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Liquid protein condensates produced by phase separation are involved in the spatiotemporal control of cellular functions, while solid fibrous aggregates (amyloids) are associated with diseases and/or manifest as infectious or heritable elements (prions). Relationships between these assemblies are poorly understood. The Saccharomyces cerevisiae release factor Sup35 can produce both fluid liquid-like condensates (e.g., at acidic pH) and amyloids (typically cross-seeded by other prions). We observed acidification-independent formation of Sup35-based liquid condensates in response to hyperosmotic shock in the absence of other prions, both at increased and physiological expression levels. The Sup35 prion domain, Sup35N, is both necessary and sufficient for condensate formation, while the middle domain, Sup35M antagonizes this process. Formation of liquid condensates in response to osmotic stress is conserved within yeast evolution. Notably, condensates of Sup35N/NM protein originated from the distantly related yeast Ogataea methanolica can directly convert to amyloids in osmotically stressed S. cerevisiae cells, providing a unique opportunity for real-time monitoring of condensate-to-fibril transition in vivo by fluorescence microscopy. Thus, cellular fate of stress-induced condensates depends on protein properties and/or intracellular environment., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Prions, the misfolding form of prion proteins, are contagious proteinaceous macromolecules. Recent studies have shown that infectious prion fibrils formed in the brain and non-infectious fibrils formed from recombinant prion protein in a partially denaturing condition have distinct structures. The amyloid core of the in vitro-prepared non-infectious fibrils starts at about residue 160, while that of infectious prion fibrils formed in the brain involves a longer sequence (residues ∼90-230) of structural conversion. The C-terminal truncated prion protein PrP(23-144) can form infectious fibrils under certain conditions and cause disease in animals. In this study, we used cryogenic electron microscopy (cryo-EM) to resolve the structure of hamster sHaPrP(23-144) fibrils prepared at pH 3.7. This 2.88 Å cryo-EM structure has an amyloid core covering residues 94-144. It comprises two protofilaments, each containing five β-strands arranged as a long hairpin plus an N-terminal β-strand. This N-terminal β-strand resides in a positively charged cluster region (named PCC2; sequence 96-111), which interacts with the turn region of the opposite protofilaments' hairpin to stabilize the fibril structure. Interestingly, this sHaPrP(23-144) fibril structure differs from a recently reported structure formed by the human or mouse counterpart at pH 6.5. Moreover, sHaPrP(23-144) fibrils have many structural features in common with infectious prions. Whether this structure is infectious remains to be determined. More importantly, the sHaPrP(23-144) structure is different from the sHaPrP(108-144) fibrils prepared in the same fibrillization buffer, indicating that the N-terminal disordered region, possibly the positively charged cluster, influences the misfolding pathway of the prion protein., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aβ amyloid fibrils from Alzheimer's brain tissue are polymorphic and structurally different from typical in vitro formed Aβ fibrils. Here, we show that brain-derived (ex vivo) fibril structures can be proliferated by seeding in vitro. The proliferation reaction is only efficient for one of the three abundant ex vivo Aβ fibril morphologies, which consists of two peptide stacks, while the inefficiently proliferated fibril morphologies contain four or six peptide stacks. In addition to the seeded fibril structures, we find that de novo nucleated fibril structures can emerge in seeded samples if the seeding reaction is continued over multiple generations. These data imply a competition between de novo nucleation and seed extension and suggest further that seeding favours the outgrowth of fibril morphologies that contain fewer peptide stacks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Methionine/valine polymorphism at position 129 of the human prion protein, huPrP, is tightly associated with the pathogenic phenotype, disease progress, and age of onset of neurodegenerative diseases such as Creutzfeldt-Jakob disease or Fatal Familial Insomnia. This raises the question of whether and how the amino acid type at position 129 influences the structural properties of huPrP, affecting its folding, stability, and amyloid formation behavior. Here, our detailed biophysical characterization of the 129M and 129V variants of recombinant full-length huPrP(23-230) by amyloid formation kinetics, CD spectroscopy, molecular dynamics simulations, and sedimentation velocity analysis reveals differences in their aggregation propensity and oligomer content, leading to deviating pathways for the conversion into amyloid at acidic pH. We determined that the 129M variant exhibits less secondary structure content before amyloid formation and higher resistance to thermal denaturation compared to the 129V variant, whereas the amyloid conformation of both variants shows similar thermal stability. Additionally, our molecular dynamics simulations and rigidity analyses at the atomistic level identify intramolecular interactions responsible for the enhanced monomer stability of the 129M variant, involving more frequent minimum distances between E196 and R156, forming a salt bridge. Removal of the N-terminal half of the 129M full-length variant diminishes its differences compared to the 129V full-length variant and highlights the relevance of the flexible N terminus in huPrP. Taken together, our findings provide insight into structural properties of huPrP and the effects of the amino acid identity at position 129 on amyloid formation behavior., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Amyloid formation continues to be a widely studied area because of its association with numerous diseases, such as Alzheimer's and Parkinson's diseases. Despite a large body of work on protein aggregation and fibril formation, there are still significant gaps in our understanding of the factors that differentiate toxic amyloid formation in vivo from alternative misfolding pathways. In addition to proteins, amyloid fibrils are often associated in their cellular context with several types of molecule, including carbohydrates, polyanions, and lipids. This review focuses in particular on evidence for the presence of lipids in amyloid fibrils and the routes by which those lipids may become incorporated. Chemical analyses of fibril composition, combined with studies to probe the lipid distribution around fibrils, provide evidence that in some cases, lipids have a strong association with fibrils. In addition, amyloid fibrils formed in the presence of lipids have distinct morphologies and material properties. It is argued that lipids are an integral part of many amyloid deposits in vivo, where their presence has the potential to influence the nucleation, morphology, and mechanical properties of fibrils. The role of lipids in these structures is therefore worthy of further study., Competing Interests: Conflict of interest The author declares no conflicts of interest with the contents of this article., (Copyright © 2022 The Author. Published by Elsevier Inc. All rights reserved.)

The study of the aggregation of amyloid proteins is challenging. A new approach to processing dynamic light scattering data was developed and tested using aggregates of the well-known model Sup35NM amyloid. After filtering and calculating the moving averages of autocorrelation functions to reduce impacts of noise, each averaged autocorrelation function is converted to the fibril length distribution via numerical modeling. The processing results were verified using atomic force and scanning electron microscopy data. Analysis of fibril length distribution changes over time gives valuable information about the aggregation process., (© 2022. European Biophysical Societies' Association.)

Amyloids are filamentous protein aggregates that are associated with a number of incurable diseases, termed amyloidoses. Amyloids can also manifest as infectious or heritable particles, known as prions. While just one prion is known in humans and animals, more than ten prion amyloids have been discovered in fungi. The propagation of fungal prion amyloids requires the chaperone Hsp104, though in excess it can eliminate some prions. Even though Hsp104 acts to disassemble prion fibrils, at normal levels it fragments them into multiple smaller pieces, which ensures prion propagation and accelerates prion conversion. Animals lack Hsp104, but disaggregation is performed by the same complement of chaperones that assist Hsp104 in yeast-Hsp40, Hsp70, and Hsp110. Exogenous Hsp104 can efficiently cooperate with these chaperones in animals and promotes disaggregation, especially of large amyloid aggregates, which indicates its potential as a treatment for amyloid diseases. However, despite the significant effects, Hsp104 and its potentiated variants may be insufficient to fully dissolve amyloid. In this review, we consider chaperone mechanisms acting to disassemble heritable protein aggregates in yeast and animals, and their potential use in the therapy of human amyloid diseases.

