Ultrasensitive Detection of Lipid-Induced Misfolding of the Prion Protein at the Aqueous-Liquid Crystal Interface.

The misfolding of the α-helical cellular prion protein into a self-propagating ß-rich aggregated form is a key pathogenic event in fatal and transmissible neurodegenerative diseases collectively known as prion diseases. Herein, we utilize the interfacial properties of liquid crystals (LCs) to monitor the lipid-membrane-induced conformational switching of prion protein (PrP) into ß-rich amyloid fibrils. The lipid-induced conformational switching resulting in aggregation occurs at the nanomolar protein concentration and is primarily mediated by electrostatic interactions between PrP and lipid headgroups. Our LC-based methodology offers a potent and sensitive tool to detect and delineate molecular mechanisms of PrP misfolding mediated by lipid-protein interactions at the aqueous interface under physiological conditions.

SERS probing of fungal HET-s fibrils formed at neutral and acidic pH conditions.

Advances in precision medical diagnostics require accurate and sensitive characterization of pathogens. In particular, health conditions associated with protein misfolding require an identification of proteinaceous amyloid fibrils or their precursors. These pathogenic entities express specific molecular structures, which require ultra-sensitive, molecular-level detection methods. A potentially transformative technique termed nanoplasmonics employs electro-optical phenomena in the vicinity of specially engineered metal nanostructures. A signature application of nanoplasmonics exploits enhancement of inelastic scattering of light in specific locations near metallic nanostructures, known as surface-enhanced Raman scattering (SERS). We applied SERS complemented with confocal microscopy imaging for ultra-sensitive, non-invasive, and label-free characterization of the fungal prion HET-s (218-289) as a model for ß-sheet rich amyloid structures. This characterization employed Au-coated dielectric supports as plasmonic substrates. After confirming the formation of HET-s fibrils at both pH 7.5 and 2.8 using negative staining transmission electron microscopy, we subjected the fibril-containing solutions to multimodal analysis using confocal microscopy and SERS. The SERS spectral fingerprints from all HET-s samples expressed vibrational markers for ß-structure, unstructured backbone, and aromatic side-chains. However, relative intensities of major SERS bands were pronouncedly different for the two pH levels. We have analyzed potential origins of the most pronounced SERS bands and proposed hypothetical mechanistic models that could explain the observed SERS fingerprints from HET-s fibrils grown at pH 7.5 and 2.8.

Travels with tau prions.

Tau was originally identified as a microtubule associated protein, and subsequently recognized to constitute the fibrillar assemblies found in Alzheimer disease and related neurodegenerative tauopathies. Point mutations in the microtubule associated protein tau (MAPT) gene cause dominantly inherited tauopathies, and most predispose it to aggregate. This indicates tau aggregation underlies pathogenesis of tauopathies. Our work has suggested that tau functions as a prion, forming unique intracellular pathological assemblies that subsequently move to other cells, inducing further aggregation that underlies disease progression. Remarkably, in simple cells tau forms stably propagating aggregates of distinct conformation, termed strains. Each strain induces a unique and, in some cases, transmissible, neuropathological phenotype upon inoculation into a mouse model. After binding heparan sulfate proteoglycans on the plasma membrane, tau assemblies enter cells via macropinocytosis. From within a vesicle, if not trafficked to the endolysosomal system, tau subsequently enters the cytoplasm, where it becomes a template for its own replication, apparently after processing by valosin containing protein. The smallest seed unit is a stable monomer, which suggests that initial folding events in tau presage subsequent pathological aggregation. The study of tau prions has raised important questions about basic cell biological processes that underlie their replication and propagation, with implications for therapy of tauopathies.

Bacteriophage-encoded chaperonins stimulate prion protein fibrillation in an ATP-dependent manner.

The pathogenesis of the various prion diseases is based on the conformational conversion of the prion protein from its physiological cellular form to the insoluble scrapie isoform. Several chaperones, including the Hsp60 family of group I chaperonins, are known to contribute to this transformation, but data on their effects are scarce and conflicting. In this work, two GroEL-like phage chaperonins, the single-ring OBP and the double-ring EL, were found to stimulate monomeric prion protein fibrillation in an ATP-dependent manner. The resulting fibrils were characterised by thioflavin T fluorescence, electron microscopy, proteinase K digestion assay and other methods. In the presence of ATP, chaperonins were found to promote the conversion of prion protein monomers into short amyloid fibrils with their further aggregation into less toxic large clusters. Fibrils generated with the assistance of phage chaperonins differ in morphology and properties from those formed spontaneously from monomeric prion in the presence of denaturants at acidic pH.

