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The transition of β-barrel proteins from a soluble to an amyloid form is biologically significant in some cases but may lead to functional activity loss. In particular, odorant-binding proteins' (OBPs) fibrils are unable to bind odorant molecules potentially contributing to olfactory dysfunction. As shown previously, OBPs' fibrillogenesis is initiated by uncoupling of protein C-terminal fragment from the β-barrel and exposing amyloidogenic sites. However, further structural transformations of OBPs are not fully understood. Here we first identified two intermediate aggregated states of OBPs: one formed by intact β-barrels, nontoxic to mammalian cells and easily dissociated by boiling and detergents; the other is prefibrillar, formed by degraded β-barrels with restructured β-strands, having almost comparable cytotoxicity but lower stability compared to mature amyloids. Obtained results revealed the β-barrel "opening" and alignment in register of its β-strands early in aggregation. Comparing the aggregation processes of bovine OBP and its mutant variant differing in the C-terminal fragment mobility showed the influence of its dynamics/orientation on the conjugation of amyloidogenic regions triggering fibril formation. The characterized properties and formation mechanisms of intermediate states in amyloidogenesis of β-barrel proteins are relevant for finding ways to prevent pathological aggregation and identifying ways to regulate the physiological fibrils assembly/disassembly., 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 B.V. All rights reserved.)

Over the past decade, the greatest promise for treating severe and currently incurable systemic and neurodegenerative diseases has turned to agents capable of effectively degrading pathological amyloid deposits without causing side effects. Specifically, amyloid destruction observed in immunotherapy is hypothesized to occur through activation of proteolytic enzymes. This study examines poorly understood effects of an immune enzyme, extracellular matrix metalloproteinase-9 (MMP9), on amyloids associated with Alzheimer's and Parkinson's diseases, lysozyme, insulin, and dialysis-related amyloidoses. The study establishes the universality of MMP9's effect on various amyloids, with its efficacy largely depending on the fibrillar cluster size. Irreversible amyloid degradation by MMP9 is attributed to the destruction of intramolecular interactions rather than intermolecular hydrogen bonds in the fibril backbone. This process results in the loss of ordered fiber structure without reducing aggregate size or increasing cytotoxicity. Thus, MMP9 can mitigate side effects of anti-amyloid therapy associated with the formation of low-molecular-weight degradation products that may accelerate fibrillogenesis and amyloid propagation between tissues and organs. MMP9 shows promise as a component of safe anti-amyloid drugs by enhancing the accessibility of binding sites through "loosening" amyloid clusters, which facilitates subsequent fragmentation and monomerization by other enzymes., 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 B.V. All rights reserved.)

Background: The accumulation of ordered protein aggregates, amyloid fibrils, accompanies various neurodegenerative diseases (such as Parkinson's, Huntington's, Alzheimer's, etc.) and causes a wide range of systemic and local amyloidoses (such as insulin, hemodialysis amyloidosis, etc.). Such pathologies are usually diagnosed when the disease is already irreversible and a large amount of amyloid plaques have accumulated. In recent years, new drugs aimed at reducing amyloid levels have been actively developed. However, although clinical trials have demonstrated a reduction in amyloid plaque size with these drugs, their effect on disease progression has been controversial and associated with significant side effects, the reasons of which are not fully understood., Aim of Review: The purpose of this review is to summarize extensive array of data on the effect of exogenous and endogenous factors (physico-mechanical effects, chemical effects of low molecular weight compounds, macromolecules and their complexes) on the structure and pathogenicity of mature amyloids for proposing future directions of the development of effective and safe anti-amyloid therapeutics., Key Scientific Concepts of Review: Our analysis show that destruction of amyloids is in most cases incomplete and degradation products often retain the properties of amyloids (including high and sometimes higher than fibrils, cytotoxicity), accelerate amyloidogenesis and promote the propagation of amyloids between cells. Probably, the appearance of protein aggregates, polymorphic in structure and properties (such as amorphous aggregates, fibril fragments, amyloid oligomers, etc.), formed because of uncontrolled degradation of amyloids, may be one of the reasons for the ambiguous effectiveness and serious side effects of the anti-amyloid drugs. This means that all medications that are supposed to be used both for degradation and slow down the fibrillogenesis must first be tested on mature fibrils: the mechanism of drug action and cytotoxic, seeding, and infectious activity of the degradation products must be analyzed., 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. Production and hosting by Elsevier B.V.)