Several studies recently showed that ex vivo fibrils from patient or animal tissue were structurally different from in vitro formed fibrils from the same polypeptide chain. Analysis of serum amyloid A (SAA) and Aβ-derived amyloid fibrils additionally revealed that ex vivo fibrils were more protease stable than in vitro fibrils. These observations gave rise to the proteolytic selection hypothesis that suggested that disease-associated amyloid fibrils were selected inside the body by their ability to resist endogenous clearance mechanisms. We here show, for more than twenty different fibril samples, that ex vivo fibrils are more protease stable than in vitro fibrils. These data support the idea of a proteolytic selection of pathogenic amyloid fibril morphologies and help to explain why only few amino acid sequences lead to amyloid diseases, although many, if not all, polypeptide chains can form amyloid fibrils in vitro .

Formation of higher-order supramolecular complexes has emerged as a common principle underlying activity of a number of immune and regulated cell-death signalling pathways in animals, plants and fungi. Some of these signalosomes employ functional amyloid motifs in their assembly process. The description of such systems in fungi finds its origin in earlier studies on a fungal prion termed [Het-s], originally identified as a non-Mendelian cytoplasmic infectious element. Janine Beisson has been a key contributor to such early studies. Recent work on this and related systems offers a more integrated view framing this prion in a broader picture including related signalling systems described in animals. We propose here an auto-commentary centred on three recent studies on amyloid signalling in microbes. Collectively, these studies increase our understanding of fold conservation in functional amyloids and the structural basis of seeding, highlight the relation of fungal amyloid motifs to mammalian RHIM (RIP homotypic interaction motif) and expand the concept of Nod-like receptor-based amyloid signalosomes to the prokaryote reign.

The formation of amyloid fibrils is critical for neurodegenerative diseases. Some physiochemical conditions can promote the conversion of proteins from soluble globular shapes into insoluble well-organized amyloid fibrils. The aim of this study was to investigate the effect of temperatures on amyloid fibrils formation in vitro using the protein model of hen egg-white lysozyme (HEWL). The HEWL fibrils were prepared at temperatures of 37, 45, 50 and 57°C in glycine solution of pH 2.2. Under transmission electron microscopy, we found the well-organized HEWL amyloid fibrils at temperatures of 45, 50 and 57°C after 10 days of incubation. Thioflavin T and Congo red florescence assays confirmed that the formation and growth of HEWL fibrils displayed a temperature-dependent increase, and 57°C produced the most amounts. Meanwhile, the surface hydrophobicity of aggregates was greatly increased by ANS binding assay, and β-sheet contents by circular dichroism analysis were increased by 17.8%, 22.0% and 34.9%, respectively. Furthermore, the HEWL fibrils formed at 57°C caused significant cytotoxicity in SH-SY5Y cells after 48 hr exposure, and the cell viability determined by MTT assay was decreased, with 81.35 ± 0.29% for 1 μM, 61.45 ± 2.62% for 2 μM, and 11.58 ± 0.39% (p < .01) for 3 μM. Nuclear staining results also confirmed the apoptosis features. These results suggest that the elevated temperatures could accelerate protein unfolding of the native structure and formation of toxic amyloid fibrils, which can improve understanding the mechanisms of the unfolding and misfolding process of prion protein., (© 2021 The Authors Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

Amyloidosis is a term referring to a group of various protein-misfolding diseases wherein normally soluble proteins form aggregates as insoluble amyloid fibrils. How, or whether, amyloid fibrils contribute to tissue damage in amyloidosis has been the topic of debate. In vitro studies have demonstrated the appearance of small globular oligomeric species during the incubation of amyloid beta peptide (Aβ). Nerve biopsy specimens from patients with systemic amyloidosis have suggested that globular structures similar to Aβ oligomers were generated from amorphous electron-dense materials and later developed into mature amyloid fibrils. Schwann cells adjacent to amyloid fibrils become atrophic and degenerative, suggesting that the direct tissue damage induced by amyloid fibrils plays an important role in systemic amyloidosis. In contrast, there is increasing evidence that oligomers, rather than amyloid fibrils, are responsible for cell death in neurodegenerative diseases, particularly Alzheimer's disease. Disease-modifying therapies based on the pathophysiology of amyloidosis have now become available. Aducanumab, a human monoclonal antibody against the aggregated form of Aβ, was recently approved for Alzheimer's disease, and other monoclonal antibodies, including gantenerumab, solanezumab, and lecanemab, could also be up for approval. As many other agents for amyloidosis will be developed in the future, studies to develop sensitive clinical scales for identifying improvement and markers that can act as surrogates for clinical scales should be conducted.

Prions, proteins that can convert between structurally and functionally distinct states and serve as non-Mendelian mechanisms of inheritance, were initially discovered and only known in eukaryotes, and consequently considered to likely be a relatively late evolutionary acquisition. However, the recent discovery of prions in bacteria and viruses has intimated a potentially more ancient evolutionary origin. Here, we provide evidence that prion-forming domains exist in the domain archaea, the last domain of life left unexplored with regard to prions. We searched for archaeal candidate prion-forming protein sequences computationally, described their taxonomic distribution and phylogeny, and analyzed their associated functional annotations. Using biophysical in vitro assays, cell-based and microscopic approaches, and dye-binding analyses, we tested select candidate prion-forming domains for prionogenic characteristics. Out of the 16 tested, eight formed amyloids, and six acted as protein-based elements of information transfer driving non-Mendelian patterns of inheritance. We also identified short peptides from our archaeal prion candidates that can form amyloid fibrils independently. Lastly, candidates that tested positively in our assays had significantly higher tyrosine and phenylalanine content than candidates that tested negatively, an observation that may help future archaeal prion predictions. Taken together, our discovery of functional prion-forming domains in archaea provides evidence that multiple archaeal proteins are capable of acting as prions-thus expanding our knowledge of this epigenetic phenomenon to the third and final domain of life and bolstering the possibility that they were present at the time of the last universal common ancestor., (Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2021.)