Copper drives prion protein phase separation and modulates aggregation.

Prion diseases are characterized by prion protein (PrP) transmissible aggregation and neurodegeneration, which has been linked to oxidative stress. The physiological function of PrP seems related to sequestering of redox-active Cu2+, and Cu2+ dyshomeostasis is observed in prion disease brain. It is unclear whether Cu2+ contributes to PrP aggregation, recently shown to be mediated by PrP condensation. This study indicates that Cu2+ promotes PrP condensation in live cells at the cell surface and in vitro through copartitioning. Molecularly, Cu2+ inhibited PrP ß-structure and hydrophobic residues exposure. Oxidation, induced by H2O2, triggered liquid-to-solid transition of PrP:Cu2+ condensates and promoted amyloid-like PrP aggregation. In cells, overexpression of PrPC initially protected against Cu2+ cytotoxicity but led to PrPC aggregation upon extended copper exposure. Our data suggest that PrP condensates function as a buffer for copper that prevents copper toxicity but can transition into PrP aggregation at prolonged oxidative stress.

Prion-like Aggregation of the Heptapeptide GNNQQNY into Amyloid Nanofiber Is Governed by Configuration Entropy.

A major cause of prion infectivity is the early formation of small, fibril-like aggregates consisting of the heptapeptide GNNQQNY. The prion aggregates exhibit a unique stacking mode in which the hydrophobic tyrosine (Y) is exposed outward, forming a bilayer ß-sheet-stacking zipper structure. This stacking mode of the prion peptides, termed "Y-outward" structure for convenience, goes against the common understanding that, for other amyloid-forming peptides, the hydrophobic residues should be hidden within the peptide fibril, referred to as "Y-inward" structure. To explore the extraordinary stacking behaviors of the prion GNNQQNY peptides, two fibril models are constructed in a fashion of "Y-outward" and "Y-inward" stackings and then studied in silico to examine their thermodynamic stabilities and disaggregation pathways. The "Y-inward" structure indeed exhibits stronger thermodynamic stability than the "Y-outward" structure, according to potential energy and stacking energy calculations. To show how the peptide fibrils dissociate, we illustrated two disaggregation pathways. A dihedral-based free energy landscape was then calculated to examine the conformational degrees of freedom of the GNNQQNY chains in the "Y-outward" and "Y-inward" structures. Peptide chains lose more configurational entropy in the "Y-inward" structure than in the "Y-outward" structure, indicating that the prion peptides are prone to aggregate in a fashion of "Y-outward" stacking pattern due to its low conformational constraints. The prion-like aggregation of the GNNQQNY peptides into amyloid fibrils is primarily governed by the configuration entropy.

Prions in Microbes: The Least in the Most.

Prions are infectious proteins that mostly replicate in self-propagating amyloid conformations (filamentous protein polymers) and consist of structurally altered normal soluble proteins. Prions can arise spontaneously in the cell without any clear reason and are generally considered fatal disease-causing agents that are only present in mammals. However, after the seminal discovery of two prions, [PSI+] and [URE3], in the eukaryotic model microorganism Saccharomyces cerevisiae, at least ten more prions have been discovered, and their biological and pathological effects on the host, molecular structure, and the relationship between prions and cellular components have been studied. In a filamentous fungus model, Podospora anserina, a vegetative incomparability-related [Het-s] prion that directly triggers cell death during anastomosis (hyphal fusion) was discovered. These prions in eukaryotic microbes have extended our understanding to overcome most fatal human prion/amyloid diseases. A prokaryotic microorganism (Clostridium botulinum) was reported to have a prion analog. The transcriptional regulators of C. botulinum-Rho can be converted into the self-replicating prion form ([RHO-X-C+]), which may affect global transcription. Here, we outline the major issues with prions in microbes and the lessons learned from the relatively uncovered microbial prion world.

Effect of phospholipid liposomes on prion fragment (106-128) amyloid formation.