The formation of amyloid fibrils is associated with many severe pathologies as well as the execution of essential physiological functions by proteins. Despite the diversity, all amyloids share a similar morphology and consist of stacked β-strands, suggesting high amyloidogenicity of native proteins enriched with β-structure. Such proteins include those with a β-barrel-like structure with β-strands arranged into a cylindrical β-sheet. However, the mechanisms responsible for destabilization of the native state and triggering fibrillogenesis have not thoroughly explored yet. Here we analyze the structural determinants of fibrillogenesis in proteins with β-barrel structures on the example of odorant-binding protein (OBP), whose amyloidogenicity was recently demonstrated in vitro. We reveal a crucial role in the fibrillogenesis of OBPs for the "open" conformation of the molecule. This conformation is achieved by disrupting the interaction between the β-barrel and the C-terminus of protein monomers or dimers, which exposes "sticky" amyloidogenic sites for interaction. The data suggest that the "open" conformation of OBPs can be induced by destabilizing the native β-barrel structure through the disruption of: 1) intramolecular disulfide cross-linking and non-covalent contacts between the C-terminal fragment and β-barrel in the protein's monomeric form, or 2) intermolecular contacts involved in domain swapping in the protein's dimeric form., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ordered protein aggregates, amyloid fibrils, form toxic plaques in the human body in amyloidosis and neurodegenerative diseases and provide adaptive benefits to pathogens and to reduce the nutritional value of legumes. To identify the amyloidogenic properties of proteins and study the processes of amyloid fibril formation and degradation, the cationic dye thioflavin T (ThT) is the most commonly used. However, its use in acidic environments that induce amyloid formation in vitro can sometimes lead to misinterpretation of experimental results due to electrostatic repulsion. In this work, we show that calculating the net charge per residue of amyloidogenic proteins or peptides is a simple and effective approach for predicting whether their fibrils will interact with ThT at acidic pH. In particular, it was shown that at pH 2, proteins and peptides with a net charge per residue > +0.18 are virtually unstained by this fluorescent probe. The applicability of the proposed approach was demonstrated by predicting and experimentally confirming the absence of ThT interaction with amyloids formed from green fluorescent (sfGFP) and odorant-binding (bOBP) proteins, whose fibrillogenesis was first carried out in an acidic environment. Correct experimental evidence that the inability to detect these fibrils under acidic conditions is precisely because of the lack of dye binding to amyloids (and not their specific structure or the low fluorescence quantum yield of the bound dye) and that the number of ThT molecules associated with fibrils increases with decreasing acidity of the medium was obtained by using the equilibrium microdialysis approach.

Odorant-binding proteins are involved in perceiving smell by capturing odorants within the protein's β-barrel. On the example of bovine odorant-binding protein (bOBP), the structural organization of such proteins and their ability to bind ligands under various conditions in vitro were examined. We found a tendency of bOBP to form oligomers and small amorphous aggregates without disturbing the integrity of protein monomers at physiological conditions. Changes in environmental parameters (increased temperature and pH) favored the formation of larger and dense supramolecular complexes that significantly reduce the binding of ligands by bOBP. The ability of bOBP to form fibrillar aggregates with the properties of amyloids, including high cytotoxicity, was revealed at sample stirring (even at physiological temperature and pH), at medium acidification or pre-solubilization with hexafluoroisopropanol. Fibrillogenesis of bOBP was initiated by the dissociation of the protein's supramolecular complexes into monomers and the destabilization of the protein's β-barrels without a significant destruction of its native β-strands., 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 © 2023 Elsevier B.V. All rights reserved.)