Systemic amyloidosis is defined as a protein misfolding disease in which the amyloid is not necessarily deposited within the same organ that produces the fibril precursor protein. There are different types of systemic amyloidosis, depending on the protein constructing the fibrils. This review will focus on recent advances made in the understanding of the structural basis of three major forms of systemic amyloidosis: systemic AA, AL and ATTR amyloidosis. The three diseases arise from the misfolding of serum amyloid A protein, immunoglobulin light chains or transthyretin. The presented advances in understanding were enabled by recent progress in the methodology available to study amyloid structures and protein misfolding, in particular concerning cryo-electron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy. An important observation made with these techniques is that the structures of previously described in vitro formed amyloid fibrils did not correlate with the structures of amyloid fibrils extracted from diseased tissue, and that in vitro fibrils were typically more protease sensitive. It is thus possible that ex vivo fibrils were selected in vivo by their proteolytic stability., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

In filamentous fungi, NLR-based signalosomes activate downstream membrane-targeting cell death-inducing proteins by a mechanism of amyloid templating. In the species Podospora anserina , two such signalosomes, NWD2/HET-S and FNT1/HELLF, have been described. An analogous system involving a distinct amyloid signaling motif, termed PP, was also identified in the genome of the species Chaetomium globosum and studied using heterologous expression in Podospora anserina The PP motif bears resemblance to the RIP homotypic interaction motif (RHIM) and to RHIM-like motifs controlling necroptosis in mammals and innate immunity in flies. We identify here a third NLR signalosome in Podospora anserina comprising a PP motif and organized as a two-gene cluster encoding an NLR and an HELL domain cell death execution protein termed HELLP. We show that the PP motif region of HELLP forms a prion we term [π] and that [π] prions trigger the cell death-inducing activity of full-length HELLP. We detect no prion cross-seeding between HET-S, HELLF, and HELLP amyloid motifs. In addition, we find that, like PP motifs, RHIMs from human RIP1 and RIP3 kinases are able to form prions in Podospora and that [π] and [Rhim] prions partially cross-seed. Our study shows that Podospora anserina displays three independent cell death-inducing amyloid signalosomes. Based on the described functional similarity between RHIM and PP, it appears likely that these amyloid motifs constitute evolutionarily related cell death signaling modules. IMPORTANCE Amyloids are β-sheet-rich protein polymers that can be pathological or display a variety of biological roles. In filamentous fungi, specific immune receptors activate programmed cell death execution proteins through a process of amyloid templating akin to prion propagation. Among these fungal amyloid signaling sequences, the PP motif stands out because it shows similarity to the RHIM, an amyloid sequence controlling necroptotic cell death in mammals. We characterized an amyloid signaling system comprising a PP motif in the model species Podospora anserina , thus bringing to three the number of independent amyloid signaling cell death pathways described in that species. We then showed that human RHIMs not only propagate as prions in P. anserina but also partially cross-seed with fungal PP prions. These results indicate that, in addition to showing sequence similarity, the PP and RHIM motifs are at least partially functionally related, supporting a model of long-term evolutionary conservation of amyloid signaling mechanisms from fungi to mammals., (Copyright © 2021 Bardin et al.)

Amyloid formation is associated with many incurable diseases. For some of these, sporadic cases are much more common than familial ones. Some reports point to the role of somatic cell mosaicism in these cases via origination of amyloids in a limited number of cells, which can then spread through tissues. However, specific types of sporadic mutations responsible for such effects are unknown. In order to identify mutations capable of increasing the de novo appearance of amyloids, we searched for such mutants in the yeast prionogenic protein Sup35. We introduced to yeast cells an additional copy of the SUP35 gene with mutated amyloidogenic domain and observed that some nonsense mutations increased the incidence of prions by several orders of magnitude. This effect was related to exposure at the C-terminus of an internal amyloidogenic region of Sup35. We also discovered that SUP35 mRNA could undergo splicing, although inefficiently, causing appearance of a shortened Sup35 isoform lacking its functional domain, which was also highly prionogenic. Our data suggest that truncated forms of amyloidogenic proteins, resulting from nonsense mutations or alternative splicing in rare somatic cells, might initiate spontaneous localized formation of amyloids, which can then spread, resulting in sporadic amyloid disease.

Neurodegenerative disorders are frequently associated with β-sheet-rich amyloid deposits. Amyloid-forming proteins can aggregate under different structural conformations known as strains, which can exhibit a prion-like behavior and distinct pathophenotypes. Precise molecular determinants defining strain specificity and cross-strain interactions (cross-seeding) are currently unknown. The HET-s prion protein from the fungus Podospora anserina represents a model system to study the fundamental properties of prion amyloids. Here, we report the amyloid prion structure of HELLF, a distant homolog of the model prion HET-s. We find that these two amyloids, sharing only 17% sequence identity, have nearly identical β-solenoid folds but lack cross-seeding ability in vivo, indicating that prion specificity can differ in extremely similar amyloid folds. We engineer the HELLF sequence to explore the limits of the sequence-to-fold conservation and to pinpoint determinants of cross-seeding and prion specificity. We find that amyloid fold conservation occurs even at an exceedingly low level of identity to HET-s (5%). Next, we derive a HELLF-based sequence, termed HEC, able to breach the cross-seeding barrier in vivo between HELLF and HET-s, unveiling determinants controlling cross-seeding at residue level. These findings show that virtually identical amyloid backbone structures might not be sufficient for cross-seeding and that critical side-chain positions could determine the seeding specificity of an amyloid fold. Our work redefines the conceptual boundaries of prion strain and sheds light on key molecular features concerning an important class of pathogenic agents., Competing Interests: The authors declare no competing interest.

Amyloids and their infectious subset, prions, represent fibrillary aggregates with regular structure. They are formed by proteins that are soluble in their normal state. In amyloid form, all or part of the polypeptide sequence of the protein is resistant to treatment with proteinase K (PK). Amyloids can have structural variants, which can be distinguished by the patterns of their digestion by PK. In this review, we describe and compare studies of the resistant cores of various amyloids from different organisms. These data provide insight into the fine structure of amyloids and their variants as well as raise interesting questions, such as those concerning the differences between amyloids obtained ex vivo and in vitro , as well as the manner in which folding of one region of the amyloid can affect other regions.

The yeast prion [URE3] propagates as a misfolded amyloid form of the Ure2 protein. Propagation of amyloid-based yeast prions requires protein quality control (PQC) factors, and altering PQC abundance or activity can cure cells of prions. Yeast antiprion systems composed of PQC factors act at normal abundance to restrict establishment of the majority of prion variants that arise de novo While these systems are well described, how they or other PQC factors interact with prion proteins remains unclear. To gain insight into such interactions, we identified mutations outside the Ure2 prion-determining region that destabilize [URE3]. Despite residing in the functional domain, 16 of 17 mutants retained Ure2 activity. Four characterized mutations caused rapid loss of [URE3] yet allowed [URE3] to propagate under prion-selecting conditions. Two sensitized [URE3] to Btn2, Cur1, and Hsp42, but in different ways. Two others reduced amyloid formation in vitro Of these, one impaired prion replication and the other apparently impaired transmission. Thus, widely dispersed sites outside a prion's amyloid-forming region can contribute to prion character, and altering such sites can disrupt prion propagation by altering interactions with PQC factors.

Prion protein amyloid aggregates are associated with infectious neurodegenerative diseases, known as transmissible spongiform encephalopathies. Self-replication of amyloid structures by refolding of native protein molecules is the probable mechanism of disease transmission. Amyloid fibril formation and self-replication can be affected by many different factors, including other amyloid proteins and peptides. Mouse prion protein fragments 107-143 (PrP(107-143)) and 89-230 (PrP(89-230)) can form amyloid fibrils. β-sheet core in PrP(89-230) amyloid fibrils is limited to residues ∼160-220 with unstructured N-terminus. We employed chemical kinetics tools, atomic force microscopy and Fourier-transform infrared spectroscopy, to investigate the effects of mouse prion protein fragment 107-143 fibrils on the aggregation of PrP(89-230). The data suggest that amyloid aggregates of a short prion-derived peptide are not able to seed PrP(89-230) aggregation; however, they accelerate the self-replication of PrP(89-230) amyloid fibrils. We conclude that PrP(107-143) fibrils could facilitate the self-replication of PrP(89-230) amyloid fibrils in several possible ways, and that this process deserves more attention as it may play an important role in amyloid propagation.