Misfolding and aggregation of cellular prion protein (PrPc) is a major molecular process involved in the pathogenesis of prion diseases. Here, we studied the aggregation properties of a prion fragment peptide PrP(106-128). The results show that the peptide aggregates in a concentration-dependent manner in an aqueous solution and that the aggregation is sensitive to pH and the preformed amyloid seeds. Furthermore, we show that the zwitterionic POPC liposomes moderately inhibit the aggregation of PrP(106-128), whereas POPC/cholesterol (8:2) vesicles facilitate peptide aggregation likely due to the increase of the lipid packing order and membrane rigidity in the presence of cholesterol. In addition, anionic lipid vesicles of POPG and POPG/cholesterol above a certain concentration accelerate the aggregation of the peptide remarkably. The strong electrostatic interactions between the N-terminal region of the peptide and POPG may constrain the conformational plasticity of the peptide, preventing insertion of the peptide into the inner side of the membrane and thus promoting fibrillation on the membrane surface. The results suggest that the charge properties of the membrane, the composition of the liposomes, and the rigidity of lipid packing are critical in determining peptide adsorption on the membrane surface and the efficiency of the membrane in catalyzing peptide oligomeric nucleation and amyloid formation. The peptide could be used as an improved model molecule to investigate the mechanistic role of the crucial regions of PrP in aggregation in a membrane-rich environment and to screen effective inhibitors to block key interactions between these regions and membranes for preventing PrP aggregation.

Vanadium-Substituted Polyoxometalates Regulate Prion Protein Fragment 106-126 Misfolding by an Oxidation Strategy.

Prion disorders are a group of lethal infectious neurodegenerative diseases caused by the spontaneous aggregation of misfolded prion proteins (PrPSc). The oxidation of such proteins by chemical reagents can significantly modulate their aggregation behavior. Herein, we exploit a series of vanadium-substituted Keggin-type tungsten and molybdenum POMs (W- and Mo-POMs) as chemical tools to oxidize PrP106-126 (denoted as PrP), an ideal model for studying PrPSc. Due to the band gaps being larger than that of Mo-POMs, W-POMs possess higher structural stability and show stronger binding and oxidation effect on PrP. Additionally, the substitution of W/Mo by vanadium elevates the local electron distribution on the bridged O(26) atom, thereby strengthening the hydrogen bonding of POMs with the histidine site. Most importantly, with the number of substituted vanadium increases, the LUMO energy level of POMs decreases, making it easier to accept electrons from methionine. As a result, PW10V2 displays the strongest oxidation on the methionine residue of PrP, leading to an excellent inhibitory effect on PrP aggregation and a significant attenuation on its neurotoxicity.

Eco-evolutionary trade-offs in the dynamics of prion strain competition.

Prion and prion-like molecules are a type of self-replicating aggregate protein that have been implicated in a variety of neurodegenerative diseases. Over recent decades, the molecular dynamics of prions have been characterized both empirically and through mathematical models, providing insights into the epidemiology of prion diseases and the impact of prions on the evolution of cellular processes. At the same time, a variety of evidence indicates that prions are themselves capable of a form of evolution, in which changes to their structure that impact their rate of growth or fragmentation are replicated, making such changes subject to natural selection. Here we study the role of such selection in shaping the characteristics of prions under the nucleated polymerization model (NPM). We show that fragmentation rates evolve to an evolutionary stable value which balances rapid reproduction of PrPSc aggregates with the need to produce stable polymers. We further show that this evolved fragmentation rate differs in general from the rate that optimizes transmission between cells. We find that under the NPM, prions that are both evolutionary stable and optimized for transmission have a characteristic length of three times the critical length below which they become unstable. Finally, we study the dynamics of inter-cellular competition between strains, and show that the eco-evolutionary trade-off between intra- and inter-cellular competition favours coexistence.

Cryo-EM Structure of the Full-length hnRNPA1 Amyloid Fibril.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is a multifunctional RNA-binding protein that is associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis and multisystem proteinopathy. In this study, we have used cryo-electron microscopy to investigate the three-dimensional structure of amyloid fibrils from full-length hnRNPA1 protein. We find that the fibril core is formed by a 45-residue segment of the prion-like low-complexity domain of the protein, whereas the remaining parts of the protein (275 residues) form a fuzzy coat around the fibril core. The fibril consists of two fibril protein stacks that are arranged into a pseudo-21 screw symmetry. The ordered core harbors several of the positions that are known to be affected by disease-associated mutations, but does not encompass the most aggregation-prone segments of the protein. These data indicate that the structures of amyloid fibrils from full-length proteins may be more complex than anticipated by current theories on protein misfolding.

Cellular processing of α-synuclein fibrils results in distinct physiological C-terminal truncations with a major cleavage site at residue Glu 114.

