensional structures of prion fibrils show at least two distinct populations: PIRIBS and 4-rungB. Although these structures provide valuable insight into fibril morphology, the mechanism responsible for templating prion protein misfolding largely remains unknown. In our project, we aim to characterize the modes of recognition between the prion proteins and fibrils on the surface of the cell membrane, using computational structural bioinformatics techniques. The results suggest that fibril-cell surface interactions are differentially driven by the electrostatics of the unique fibril morphologies. Our mapping of the binding interface of prion protein and fibril indicates a distribution of recognition sites, including the lateral surface and edge of the fibril. We will discuss our results considering the hypothesis that a distribution of protein-fibril recognition events leads to distinct outcomes, either scaffolding templated misfolding or triggering neurotoxic signaling. We will propose implications in terms of anti-prion druggable pathways as they relate to the potential mechanisms of neurotoxicity.

Probing the role of RNA in prion protein and prion fibril recognition.

The conversion of the cellular form of the prion protein PrPC to the misfolded and aggregated form PrPSc is the key pathological event in prion diseases, yet the mechanism of the conformational change remains unknown. Evidence suggests that prion protein misfolding initiates on the cell surface, where the prion protein is GPI anchored to the outer leaflet of the plasma membrane. Compelling results document that extracellularly located nucleic acids scaffold prion protein aggregation; however, whether nucleic acids interact directly with the monomer prion protein, a misfolded intermediate, or PrPSc fibrils remains unknown. Recent studies indicate upon RNA binding, a pincer-like structure is formed between the N-terminus and a-helix 1 of the prion protein that traps the RNA and induces misfolding of the helix. However, it is unknown how the initial misfolding of a-helix 1 affects the other helices. To fill this gap, we aim to examine the changes in protein and fibril connectivity as well as identify key residues involved in RNA mediated aggregation of the prion protein. We used structural bioinformatics techniques to model and examine the connectivity of the prion protein and prion fibrils when bound to RNA. Our results suggest that the binding of RNA to a-helix 1 modulates the long-distance coupling to other helices. We will also probe the modulatory effects of RNA on the interactions between prion protein and fibrils. We will use these calculations to infer the role of RNA on prion protein misfolding and protein-fibril recognition.

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

Mammalian prions propagate as distinct strains and are composed of multichain assemblies of misfolded host-encoded prion protein (PrP). Here, we present a near-atomic resolution cryo-EM structure of PrP fibrils present in highly infectious prion rod preparations isolated from the brains of RML prion-infected mice. We found that prion rods comprise single-protofilament helical amyloid fibrils that coexist with twisted pairs of the same protofilaments. Each rung of the protofilament is formed by a single PrP monomer with the ordered core comprising PrP residues 94–225, which folds to create two asymmetric lobes with the N-linked glycans and the glycosylphosphatidylinositol anchor projecting from the C-terminal lobe. The overall architecture is comparable to that of recently reported PrP fibrils isolated from the brain of hamsters infected with the 263K prion strain. However, there are marked conformational variations that could result from differences in PrP sequence and/or represent distinguishing features of the distinct prion strains.

Effect of point mutations on the network of side chain interactions of prion protein fibrils with distinct quaternary structures

Prion diseases are fatal neurodegenerative disorders that occur in mammals, including humans. At the molecular level, prion diseases are characterized by the presence of prion aggregates that are fibrillar and b-sheet rich. Intriguingly, prion protein fibrils display multiple morphologies, each characterized by unique biochemical properties and linked to distinct disease phenotypes. Few quaternary structures of prion protein fibrils from mice have been proposed based on cryoEM studies. However, the extent to which those structures represent fibrils formed by the prion protein of differentially disease-susceptible species remains unresolved.

Superoxide dismutase-1 alters the rate of prion protein aggregation and resulting fibril conformation

The assembly of amyloidogenic proteins into highly-structured fibrillar aggregates is related to the onset and progression of several amyloidoses, including neurodegenerative Alzheimer's or Parkinson's diseases. Despite years of research and a general understanding of the process of such aggregate formation, there are currently still very few drugs and treatment modalities available. One of the factors that is relatively insufficiently understood is the cross-interaction between different amyloid-forming proteins. In recent years, it has been shown that several of these proteins or their aggregates can alter each other's fibrillization properties, however, there are still many unknowns in the amyloid interactome. In this work, we examine the interaction between amyloid disease-related prion protein and superoxide dismutase-1. We show that not only does superoxide dismutase-1 increase the lag time of prion protein fibril formation, but it also changes the conformation of the resulting aggregates.

The Unifying Hypothesis of Alzheimer's Disease: Heparan Sulfate Proteoglycans/Glycosaminoglycans Are Key as First Hypothesized Over 30 Years Ago.