Amyloids are implicated in many protein misfolding diseases. Amyloid folds, however, also display a range of functional roles particularly in the microbial world. The templating ability of these folds endows them with specific properties allowing their self-propagation and protein-to-protein transmission in vivo. This property, the prion principle, is exploited by specific signaling pathways that use transmission of the amyloid fold as a way to convey information from a receptor to an effector protein. I describe here amyloid signaling pathways involving fungal nucleotide binding and oligomerization domain (NOD)-like receptors that were found to control nonself recognition and programmed cell death processes. Studies on these fungal amyloid signaling motifs stem from the characterization of the fungal [Het-s] prion protein and have led to the identification in fungi but also in multicellular bacteria of several distinct families of signaling motifs, one of which is related to RHIM [receptor-interacting protein (RIP) homotypic interaction motif], an amyloid motif regulating mammalian necroptosis.

Classical views of hereditary mechanisms consider linear nucleic acids, DNA and RNA, as template molecules wherein genetic information is encoded by the sequence of nitrogenous bases. The template principle embodied in the central dogma of molecular biology describes the allowed paths of genetic information transfer from nucleic acids to proteins. The discovery of prions revealed an additional hereditary mechanism whereby the spatial structure is transmitted from one protein molecule to another independently of the sequence of nitrogenous bases in their structural genes. The simultaneous existence of linear (type I) and conformational (type II) templates in one cell inevitably implies their interaction. The review analyzes the current data confirming the idea that protein amyloid transformation may influence the genome stability and considers potential mechanisms of interactions between type I and type II template processes. Special attention is paid to the joint contribution of the two process to tumor "evolution" and the mechanisms of genome destabilization due to amyloid transformation of proteins in Alzheimer's and Parkinson's diseases and Down syndrome.

The accumulation of amyloid Tau aggregates is implicated in Alzheimer's disease (AD) and other tauopathies. Molecular chaperones are known to maintain protein homeostasis. Here, we show that an ATP-dependent human chaperone system disassembles Tau fibrils in vitro We found that this function is mediated by the core chaperone HSC70, assisted by specific cochaperones, in particular class B J-domain proteins and a heat shock protein 110 (Hsp110)-type nucleotide exchange factor (NEF). The Hsp70 disaggregation machinery processed recombinant fibrils assembled from all six Tau isoforms as well as Sarkosyl-resistant Tau aggregates extracted from cell cultures and human AD brain tissues, demonstrating the ability of the Hsp70 machinery to recognize a broad range of Tau aggregates. However, the chaperone activity released monomeric and small oligomeric Tau species, which induced the aggregation of self-propagating Tau conformers in a Tau cell culture model. We conclude that the activity of the Hsp70 disaggregation machinery is a double-edged sword, as it eliminates Tau amyloids at the cost of generating new seeds., Competing Interests: Conflict of interest—No author has an actual or perceived conflict of interest with the contents of this article., (© 2020 Nachman et al.)

Pseudomonas aeruginosa infection elicits the production of cytotoxic amyloids from lung endothelium, yet molecular mechanisms of host-pathogen interaction that underlie the amyloid production are not well understood. We examined the importance of type III secretion system (T3SS) effectors in the production of cytotoxic amyloids. P aeruginosa possessing a functional T3SS and effectors induced the production and release of cytotoxic amyloids from lung endothelium, including beta amyloid, and tau. T3SS effector intoxication was sufficient to generate cytotoxic amyloid release, yet intoxication with exoenzyme Y (ExoY) alone or together with exoenzymes S and T (ExoS/T/Y) generated the most virulent amyloids. Infection with lab and clinical strains engendered cytotoxic amyloids that were capable of being propagated in endothelial cell culture and passed to naïve cells, indicative of a prion strain. Conversely, T3SS-incompetent P aeruginosa infection produced non-cytotoxic amyloids with antimicrobial properties. These findings provide evidence that (1) endothelial intoxication with ExoY is sufficient to elicit self-propagating amyloid cytotoxins during infection, (2) pulmonary endothelium contributes to innate immunity by generating antimicrobial amyloids in response to bacterial infection, and (3) ExoY contributes to the virulence arsenal of P aeruginosa through the subversion of endothelial amyloid host-defense to promote a lung endothelial-derived cytotoxic proteinopathy., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The unseeded aggregation of superoxide dismutase-1 (SOD1) into amyloid-like fibrils occurs stochastically in vitro and in vivo , that is, isolated populations of SOD1 proteins (within microplate wells or living cells) self-assemble into amyloid at rates that span a probability distribution. This stochasticity has been attributed to variable degrees of monomer depletion by competing pathways of amorphous and fibrillar aggregation (inter alia). Here, microplate-based thioflavin-T (ThT) fluorescence assays were performed at high iteration (∼300) to establish whether this observed stochasticity persists when progenitor ("parent") SOD1 fibrils are used to seed the formation of multiple generations of progeny fibrils (daughter, granddaughter, and great-granddaughter fibrils). Populations of progenitor fibrils formed stochastically at different rates and fluorescence intensity, however, progeny fibrils formed at more similar rates regardless of the formation rate of the progenitor fibril. For example, populations of progenitor fibrils that formed with a lag time of ∼30 h or ∼15 h both produced progeny fibrils with lag times of ∼8 h. Likewise, populations of progenitor fibrils with high or low maximum fluorescence (e.g., ∼450 or ∼75 A.U.) both produced progeny fibrils with more similar maximum fluorescence (∼125 A.U.). The rate of propagation was found to be more dependent on monomer concentration than seed concentration. These results can be rationalized by classical rate laws for primary nucleation and monomer-dependent secondary nucleation. We also find that the seeding propensity of some "families" of in vitro grown fibrils exhibit a finite lifetime (similar to that observed in the seeding of small molecule crystals and colloids). The single biological takeaway of this study is that the concentration of native SOD1 in a cell can have a stronger effect on rates of seeded aggregation than the concentration of prion-like seed that infected the cell.

The prion-like spreading and accumulation of specific protein aggregates appear to be central to the pathogenesis of many human diseases, including Alzheimer's and Parkinson's. Accumulating evidence indicates that inoculation of tissue extracts from diseased individuals into suitable experimental animals can in many cases induce the aggregation of the disease-associated protein, as well as related pathological lesions. These findings, together with the history of the prion field, have raised the questions about whether such disease-associated protein aggregates are transmissible between humans by casual or iatrogenic routes, and, if so, do they propagate enough in the new host to cause disease? These practical considerations are important because real, and perhaps even only imagined, risks of human-to-human transmission of diseases such as Alzheimer's and Parkinson's may force costly changes in clinical practice that, in turn, are likely to have unintended consequences. The prion field has taught us that a single protein, PrP, can aggregate into forms that can propagate exponentially in vitro, but range from being innocuous to deadly when injected into experimental animals in ways that depend strongly on factors such as conformational subtleties, routes of inoculation, and host responses. In assessing the hazards posed by various disease-associated, self-propagating protein aggregates, it is imperative to consider both their actual transmissibilities and the pathological consequences of their propagation, if any, in recipient hosts.