α-synuclein (αS) is an abundant, neuronal protein that assembles into fibrillar pathological inclusions in a spectrum of neurodegenerative diseases that include Lewy body diseases (LBD) and Multiple System Atrophy (MSA). The cellular and regional distributions of pathological inclusions vary widely between different synucleinopathies contributing to the spectrum of clinical presentations. Extensive cleavage within the carboxy (C)-terminal region of αS is associated with inclusion formation, although the events leading to these modifications and the implications for pathobiology are of ongoing study. αS preformed fibrils can induce prion-like spread of αS pathology in both in vitro and animal models of disease. Using C truncation-specific antibodies, we demonstrated here that prion-like cellular uptake and processing of αS preformed fibrils resulted in two major cleavages at residues 103 and 114. A third cleavage product (122 αS) accumulated upon application of lysosomal protease inhibitors. In vitro, both 1-103 and 1-114 αS polymerized rapidly and extensively in isolation and in the presence of full-length αS. 1-103 αS also demonstrated more extensive aggregation when expressed in cultured cells. Furthermore, we used novel antibodies to αS cleaved at residue Glu114, to assess x-114 αS pathology in postmortem brain tissue from patients with LBD and MSA, as well as three different transgenic αS mouse models of prion-like induction. The distribution of x-114 αS pathology was distinct from that of overall αS pathology. These studies reveal the cellular formation and behavior of αS C-truncated at residues 114 and 103 as well as the disease dependent distribution of x-114 αS pathology.

Full-length prion protein incorporated into prion aggregates is a marker for prion strain-specific destabilization of aggregate structure following cellular uptake.

Accumulation of insoluble aggregates of infectious, partially protease-resistant prion protein (PrPD) generated via the misfolding of protease sensitive prion protein (PrPC) into the same infectious conformer, is a hallmark of prion diseases. Aggregated PrPD is taken up and degraded by cells, a process likely involving changes in aggregate structure that can be monitored by accessibility of the N-terminus of full-length PrPD to cellular proteases. We therefore tracked the protease sensitivity of full-length PrPD before and after cellular uptake for two murine prion strains, 22L and 87V. For both strains, PrPD aggregates were less stable following cellular uptake with increased accessibility of the N-terminus to cellular proteases across most aggregate sizes. However, a limited size range of aggregates was able to better protect the N-termini of full-length PrPD, with the N-terminus of 22L-derived PrPD more protected than that of 87V. Interestingly, changes in aggregate structure were associated with minimal changes to the protease-resistant core of PrPD. Our data show that cells destabilize the aggregate quaternary structure protecting PrPD from proteases in a strain-dependent manner, with structural changes exposing protease sensitive PrPD having little effect on the protease-resistant core, and thus conformation, of aggregated PrPD.

N-terminal region of Drosophila melanogaster Argonaute2 forms amyloid-like aggregates.

BACKGROUND: Argonaute proteins play a central role in RNA silencing by forming protein-small RNA complexes responsible for the silencing process. While most Argonaute proteins have a short N-terminal region, Argonaute2 in Drosophila melanogaster (DmAgo2) harbors a long and unique N-terminal region. Previous in vitro biochemical studies have shown that the loss of this region does not impair the RNA silencing activity of the complex. However, an N-terminal mutant of Drosophila melanogaster has demonstrated abnormal RNA silencing activity. To explore the causes of this discrepancy between in vitro and in vivo studies, we investigated the biophysical properties of the region. The N-terminal region is highly rich in glutamine and glycine residues, which is a well-known property for prion-like domains, a subclass of amyloid-forming peptides. Therefore, the possibility of the N-terminal region functioning as an amyloid was tested. RESULTS: Our in silico and biochemical assays demonstrated that the N-terminal region exhibits amyloid-specific properties. The region indeed formed aggregates that were not dissociated even in the presence of sodium dodecyl sulfate. Also, the aggregates enhanced the fluorescence intensity of thioflavin-T, an amyloid detection reagent. The kinetics of the aggregation followed that of typical amyloid formation exhibiting self-propagating activity. Furthermore, we directly visualized the aggregation process of the N-terminal region under fluorescence microscopy and found that the aggregations took fractal or fibril shapes. Together, the results indicate that the N-terminal region can form amyloid-like aggregates. CONCLUSIONS: Many other amyloid-forming peptides have been reported to modulate the function of proteins through their aggregation. Therefore, our findings raise the possibility that aggregation of the N-terminal region regulates the RNA silencing activity of DmAgo2.

The seeding barrier between human and Syrian hamster prion protein amyloid fibrils is determined by ß2-α2 loop sequence elements.