The karyosphere (karyosome) is a structure that forms in the oocyte nucleus-germinal vesicle (GV)-at the diplotene stage of meiotic prophase due to the assembly of all chromosomes in a limited portion of the GV. In some organisms, the karyosphere has an extrachromosomal external capsule, the marker protein of which is nuclear F-actin. Despite many years of theories about the formation of the karyosphere capsule (KC) in the GV of the common frog Rana temporaria , we present data that cast doubt on its existence, at least in this species. Specific extrachromosomal strands, which had been considered the main elements of the frog's KC, do not form a continuous layer around the karyosphere and, according to immunogold labeling, do not contain structural proteins, such as actin and lamin B. At the same time, F-actin is indeed noticeably concentrated around the karyosphere, creating the illusion of a capsule at the light microscopy/fluorescence level. The barrier-to-autointegration factor (BAF) and one of its functional partners-LEMD2, an inner nuclear membrane protein-are not localized in the strands, suggesting that the strands are not functional counterparts of the nuclear envelope. The presence of characteristic strands in the GV of R. temporaria late oocytes may reflect an excess of SMC1 involved in the structural maintenance of diplotene oocyte chromosomes at the karyosphere stage, since SMC1 has been shown to be the most abundant protein in the strands. Other characteristic microstructures-the so-called annuli , very similar in ultrastructure to the nuclear pore complexes-do not contain nucleoporins Nup35 and Nup93, and, therefore, they cannot be considered autonomous pore complexes, as previously thought. Taken together, our data indicate that traditional ideas about the existence of the R. temporaria KC as a special structural compartment of the GV are to be revisited.

Outer membrane proteins (Omps) of Gram-negative bacteria represent porins involved in a wide range of virulence- and pathogenesis-related cellular processes, including transport, adhesion, penetration, and the colonization of host tissues. Most outer membrane porins share a specific spatial structure called the β-barrel that provides their structural integrity within the membrane lipid bilayer. Recent data suggest that outer membrane proteins from several bacterial species are able to adopt the amyloid state alternative to their β-barrel structure. Amyloids are protein fibrils with a specific spatial structure called the cross-β that gives them an unusual resistance to different physicochemical influences. Various bacterial amyloids are known to be involved in host-pathogen and host-symbiont interactions and contribute to colonization of host tissues. Such an ability of outer membrane porins to adopt amyloid state might represent an important mechanism of bacterial virulence. In this work, we investigated the amyloid properties of the OmpC and OmpF porins from two species belonging to Enterobacteriaceae family, Escherichia coli , and Salmonella enterica. We demonstrated that OmpC and OmpF of E. coli and S. enterica form toxic fibrillar aggregates in vitro. These aggregates exhibit birefringence upon binding Congo Red dye and show characteristic reflections under X-ray diffraction. Thus, we confirmed amyloid properties for OmpC of E. coli and demonstrated bona fide amyloid properties for three novel proteins: OmpC of S. enterica and OmpF of E. coli and S. enterica in vitro. All four studied porins were shown to form amyloid fibrils at the surface of E. coli cells in the curli-dependent amyloid generator system. Moreover, we found that overexpression of recombinant OmpC and OmpF in the E. coli BL21 strain leads to the formation of detergent- and protease-resistant amyloid-like aggregates and enhances the birefringence of bacterial cultures stained with Congo Red. We also detected detergent- and protease-resistant aggregates comprising OmpC and OmpF in S. enterica culture. These data are important in the context of understanding the structural dualism of Omps and its relation to pathogenesis.

Although incurable pathologies associated with the formation of highly ordered fibrillar protein aggregates called amyloids have been known for about two centuries, functional roles of amyloids have been studied for only two decades. Recently, we identified functional amyloids in plants. These amyloids formed using garden pea Pisum sativum L. storage globulin and vicilin, accumulated during the seed maturation and resisted treatment with gastric enzymes and canning. Thus, vicilin amyloids ingested with food could interact with mammalian proteins. In this work, we analyzed the effects of vicilin amyloids on the fibril formation of proteins that form pathological amyloids. We found that vicilin amyloids inhibit the fibrillogenesis of these proteins. In particular, vicilin amyloids decrease the number and length of lysozyme amyloid fibrils; the length and width of β-2-microglobulin fibrils; the number, length and the degree of clustering of β-amyloid fibrils; and, finally, they change the structure and decrease the length of insulin fibrils. Such drastic influences of vicilin amyloids on the pathological amyloids' formation cause the alteration of their toxicity for mammalian cells, which decreases for all tested amyloids with the exception of insulin. Taken together, our study, for the first time, demonstrates the anti-amyloid effect of vicilin fibrils and suggests the mechanisms underlying this phenomenon.