Amyloids are self-perpetuating protein aggregates causing neurodegenerative diseases in mammals. Prions are transmissible protein isoforms (usually of amyloid nature). Prion features were recently reported for various proteins involved in amyloid and neural inclusion disorders. Heritable yeast prions share molecular properties (and in the case of polyglutamines, amino acid composition) with human disease-related amyloids. Fundamental protein quality control pathways, including chaperones, the ubiquitin proteasome system and autophagy are highly conserved between yeast and human cells. Crucial cellular proteins and conditions influencing amyloids and prions were uncovered in the yeast model. The treatments available for neurodegenerative amyloid-associated diseases are few and their efficiency is limited. Yeast models of amyloid-related neurodegenerative diseases have become powerful tools for high-throughput screening for chemical compounds and FDA-approved drugs that reduce aggregation and toxicity of amyloids. Although some environmental agents have been linked to certain amyloid diseases, the molecular basis of their action remains unclear. Environmental stresses trigger amyloid formation and loss, acting either via influencing intracellular concentrations of the amyloidogenic proteins or via heterologous inducers of prions. Studies of environmental and physiological regulation of yeast prions open new possibilities for pharmacological intervention and/or prophylactic procedures aiming on common cellular systems rather than the properties of specific amyloids.

Prion diseases are transmissible fatal neurodegenerative disorders affecting humans and other mammals. The disease transmission can occur between different species but is limited by the sequence homology between host and inoculum. The crucial molecular event in the progression of this disease is prion formation, starting from the conformational conversion of the normal, membrane-anchored prion protein (PrP C ) into the misfolded, β-sheet-rich and aggregation-prone isoform (PrP Sc ), which then self-associates into the infectious amyloid form called prion. Amyloid is the aggregate formed from one-dimensional protein association. As amyloid formation is a key hallmark in prion pathogenesis, studying which segments in prion protein are involved in the amyloid formation can provide molecular details in the cross-species transmission barrier of prion diseases. However, due to the difficulties of studying protein aggregates, very limited knowledge about prion structure or prion formation was disclosed by now. In this study, cross-seeding assay was used to identify the segments involved in the amyloid fibril formation of full-length hamster prion protein, SHaPrP(23-231). Our results showed that the residues in the segments 108-127, 172-194 (helix 2 in PrP C ) and 200-227 (helix 3 in PrP C ) are in the amyloid core of hamster prion fibrils. The segment 127-143, but not 107-126 (which corresponds to hamster sequence 108-127), was previously reported to be involved in the amyloid core of full-length mouse prion fibrils. Our results indicate that hamster prion protein and mouse prion protein use different segments to form the amyloid core in amyloidogenesis. The sequence-dependent core formation can be used to explain the seeding barrier between mouse and hamster.

The abnormal protein aggregates in progressive neurodegenerative disorders, such as Alzheimer's, Parkinson's and prion diseases, adopt a generic structural form called amyloid fibrils. The precise amyloid fold can differ between patients and these differences are related to distinct neuropathological phenotypes of the diseases. A key focus in current research is the molecular mechanism governing such structural diversity, known as amyloid polymorphism. In this review, we focus on our recent work on recombinant prion protein (recPrP) and the use of pressure as a variable for perturbing protein structure. We suggest that the amyloid polymorphism is based on volumetric features. Accordingly, pressure is the thermodynamic parameter that fits best to exploit volume differences within the states of a chemical reaction, since it shifts the equilibrium constant to the state that has the smaller volume. In this context, there are analogies with the process of correct protein folding, the high pressure-induced effects of which have been studied for more than a century and which provides a valuable source of inspiration. We present a short overview of this background and review our recent results regarding the folding, misfolding, and aggregation-disaggregation of recPrP under pressure. We present preliminary experiments aimed at identifying how prion protein fibril diversity is related to the quaternary structure by using pressure and varying protein sequences. Finally, we consider outstanding questions and testable mechanistic hypotheses regarding the multiplicity of states in the amyloid fold.

The mechanism of prion strain diversity remains unsolved. Investigation of inheritance and diversification of protein-based pathogenic information demands the identification of the detailed structures of abnormal isoforms of the prion protein (PrP Sc ); however, achieving purification is difficult without affecting infectivity. Similar prion-like properties are recognized also in other disease-associated in-register parallel β-sheet amyloids including Tau and α-synuclein (αSyn) amyloids. Investigations into structures of those amyloids via solid-state nuclear magnetic resonance spectroscopy and cryo-electron microscopy recently made remarkable advances due to their relatively small sizes and lack of post-translational modifications. Herein, we review advances regarding pathogenic amyloids, particularly Tau and αSyn, and discuss implications about strain diversity mechanisms of prion/PrP Sc from the perspective that PrP Sc is an in-register parallel β-sheet amyloid. Additionally, we present our recent data of molecular dynamics simulations of αSyn amyloid, which suggest significance of compatibility between β-sheet propensities of the substrate and local structures of the template for stability of amyloid structures. Detailed structures of αSyn and Tau amyloids are excellent models of pathogenic amyloids, including PrP Sc , to elucidate strain diversity and pathogenic mechanisms.

The fold of a protein determines its function and its misfolding can result in loss-of-function defects. In addition, for certain proteins their misfolding can lead to gain-of-function toxicities resulting in protein misfolding diseases such as Alzheimer's, Parkinson's, or the prion diseases. In all of these diseases one or more proteins misfold and aggregate into disease-specific assemblies, often in the form of fibrillar amyloid deposits. Most, if not all, protein misfolding diseases share a fundamental molecular mechanism that governs the misfolding and subsequent aggregation. A wide variety of experimental methods have contributed to our knowledge about misfolded protein aggregates, some of which are briefly described in this review. The misfolding mechanism itself is difficult to investigate, as the necessary timescale and resolution of the misfolding events often lie outside of the observable parameter space. Molecular dynamics simulations fill this gap by virtue of their intrinsic, molecular perspective and the step-by-step iterative process that forms the basis of the simulations. This review focuses on molecular dynamics simulations and how they combine with experimental analyses to provide detailed insights into protein misfolding and the ensuing diseases., (Copyright © 2020 Elsevier Inc. All rights reserved.)

α-Synuclein (αsyn) aggregates into toxic fibrils in multiple neurodegenerative diseases where these fibrils form characteristic pathological inclusions such as Lewy bodies (LBs). The mechanisms initiating αsyn aggregation into fibrils are unclear, but ubiquitous post-translational modifications of αsyn present in LBs may play a role. Specific C-terminally (C)-truncated forms of αsyn are present within human pathological inclusions and form under physiological conditions likely in lysosome-associated pathways, but the roles for these C-truncated forms of αsyn in inclusion formation and disease are not well understood. Herein, we characterized the in vitro aggregation properties, amyloid fibril structures, and ability to induce full-length (FL) αsyn aggregation through prion-like mechanisms for eight of the most common physiological C-truncated forms of αsyn (1-115, 1-119, 1-122, 1-124, 1-125, 1-129, 1-133, and 1-135). In vitro , C-truncated αsyn aggregated more readily than FL αsyn and formed fibrils with unique morphologies. The presence of C-truncated αsyn potentiated aggregation of FL αsyn in vitro through co-polymerization. Specific C-truncated forms of αsyn in cells also exacerbated seeded aggregation of αsyn. Furthermore, in primary neuronal cultures, co-polymers of C-truncated and FL αsyn were potent prion-like seeds, but polymers composed solely of the C-truncated protein were not. These experiments indicated that specific physiological C-truncated forms of αsyn have distinct aggregation properties, including the ability to modulate the prion-like aggregation and seeding activity of FL αsyn. Proteolytic formation of these C-truncated species may have an important role in both the initiation of αsyn pathological inclusions and further progression of disease with strain-like properties., (© 2018 Sorrentino et al.)