The updated “Unifying Hypothesis of Alzheimer’s disease” (AD) is described that links all the observed neuropathology in AD brain (i.e., plaques, tangles, and cerebrovascular amyloid deposits), as well as inflammation, genetic factors (involving ApoE), “AD-in-a-Dish” studies, beta-amyloid protein (Aβ) as a microbial peptide; and theories that bacteria, gut microflora, gingivitis and viruses all play a role in the cause of AD. The common link is the early accumulation of heparan sulfate proteoglycans (HSPGs) and heparan sulfate glycosaminoglycans (GAGs). HS GAG accumulation and/or decreased HS GAG degradation is postulated to be the key initiating event. HS GAGs and highly sulfated macromolecules induce Aβ 1–40 (but not 1–42) to form spherical congophilic maltese-cross star-like amyloid core deposits identical to those in the AD brain. Heparin/HS also induces tau protein to form paired helical filaments (PHFs). Increased sulfation and/or decreased degradation of HSPGs and HS GAGs that occur due to brain aging leads to the formation of plaques and tangles in AD brain. Knockout of HS genes markedly reduce the accumulation of Aβ fibrils in the brain demonstrating that HS GAGs are key. Bacteria and viruses all use cell surface HS GAGs for entry into cells, including SARS-CoV-2. Bacteria and viruses cause HS GAGs to rapidly increase to cause near-immediate aggregation of Aβ fibrils. “AD-in-a-dish” studies use “Matrigel” as the underlying scaffold that spontaneously causes plaque, and then tangle formation in a dish. Matrigel mostly contains large amounts of perlecan, the same specific HSPG implicated in AD and amyloid disorders. Mucopolysaccharidoses caused by lack of specific HS GAG enzymes lead to massive accumulation of HS in lysosomal compartments in neurons and contribute to cognitive impairment in children. Neurons full of HS demonstrate marked accumulation and fibrillization of Aβ, tau, α-synuclein, and prion protein (PrP) in mucopolysaccharidosis animal models demonstrating that HS GAG accumulation is a precursor to Aβ accumulation in neurons. Brain aging leads to changes in HSPGs, including newly identified splice variants leading to increased HS GAG sulfation in the AD brain. All of these events lead to the new “Unifying Hypothesis of Alzheimer’s disease” that further implicates HSPGs /HS GAGs as key (as first hypothesized by Snow and Wight in 1989).

Aggregation Condition-Structure Relationship of Mouse Prion Protein Fibrils.

Prion diseases are associated with conformational conversion of cellular prion protein into a misfolded pathogenic form, which resembles many properties of amyloid fibrils. The same prion protein sequence can misfold into different conformations, which are responsible for variations in prion disease phenotypes (prion strains). In this work, we use atomic force microscopy, FTIR spectroscopy and magic-angle spinning NMR to devise structural models of mouse prion protein fibrils prepared in three different denaturing conditions. We find that the fibril core region as well as the structure of its N- and C-terminal parts is almost identical between the three fibrils. In contrast, the central part differs in length of β-strands and the arrangement of charged residues. We propose that the denaturant ionic strength plays a major role in determining the structure of fibrils obtained in a particular condition by stabilizing fibril core interior-facing glutamic acid residues.

Temperature-Dependent Structural Variability of Prion Protein Amyloid Fibrils.

Prion protein aggregation into amyloid fibrils is associated with the onset and progression of prion diseases—a group of neurodegenerative amyloidoses. The process of such aggregate formation is still not fully understood, especially regarding their polymorphism, an event where the same type of protein forms multiple, conformationally and morphologically distinct structures. Considering that such structural variations can greatly complicate the search for potential antiamyloid compounds, either by having specific propagation properties or stability, it is important to better understand this aggregation event. We have recently reported the ability of prion protein fibrils to obtain at least two distinct conformations under identical conditions, which raised the question if this occurrence is tied to only certain environmental conditions. In this work, we examined a large sample size of prion protein aggregation reactions under a range of temperatures and analyzed the resulting fibril dye-binding, secondary structure and morphological properties. We show that all temperature conditions lead to the formation of more than one fibril type and that this variability may depend on the state of the initial prion protein molecules.