Transmissive spongiform encephalopathies (TSE) are a group of neurodegenerative diseases caused by infectious protein particles, known as prions. Prions are formed from cellular prion proteins (PrP) and can be transmitted between different mammalian species. Subsequently, the host's PrPs are then converted to prions, followed by the onset of TSE. Interspecies prion infectivity is governed by the amino acid sequence differences of PrPs and prions' inability to replicate in a host is termed a species barrier. Here, we investigated the amino acid sequence determinants of species barrier between recombinant human (rHuPrP) and hamster (rShaPrP) prion protein amyloid fibrils. We discovered that a unidirectional species barrier between rShaPrP and rHuPrP amyloid fibrils exists. This barrier stems from the difference of amino acid sequences in the conserved ß2-α2 loop region. Our results revealed that individual amino acids in the ß2-α2 loop region are critical for overcoming the barrier between human and hamster prion protein amyloid fibrils in vitro. Furthermore, the barrier was only possible to observe through aggregation kinetics, as the secondary structure rHuPrP fibrils was not affected by the cross-seeding. Overall, we demonstrated the mechanistic pathway behind this interspecies barrier phenomenon, which increases our understanding of prion-related disease development.

ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification.

Prion-like self-perpetuating conformational conversion of proteins into amyloid aggregates is associated with both transmissible neurodegenerative diseases and non-Mendelian inheritance. The cellular energy currency ATP is known to indirectly regulate the formation, dissolution, or transmission of amyloid-like aggregates by providing energy to the molecular chaperones that maintain protein homeostasis. In this work, we demonstrate that ATP molecules, independent of any chaperones, modulate the formation and dissolution of amyloids from a yeast prion domain (NM domain of Saccharomyces cerevisiae Sup35) and restricts autocatalytic amplification by controlling the amount of fragmentable and seeding-competent aggregates. ATP, at (high) physiological concentrations in the presence of Mg2+, kinetically accelerates NM aggregation. Interestingly, ATP also promotes phase separation-mediated aggregation of a human protein harboring a yeast prion-like domain. We also show that ATP disaggregates preformed NM fibrils in a dose-independent manner. Our results indicate that ATP-mediated disaggregation, unlike the disaggregation by the disaggregase Hsp104, yields no oligomers that are considered one of the critical species for amyloid transmission. Furthermore, high concentrations of ATP delimited the number of seeds by giving rise to compact ATP-bound NM fibrils that exhibited nominal fragmentation by either free ATP or Hsp104 disaggregase to generate lower molecular weight amyloids. In addition, (low) pathologically relevant ATP concentrations restricted autocatalytic amplification by forming structurally distinct amyloids that are found seeding inefficient because of their reduced ß-content. Our results provide key mechanistic underpinnings of concentration-dependent chemical chaperoning by ATP against prion-like transmissions of amyloids.

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.

Shapeshifter TDP-43: Molecular mechanism of structural polymorphism, aggregation, phase separation and their modulators.

TDP-43 is a nucleic acid-binding protein that performs physiologically essential functions and is known to undergo phase separation and aggregation during stress. Initial observations have shown that TDP-43 forms heterogeneous assemblies, including monomer, dimer, oligomers, aggregates, phase-separated assemblies, etc. However, the significance of each assembly of TDP-43 concerning its function, phase separation, and aggregation is poorly known. Furthermore, how different assemblies of TDP-43 are related to each other is unclear. In this review, we focus on the various assemblies of TDP-43 and discuss the plausible origin of the structural heterogeneity of TDP-43. TDP-43 is involved in multiple physiological processes like phase separation, aggregation, prion-like seeding, and performing physiological functions. However, the molecular mechanism behind the physiological process performed by TDP-43 is not well understood. The current review discusses the plausible molecular mechanism of phase separation, aggregation, and prion-like propagation of TDP-43.

A structural basis for prion strain diversity.

Recent cryogenic electron microscopy (cryo-EM) studies of infectious, ex vivo, prion fibrils from hamster 263K and mouse RML prion strains revealed a similar, parallel in-register intermolecular ß-sheet (PIRIBS) amyloid architecture. Rungs of the fibrils are composed of individual prion protein (PrP) monomers that fold to create distinct N-terminal and C-terminal lobes. However, disparity in the hamster/mouse PrP sequence precludes understanding of how divergent prion strains emerge from an identical PrP substrate. In this study, we determined the near-atomic resolution cryo-EM structure of infectious, ex vivo mouse prion fibrils from the ME7 prion strain and compared this with the RML fibril structure. This structural comparison of two biologically distinct mouse-adapted prion strains suggests defined folding subdomains of PrP rungs and the way in which they are interrelated, providing a structural definition of intra-species prion strain-specific conformations.