Prion diseases are fatal neurodegenerative diseases associated with structural conversion of α-helical prion protein (PrP) into its β-sheet rich isoform (PrP Sc ). Previous genetic analyses have indicated that several amino acid residues involved in the hydrophobic core of PrP (such as V180, F198, and V210) play a critical role in the development of prion diseases. To understand how these hydrophobic residues would contribute to the α-to-β conversion process of PrP, we substituted the V210 residue with bulkier (V210F, V210I, and V210L), smaller (V210A), and charged amino acids (V210K) and characterized its effects. Interestingly, although most of the mutations had little or no effect on the biochemical properties of PrP, the V210K mutation induced structural conversion of PrP into a β-structure. The β-inducing effect was prominent and observed even under a physiological condition (i.e., in the absence of denaturant, acidic pH, reducing agent, and high temperature) in contrast to the disease-associated mutations in the PrP gene. We also examined structural features of V210K PrP using guanidine-hydrochloride unfolding, dynamic light scattering, 8-anilino-1-naphthalene sulfonate fluorescence, and electron microscopy, and revealed that V210K PrP assembles into a non-fibrillar β-rich oligomer. Thus, the α-to-β conversion can be induced by introduction of a charged residue into the hydrophobic core, which provide novel insight into the structural dynamics of PrP., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The cellular prion protein (PrP C ) can act as a cell-surface receptor for β-amyloid (Aβ) peptide; however, a role for PrP C in the pathogenesis of Alzheimer's disease (AD) is contested. Here, we expressed a range of Aβ isoforms and PrP C in the Drosophila brain. We found that co-expression of Aβ and PrP C significantly reduces the lifespan, disrupts circadian rhythms, and increases Aβ deposition in the fly brain. In contrast, under the same conditions, expression of Aβ or PrP C individually did not lead to these phenotypic changes. In vitro studies revealed that substoichiometric amounts of PrP C trap Aβ as oligomeric assemblies and fragment-preformed Aβ fibers. The ability of membrane-anchored PrP C to trap Aβ as cytotoxic oligomers at the membrane surface and fragment inert Aβ fibers suggests a mechanism by which PrP C exacerbates Aβ deposition and pathogenic phenotypes in the fly, supporting a role for PrP C in AD. This study provides a second animal model linking PrP C expression with Aβ toxicity and supports a role for PrP C in AD pathogenesis. Blocking the interaction of Aβ and PrP C represents a potential therapeutic strategy., (© 2018 Younan et al.)

Amyloids are unbranched protein fibrils with a characteristic spatial structure. Although the amyloids were first described as protein deposits that are associated with the diseases, today it is becoming clear that these protein fibrils play multiple biological roles that are essential for different organisms, from archaea and bacteria to humans. The appearance of amyloid, first of all, causes changes in the intracellular quantity of the corresponding soluble protein(s), and at the same time the aggregate can include other proteins due to different molecular mechanisms. The co-aggregation may have different consequences even though usually this process leads to the depletion of a functional protein that may be associated with different diseases. The protein co-aggregation that is related to functional amyloids may mediate important biological processes and change of protein functions. In this review, we survey the known examples of the amyloid-related co-aggregation of proteins, discuss their pathogenic and functional roles, and analyze methods of their studies from bacteria and yeast to mammals. Such analysis allow for us to propose the following co-aggregation classes: (i) titration: deposition of soluble proteins on the amyloids formed by their functional partners, with such interactions mediated by a specific binding site; (ii) sequestration: interaction of amyloids with certain proteins lacking a specific binding site; (iii) axial co-aggregation of different proteins within the same amyloid fibril; and, (iv) lateral co-aggregation of amyloid fibrils, each formed by different proteins.

Tauopathies feature progressive accumulation of tau amyloids. Pathology may begin when these amplify from a protein template, or seed, whose structure is unknown. We have purified and characterized distinct forms of tau monomer-inert (M i ) and seed-competent (M s ). Recombinant M s triggered intracellular tau aggregation, induced tau fibrillization in vitro, and self-assembled. M s from Alzheimer's disease also seeded aggregation and self-assembled in vitro to form seed-competent multimers. We used crosslinking with mass spectrometry to probe structural differences in M i vs. M s . Crosslinks informed models of local peptide structure within the repeat domain which suggest relative inaccessibility of residues that drive aggregation (VQIINK/VQIVYK) in M i , and exposure in M s . Limited proteolysis supported this idea. Although tau monomer has been considered to be natively unstructured, our findings belie this assumption and suggest that initiation of pathological aggregation could begin with conversion of tau monomer from an inert to a seed-competent form., Competing Interests: HM, DC, OM, KR, AS, XL, RP, DC, HM, LJ, MD No competing interests declared, (© 2018, Mirbaha et al.)

Amyloid fibrils are filamentous protein aggregates associated with the pathogenesis of a wide variety of human diseases. The formation of such aggregates typically follows nucleation-dependent kinetics, wherein the assembly and structural conversion of amyloidogenic proteins into oligomeric aggregates (nuclei) is the rate-limiting step of the overall reaction. In this study, we sought to gain structural insights into the oligomeric nuclei of the human prion protein (PrP) by preparing a series of deletion mutants lacking 14-44 of the C-terminal 107 residues of PrP and examined the kinetics and thermodynamics of these mutants in amyloid formation. An analysis of the experimental data using the concepts of the Φ-value analysis indicated that the helix 2 region (residues 168-196) acquires an amyloid-like β-sheet during nucleation, whereas the other regions preserves a relatively disordered structure in the nuclei. This finding suggests that the helix 2 region serves as the nucleation site for the assembly of amyloid fibrils.-Honda, R., Kuwata, K. Evidence for a central role of PrP helix 2 in the nucleation of amyloid fibrils.

A crucial stage in the origin of life was the emergence of the first molecular entity that was able to replicate, transmit information, and evolve on the early Earth. The amyloid world hypothesis posits that in the pre-RNA era, information processing was based on catalytic amyloids. The self-assembly of short peptides into β-sheet amyloid conformers leads to extraordinary structural stability and novel multifunctionality that cannot be achieved by the corresponding nonaggregated peptides. The new functions include self-replication, catalytic activities, and information transfer. The environmentally sensitive template-assisted replication cycles generate a variety of amyloid polymorphs on which evolutive forces can act, and the fibrillar assemblies can serve as scaffolds for the amyloids themselves and for ribonucleotides proteins and lipids. The role of amyloid in the putative transition process from an amyloid world to an amyloid-RNA-protein world is not limited to scaffolding and protection: the interactions between amyloid, RNA, and protein are both complex and cooperative, and the amyloid assemblages can function as protometabolic entities catalyzing the formation of simple metabolite precursors. The emergence of a pristine amyloid-based in-put sensitive, chiroselective, and error correcting information-processing system, and the evolvement of mutualistic networks were, arguably, of essential importance in the dynamic processes that led to increased complexity, organization, compartmentalization, and, eventually, the origin of life.