Ellagic acid and pentagalloylglucose are potential inhibitors of prion protein fibrillization

Prion diseases are fatal neurodegenerative diseases caused by the conformational transition of the cellular prion protein (PrPC) to the abnormal pathological prion protein (PrPSc). In this work, the effects of ellagic acid (EA) and pentagalloylglucose (PGG) on prion protein (PrP) fibrillization were investigated. Fluorescence quenching experiments indicated that both EA and PGG could specifically interact with native human PrP with binding affinities of 1.92 × 105 and 2.36 × 105 L·mol−1, respectively. Thioflavin-T (ThT) fluorescence assays showed that the binding of EA or PPG could effectively inhibit the nucleation and elongation of PrP fibrilization and reduce the amount of PrP fibrils generated. EA and PGG could also lead to a significant disaggregation of PrP fibrils. Circular dichroism (CD) measurements suggested that EA- or PPG-bound PrP could preserve a higher content of α-helical structures than β-sheet-rich PrP fibrils. The PrP aggregates formed in the presence of EA or PGG showed lower resistance to proteinase K (PK) digestion. Overall, the present work reported the inhibitory effect of EA and PGG on PrP fibrillization. These two natural polyphenols could be potential prodrug molecules for the prevention and treatment of prion diseases.

Accommodation of In-Register N-Linked Glycans on Prion Protein Amyloid Cores.

Although prion protein fibrils can have either parallel-in-register intermolecular β-sheet (PIRIBS) or, probably, β-solenoid architectures, the plausibility of PIRIBS architectures for the usually glycosylated natural prion strains has been questioned based the expectation that such glycans would not fit if stacked in-register on each monomer within a fibril. To directly assess this issue, we have added N-linked glycans to a recently reported cryo-electron microscopy-based human prion protein amyloid model with a PIRIBS architecture and performed in silico molecular dynamics studies to determine if the glycans can fit. Our results show that triantennary glycans can be sterically accommodated in-register on both N-linked glycosylation sites of each monomer. Additional simulations with an artificially mutated β-solenoid model confirmed that glycans can be accommodated when aligned with ∼4.8 Å spacing on every rung of a fibril. Altogether, we conclude that steric intermolecular clashes between glycans do not, in themselves, preclude PIRIBS architectures for prions.

Quercetin Disaggregates Prion Fibrils and Decreases Fibril-Induced Cytotoxicity and Oxidative Stress.

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases caused by misfolding and aggregation of prion protein (PrP). Previous studies have demonstrated that quercetin can disaggregate some amyloid fibrils, such as amyloid β peptide (Aβ) and α-synuclein. However, the disaggregating ability is unclear in PrP fibrils. In this study, we examined the amyloid fibril-disaggregating activity of quercetin on mouse prion protein (moPrP) and characterized quercetin-bound moPrP fibrils by imaging, proteinase resistance, hemolysis assay, cell viability, and cellular oxidative stress measurements. The results showed that quercetin treatment can disaggregate moPrP fibrils and lead to the formation of the proteinase-sensitive amorphous aggregates. Furthermore, quercetin-bound fibrils can reduce the membrane disruption of erythrocytes. Consequently, quercetin-bound fibrils cause less oxidative stress, and are less cytotoxic to neuroblastoma cells. The role of quercetin is distinct from the typical function of antiamyloidogenic drugs that inhibit the formation of amyloid fibrils. This study provides a solution for the development of antiamyloidogenic therapy.

Cryo-EM structure of an amyloid fibril formed by full-length human prion protein.

Prion diseases are caused by the misfolding of prion protein (PrP). Misfolded PrP forms protease-resistant aggregates in vivo (PrPSc) that are able to template the conversion of the native form of the protein (PrPC), a property shared by in vitro-produced PrP fibrils. Here we produced amyloid fibrils in vitro from recombinant, full-length human PrPC (residues 23-231) and determined their structure using cryo-EM, building a model for the fibril core comprising residues 170-229. The PrP fibril consists of two protofibrils intertwined in a left-handed helix. Lys194 and Glu196 from opposing subunits form salt bridges, creating a hydrophilic cavity at the interface of the two protofibrils. By comparison with the structure of PrPC, we propose that two α-helices in the C-terminal domain of PrPC are converted into β-strands stabilized by a disulfide bond in the PrP fibril. Our data suggest that different PrP mutations may play distinct roles in modulating the conformational conversion.

Direct Observation of Prion Protein Fibril Elongation Kinetics

It is widely accepted that prion diseases are caused by the conversion of cellular prion protein (PrPC) to the misfolded form PrPSc, which is self-propagating by seeded aggregation. The molecular mechanism and propagation kinetics are not well understood. Here, we use Total Internal Reflection Fluorescence and Transient Amyloid Binding super-resolution microscopy to investigate the elongation kinetics of mouse prion protein at single fibril level. Recombinant mouse prion protein (recMoPrP 91-231) fibrils formed under partially denaturing conditions were deposited on a glass slide, incubated with recMoPrP 91-231 monomer solution and imaged for up to 70h. We found that PrP fibrils elongated by an intermittent ‘stop-and-go’ mechanism. The elongation rates of growing phase are obtained under different temperature, various concentrations of monomer, denaturant, and sodium chloride, which are then used to calculate the kinetic parameters. This study sheds light on the mechanism of prion protein seeded propagation in vitro and provides a useful system to study the influence of PrP mutants and screening of inhibitors.