Background: The most obvious manifestation of amyloidoses is the accumulation of amyloid fibrils as plaques in tissues and organs, which always leads to a noticeable deterioration in the patients' condition and is the main marker of the disease. For this reason, early diagnosis of amyloidosis is difficult, and inhibition of fibrillogenesis, when mature amyloids are already accumulated in large quantities, is ineffective. A new direction for amyloidosis treatment is the development of approaches aimed at the degradation of mature amyloid fibrils. In the present work, we investigated possible consequences of amyloid's degradation. Methods: We analyzed the size and morphology of amyloid degradation products by transmission and confocal laser scanning microscopy, their secondary structure and spectral properties of aromatic amino acids, intrinsic chromophore sfGFP, and fibril-bound amyloid-specific probe thioflavin T (ThT) by the absorption, fluorescence and circular dichroism spectroscopy, as well as the cytotoxicity of the formed protein aggregates by MTT-test and their resistance to ionic detergents and boiling by SDS-PAGE. Results: On the example of sfGFP fibrils (model fibrils, structural rearrangements of which can be detected by a specific change in the spectral properties of their chromophore), and pathological Aβ-peptide (Aβ42) fibrils, leading to neuronal death in Alzheimer's disease, the possible mechanisms of amyloids degradation after exposure to factors of different nature (proteins with chaperone and protease activity, denaturant, and ultrasound) was demonstrated. Our study shows that, regardless of the method of fibril degradation, the resulting species retain some amyloid's properties, including cytotoxicity, which may even be higher than that of intact amyloids. Conclusion: The results of our work indicate that the degradation of amyloid fibrils in vivo should be treated with caution since such an approach can lead not to recovery, but to aggravation of the disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sulatsky, Stepanenko, Stepanenko, Mikhailova, Kuznetsova, Turoverov and Sulatskaya.)

Amyloids represent protein aggregates with highly ordered fibrillar structure associated with the development of various disorders in humans and animals and involved in implementation of different vital functions in all three domains of life. In prokaryotes, amyloids perform a wide repertoire of functions mostly attributed to their interactions with other organisms including interspecies interactions within bacterial communities and host-pathogen interactions. Recently, we demonstrated that free-living cells of Rhizobium leguminosarum , a nitrogen-fixing symbiont of legumes, produce RopA and RopB which form amyloid fibrils at cell surface during the stationary growth phase thus connecting amyloid formation and host-symbiont interactions. Here we focused on a more detailed analysis of the RopB amyloid state in vitro and in vivo , during the symbiotic interaction between R. leguminosarum bv. viciae with its macrosymbiont, garden pea ( Pisum sativum L.). We confirmed that RopB is the bona fide amyloid protein since its fibrils exhibit circular x-ray reflections indicating its cross-β structure specific for amyloids. We found that fibrils containing RopB and exhibiting amyloid properties are formed in vivo at the surface of bacteroids of R. leguminosarum extracted from pea nodules. Moreover, using pea sym31 mutant we demonstrated that formation of extracellular RopB amyloid state occurs at different stages of bacteroid development but is enhanced in juvenile symbiosomes. Proteomic screening of potentially amyloidogenic proteins in the nodules revealed the presence of detergent-resistant aggregates of different plant and bacterial proteins including pea amyloid vicilin. We demonstrated that preformed vicilin amyloids can cross-seed RopB amyloid formation suggesting for probable interaction between bacterial and plant amyloidogenic proteins in the nodules. Taken together, we demonstrate that R. leguminosarum bacteroids produce extracellular RopB amyloids in pea nodules in vivo and these nodules also contain aggregates of pea vicilin amyloid protein, which is able to cross-seed RopB fibrillogenesis in vitro. Thus, we hypothesize that plant nodules contain a complex amyloid network consisting of plant and bacterial amyloids and probably modulating host-symbiont interactions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kosolapova, Belousov, Sulatsky, Tsyganova, Sulatskaya, Bobylev, Shtark, Tsyganov, Volkov, Zhukov, Tikhonovich and Nizhnikov.)