Amyloid fibrils are protein homopolymers that adopt diverse cross-β conformations. Some amyloid fibrils are associated with the pathogenesis of devastating neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease. Conversely, functional amyloids play beneficial roles in melanosome biogenesis, long-term memory formation and release of peptide hormones. Here, we showcase advances in our understanding of amyloid assembly and structure, and how distinct amyloid strains formed by the same protein can cause distinct neurodegenerative diseases. We discuss how mutant steric zippers promote deleterious amyloidogenesis and aberrant liquid-to-gel phase transitions. We also highlight effective strategies to combat amyloidogenesis and related toxicity, including: (1) small-molecule drugs (e.g. tafamidis) to inhibit amyloid formation or (2) stimulate amyloid degradation by the proteasome and autophagy, and (3) protein disaggregases that disassemble toxic amyloid and soluble oligomers. We anticipate that these advances will inspire therapeutics for several fatal neurodegenerative diseases., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The search for novel pathological and functional amyloids represents one of the most important tasks of contemporary biomedicine. Formation of pathological amyloid fibrils in the aging brain causes incurable neurodegenerative disorders such as Alzheimer's, Parkinson's Huntington's diseases. At the same time, a set of amyloids regulates vital processes in archaea, prokaryotes and eukaryotes. Our knowledge of the prevalence and biological significance of amyloids is limited due to the lack of universal methods for their identification. Here, using our original method of proteomic screening PSIA-LC-MALDI, we identified a number of proteins that form amyloid-like detergent-resistant aggregates in Saccharomyces cerevisiae. We revealed in yeast strains of different origin known yeast prions, prion-associated proteins, and a set of proteins whose amyloid properties were not shown before. A substantial number of the identified proteins are cell wall components, suggesting that amyloids may play important roles in the formation of this extracellular protective sheath. Two proteins identified in our screen, Gas1 and Ygp1, involved in biogenesis of the yeast cell wall, were selected for detailed analysis of amyloid properties. We show that Gas1 and Ygp1 demonstrate amyloid properties both in vivo in yeast cells and using the bacteria-based system C-DAG. Taken together, our data show that this proteomic approach is very useful for identification of novel amyloids.

Fibrous cross-β aggregates (amyloids) and their transmissible forms (prions) cause diseases in mammals (including humans) and control heritable traits in yeast. Initial nucleation of a yeast prion by transiently overproduced prion-forming protein or its (typically, QN-rich) prion domain is efficient only in the presence of another aggregated (in most cases, QN-rich) protein. Here, we demonstrate that a fusion of the prion domain of yeast protein Sup35 to some non-QN-rich mammalian proteins, associated with amyloid diseases, promotes nucleation of Sup35 prions in the absence of pre-existing aggregates. In contrast, both a fusion of the Sup35 prion domain to a multimeric non-amyloidogenic protein and the expression of a mammalian amyloidogenic protein that is not fused to the Sup35 prion domain failed to promote prion nucleation, further indicating that physical linkage of a mammalian amyloidogenic protein to the prion domain of a yeast protein is required for the nucleation of a yeast prion. Biochemical and cytological approaches confirmed the nucleation of protein aggregates in the yeast cell. Sequence alterations antagonizing or enhancing amyloidogenicity of human amyloid-β (associated with Alzheimer's disease) and mouse prion protein (associated with prion diseases), respectively, antagonized or enhanced nucleation of a yeast prion by these proteins. The yeast-based prion nucleation assay, developed in our work, can be employed for mutational dissection of amyloidogenic proteins. We anticipate that it will aid in the identification of chemicals that influence initial amyloid nucleation and in searching for new amyloidogenic proteins in a variety of proteomes., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Misfolding of the normally folded prion protein of mammals (PrP C ) into infectious fibrils causes a variety of diseases, from scrapie in sheep to chronic wasting disease (CWD) in cervids. The misfolded form of PrP C , termed PrP Sc , or in this case PrP CWD , interacts with PrP C to create more PrP CWD . This process is not clearly defined but is affected by the presence and interactions of biotic and abiotic cofactors. These include nucleic acids, lipids, glycosylation, pH, and ionic character. PrP C has been shown to act as a copper-binding protein in vivo, though it also binds to other divalents as well. The significance of this action has not been conclusively elucidated. Previous reports have shown that metal binding sites occur throughout the N-terminal region of PrP C . Other cations like manganese have also been shown to affect PrP C abundance in a transcript-independent fashion. Here, we examined the ability of different divalent cations to influence the stability and in vitro conversion of two variants of PrP from elk (L/M132, 26-234). We find that copper and zinc de-stabilize PrP. We also find that PrP M132 exhibits a greater degree of divalent cation induced destabilization than L132. This supports findings that leucine at position 132 confers resistance to CWD, while M132 is susceptible. However, in vitro conversion of PrP is equally suppressed by either copper or zinc, in both L132 and M132 backgrounds. This report demonstrates the complex importance of ionic character on the PrP C folding pathway selection on the route to PrP Sc formation.

Lipid membranes play a fundamental role in the pathological development of protein misfolding diseases. Several pieces of evidence suggest that the lipid membrane could act as a catalytic surface for protein aggregation. Furthermore, a leading theory indicates the interaction between the cell membrane and misfolded oligomer species as the responsible for cytotoxicity, hence, for neurodegeneration in disorders such as Alzheimer's and Parkinson's disease. The definition of the mechanisms that drive the interaction between pathological protein aggregates and plasma membrane is fundamental for the development of effective therapies for a large class of diseases. Atomic force microscopy (AFM) has been employed to study how amyloid aggregates affect the cell physiological properties. Considerable efforts were spent to characterize the interaction with model systems, i.e., planar supported lipid bilayers, but some works also addressed the problem directly on living cells. Here, an overview of the main works involving the use of the AFM on both model system and living cells will be provided. Different kind of approaches will be presented, as well as the main results derived from the AFM analysis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Prion and some other incurable human neurodegenerative diseases are associated with misfolding of specific proteins, followed by the formation of amyloids. Despite the widespread usage of the transmission electron and of the atomic force microscopy for studing such amyloids, many related methodological issues still have not been studied until now. Here, we consider one of the first amyloids found in Saccharomyces cerevisiae yeast, i.e. Sup35NMp, to study the adsorption of monomeric protein and its fibrils on the surface of mica, silica, gold and on formvar film. Comparison of linear characteristics of these units calculated by processing of images obtained by the atomic force, transmission and scanning electron microscopy was carried out. The minimal number of measurements of fibril diameters to obtain the values in a given confidence interval were determined. We investigated the film formed by monomeric protein on mica surface, which veiled some morphology features of fibrils. Besides, we revealed that parts of the Sup35NMp excluded from the fibril core can form a wide "coat". The length of the protein forming the core of the fibrils was estimated., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Resveratrol and its derivatives have been shown to display beneficial effects to neurodegenerative diseases. However, the molecular mechanism of resveratrol and its derivatives on prion conformational conversion is poorly understood. In this work, the interaction mechanism between prion and resveratrol as well as its derivatives was investigated using steady-state fluorescence quenching, Thioflavin T binding assay, Western blotting, and molecular dynamics simulation. Protein fluorescence quenching method and Thioflavin T assay revealed that resveratrol and its derivatives could interact with prion and interrupt prion fibril formation. Molecular dynamics simulation results indicated that resveratrol can stabilize the PrP 127-147 peptide mainly through π-π stacking interactions between resveratrol and Tyr128. The hydrogen bonds interactions between resveratrol and the PrP 127-147 peptide could further reduce the flexibility and the propensity to aggregate. The results of this study not only can provide useful information about the interaction mechanism between resveratrol and prion, but also can provide useful clues for further design of new inhibitors inhibiting prion aggregation.