Neutralizing Mutations Significantly Inhibit Amyloid Formation by Human Prion Protein and Decrease Its Cytotoxicity

Prion diseases, such as Creutzfeldt–Jakob disease and bovine spongiform encephalopathy, are fatal neurodegenerative diseases that affect many mammals including humans and are caused by the misfolding of prion protein (PrP). A naturally occurring protective polymorphism G127V in human PrP has recently been found to significantly attenuate prion diseases, but the mechanism has remained elusive. We herein report that the hydrophobic chain introduced in G127V significantly inhibits amyloid fibril formation by human PrP, highlighting the protective effect of the G127V polymorphism. We further introduce an amino acid with a different hydrophobic chain (Ile) at the same position and find that G127I has similar protective effects as G127V. Moreover, we show that these two neutralizing mutations, G127V and G127I, significantly decrease the human PrP cytotoxicity resulting from PrP fibril formation, mitochondrial damage, and elevated reactive oxygen species production enhanced by a strong prion-prone peptide PrP 106-126. These findings elucidate the molecular basis for a natural protective polymorphism in PrP and will enable the development of novel therapeutic strategies against prion diseases.

Recent Advances in Understanding Mammalian Prion Structure: A Mini Review.

Prions are lethal pathogens, which cause fatal neurodegenerative diseases in mammals. They are unique infectious agents and are composed of self-propagating multi-chain assemblies of misfolded host-encoded prion protein (PrP). Understanding prion structure is fundamental to understanding prion disease pathogenesis however to date, the high-resolution structure of authentic ex vivo infectious prions remains unknown. Advances in determining prion structure have been severely impeded by the difficulty in recovering relatively homogeneous prion particles from infected brain and definitively associating infectivity with the PrP assembly state. Recently, however, images of highly infectious ex vivo PrP rods that produce prion-strain specific disease phenotypes in mice have been obtained using cryo-electron microscopy and atomic force microscopy. These images have provided the most detailed description of ex vivo mammalian prions reported to date and have established that prions isolated from multiple strains have a common hierarchical structure. Misfolded PrP is assembled into 20 nm wide rods containing two fibers, each with double helical repeating substructure, separated by a characteristic central gap 8–10 nm in width. Irregularly structured material with adhesive properties distinct to that of the fibers is present within the central gap of the rod. Prions are clearly distinguishable from non-infectious recombinant PrP fibrils generated in vitro and from all other propagating protein structures so far described in other neurodegenerative diseases. The basic architecture of mammalian prions appears to be exceptional and fundamental to their lethal pathogenicity.

Impact of N-glycosylation site variants during human PrP aggregation and fibril nucleation

Misfolding and aggregation of the human prion protein (PrP) cause neurodegenerative transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease. Mature native PrP is composed of 209 residues and is folded into a C-terminal globular domain (residues 125–209) comprising a small two-stranded β-sheet and three α-helices. The N-terminal domain (residues 23–124) is intrinsically disordered. Expression of truncated PrP (residues 90–231) is sufficient to cause prion disease and residues 90/100–231 is comprising the amyloid-like fibril core of misfolded infectious PrP. During PrP fibril formation under native conditions in vitro, the disordered N-terminal domain slows down fibril formation likely due to a mechanism of initial aggregation forming morphologically disordered aggregates. The morphological disordered aggregate is a transient phase. Nucleation of fibrils occurs from this initial aggregate. The aggregate phase is largely circumvented by seeding with preformed PrP fibrils. In vivo PrP is N-glycosylated at positions Asn181 and Asn197. Little is known about the importance of these positions and their glycans for PrP stability, aggregation and fibril formation. We have in this study taken a step towards that goal by mutating residues 181 and 197 for cysteines to study the positional impact on these processes. We have further by organic synthetic chemistry and chemical modification generated synthetic glycosylations in these positions. Our data shows that residue 181 when mutated to a cysteine is a key residue for self-chaperoning, rendering a trap in the initial aggregate preventing conformational changes towards amyloid fibril formation. Position 197 is less involved in the aggregate trapping and is more geared towards β-sheet structure conversion within amyloid fibrils. As expected, synthetic glycosylated 197 is less affected towards fibril formation compared to glycosylated 181. Our data are rather compatible with the parallel in-register intermolecular β-sheet model structure of the PrP90–231 fibril and sheds light on the misfolding transitions of PrP in vitro. We hypothesize that glycosylation of position 181 is a key site for prion strain differentiation in vivo.