The molecular architecture of amyloids formed in vivo can be interrogated using luminescent conjugated oligothiophenes (LCOs), a unique class of amyloid dyes. When bound to amyloid, LCOs yield fluorescence emission spectra that reflect the 3D structure of the protein aggregates. Given that synthetic amyloid-β peptide (Aβ) has been shown to adopt distinct structural conformations with different biological activities, we asked whether Aβ can assume structurally and functionally distinct conformations within the brain. To this end, we analyzed the LCO-stained cores of β-amyloid plaques in postmortem tissue sections from frontal, temporal, and occipital neocortices in 40 cases of familial Alzheimer's disease (AD) or sporadic (idiopathic) AD (sAD). The spectral attributes of LCO-bound plaques varied markedly in the brain, but the mean spectral properties of the amyloid cores were generally similar in all three cortical regions of individual patients. Remarkably, the LCO amyloid spectra differed significantly among some of the familial and sAD subtypes, and between typical patients with sAD and those with posterior cortical atrophy AD. Neither the amount of Aβ nor its protease resistance correlated with LCO spectral properties. LCO spectral amyloid phenotypes could be partially conveyed to Aβ plaques induced by experimental transmission in a mouse model. These findings indicate that polymorphic Aβ-amyloid deposits within the brain cluster as clouds of conformational variants in different AD cases. Heterogeneity in the molecular architecture of pathogenic Aβ among individuals and in etiologically distinct subtypes of AD justifies further studies to assess putative links between Aβ conformation and clinical phenotype., Competing Interests: The authors declare no conflict of interest.

Transmissible amyloid particles called prions are associated with infectious prion diseases in mammals and inherited phenotypes in yeast. All amyloid aggregates can give rise to potentially infectious seeds that accelerate their growth. Why some amyloid seeds are highly infectious prion particles while others are less infectious or even inert, is currently not understood. To address this question, we analyzed the suprastructure and dimensions of synthetic amyloid fibrils assembled from the yeast ( Saccharomyces cerevisiae ) prion protein Sup35NM. We then quantified the ability of these particles to induce the [ PSI + ] prion phenotype in cells. Our results show a striking relationship between the length distribution of the amyloid fibrils and their ability to induce the heritable [ PSI + ] prion phenotype. Using a simple particle size threshold model to describe transfection activity, we explain how dimensions of amyloid fibrils are able to modulate their infectious potential as prions.

Amyloids represent protein fibrils that have highly ordered structure with unique physical and chemical properties. Amyloids have long been considered lethal pathogens that cause dozens of incurable diseases in humans and animals. Recent data show that amyloids may not only possess pathogenic properties but are also implicated in the essential biological processes in a variety of prokaryotes and eukaryotes. Functional amyloids have been identified in archaea, bacteria, fungi, and animals, including humans. Plants are one of the most poorly studied groups of organisms in the field of amyloid biology. Although amyloid properties have not been shown under native conditions for any plant protein, studies demonstrating amyloid properties for a set of plant proteins in vitro or in heterologous systems in vivo have been published in recent years. In this review, we systematize the data on the amyloidogenic proteins of plants and their functions and discuss the perspectives of identifying novel amyloids using bioinformatic and proteomic approaches.

Prions are misfolded, aggregated, infectious proteins found in a range of organisms from mammals to bacteria. In mammals, prion formation is difficult to study because misfolding and aggregation take place prior to symptom presentation. The study of the yeast prion [PSI + ], which is the misfolded infectious form of Sup35p, provides a tractable system to monitor prion formation in real time. Recently, we showed that the de novo formation of prion aggregates begins with the appearance of highly mobile cytoplasmic foci, called early foci, which assemble into larger ring or dot structures. We also observed SDS-resistant oligomers during formation, and lysates containing newly formed oligomers can convert [psi - ] cells to the [PSI + ] state, suggesting that these oligomers have infectious potential. Here, we further characterize two aspects of prion formation: spatial sequestration of early foci and oligomerization of endogenous Sup35p. Our data provides important insights into the process of prion formation and explores the minimal oligomer requirement for infectivity.

TRIT1 is a highly conserved tRNA isopentenyl transferase that modifies a subset of tRNAs in human cells and is a candidate tumor suppressor in lung cancer in certain ethnic populations. The yeast homologue, Mod5, has similar tRNA-modifying functions in the cytoplasm and is required for the transcriptional silencing activity of RNA polymerase II promoters near tRNA genes in the nucleus, a phenomenon termed tRNA gene mediated (tgm) silencing. Furthermore, Mod5 can fold into amyloid fibers in vitro and in vivo, which confers resistance to certain fungicides in yeast. Since TRIT1 complements both tRNA modifying and tgm-silencing activities in yeast where the Mod5 gene has been deleted, it seemed possible that TRIT1 might also have amyloid-forming capabilities. Here we show that TRIT1, like Mod5, directly binds to tRNAs that are both substrate and non-substrates for modification with similar affinity, and to an unstructured, non-tRNA. Binding appears to involve distinct protein-RNA multimers which decrease in electrophoretic mobility as the protein to RNA ratio increases. Furthermore, we characterize TRIT1 as a novel human amyloid fiber forming protein. We discuss these data in light of TRIT1's functional roles and possible implications for disease., (Copyright © 2016. Published by Elsevier B.V.)

Amyloid folds not only represent the underlying cause of a large class of human diseases but also display a variety of functional roles both in prokaryote and eukaryote organisms. Among these roles is a recently-described activity in signal transduction cascades functioning in host defense and programmed cell death and involving Nod-like receptors (NLRs). In different fungal species, prion amyloid folds convey activation signals from a receptor protein to an effector domain by an amyloid templating and propagation mechanism. The discovery of these amyloid signaling motifs derives from the study of [Het-s], a fungal prion of the species Podospora anserina . These signaling pathways are typically composed of two basic components encoded by adjacent genes, the NLR receptor bearing an amyloid motif at the N-terminal end and a cell death execution protein with a HeLo pore-forming domain bearing a C-terminal amyloid motif. Activation of the NLR receptor allows for amyloid folding of the N-terminal amyloid motifs which then template trans-conformation of the homologous motif in the cell death execution protein. A variety of such motifs, which differ by their sequence signature, have been described in fungi. Among them, the PP-motif bears resemblance with the RHIM amyloid motif involved in the necroptosis pathway in mammals suggesting an evolutionary conservation of amyloid signaling from fungi to mammals.