The accumulation of β-sheet-rich protein aggregates, amyloid fibrils, accompanies severe pathologies (Alzheimer's, Parkinson's diseases, ALS, etc.). The high amyloidogenicity of proteins with a native β-barrel structure, and the amyloidogenic peptides ability to form a universal cylindrin-like oligomeric state were proven. The mechanisms for the proteins' transformation from this state to a fibrillar one are still not fully understood. We defined the structural rearrangements of the amyloidogenic β-barrel superfolder GFP (sfGFP) prior to fibrillogenesis using its tryptophan and chromophore fluorescence. We characterized the early intermediate "native-like" state preserving the integrity of the sfGFP β-barrel scaffold despite the partial distortion of the three β-strands closing it. The interaction between the "melted" regions of the protein leads to the assembly of high molecular weight complexes, which are not dynamic structures but are less stable and less cytotoxic than mature amyloids. Additional contacts of sfGFP monomers facilitate the global reorganization of its structure and stabilization of the second intermediate state in which the β-barrel opens and some of the native α-helices and disordered regions refold into non-native β-strands, which, along with native β-strands, form an amyloid fiber. Reported sfGFP structural transformations may occur during the fibrillogenesis of other β-barrel proteins, and the identified intermediate states are likely universal. Thus sfGFP can be used as a sensing platform to develop therapeutic agents inhibiting amyloidogenesis through interaction with protein intermediates and destroying low-stable aggregates formed at the early stages of fibrillogenesis., (Copyright © 2022. Published by Elsevier B.V.)

The relative abundance of two main Abeta-peptide types with different lengths, Aβ40 and Aβ42, determines the severity of the Alzheimer's disease progression. However, the factors responsible for different behavior patterns of these peptides in the amyloidogenesis process remain unknown. In this comprehensive study, new evidence on Aβ40 and Aβ42 amyloid polymorphism was obtained using a wide range of experimental approaches, including custom-designed approaches. We have for the first time determined the number of modes of thioflavin T (ThT) binding to Aβ40 and Aβ42 fibrils and their binding parameters using a specially developed approach based on the use of equilibrium microdialysis, which makes it possible to distinguish between the concentration of the injected dye and the concentration of dye bound to fibrils. The binding sites of one of these modes located at the junction of adjacent fibrillar filaments were predicted by molecular modeling techniques. We assumed that the sites of the additional mode of ThT-Aβ42 amyloid binding observed experimentally (which are not found in the case of Aβ40 fibrils) are localized in amyloid clots, and the number of these sites could be used for estimation of the level of fiber clustering. We have shown the high tendency of Aβ42 fibers to form large clots compared to Aβ40 fibrils. It is probable that this largely determines the high resistance of Aβ42 amyloids to destabilizing effects (denaturants, ionic detergents, ultrasonication) and their explicit cytotoxic effect, which we have shown. Remarkably, cross-seeding of Aβ40 fibrillogenesis using the preformed Aβ42 fibrils changes the morphology and increases the stability and cytotoxicity of Aβ40 fibrils. The differences in the tendency to cluster and resistance to external factors of Aβ40 and Aβ42 fibrils revealed here may be related to the distinct role they play in the deposition of amyloids and, therefore, differences in pathogenicity in Alzheimer's disease.

Green fluorescent proteins (GFP) are commonly used as fluorescent tags and biosensors in cell biology and medicine. However, the propensity of GFP-like proteins to aggregate and the consequence of intermolecular interaction for their application have not been thoroughly examined. In this work, alternative aggregation pathways of superfolder green fluorescent protein (sfGFP) were demonstrated using a spectroscopic and microscopic study of the samples prepared by equilibrium microdialysis. Besides oligomerization of native monomers, we showed for the first time the condition-specific formation by sfGFP of either amyloid fibrils (at increased temperature or acidity) or amorphous aggregates (at physiological conditions). Both types of sfGFP aggregates had lost green fluorescence and were toxic to cells. Thus, when using GFP-like proteins as fluorescent tags, one should take into account their high ability to form aggregates with lost unique visible fluorescence in the cellular environment, which affects cell viability. Moreover, the results of this work cast doubt on the correctness of the data on the fibrillogenesis of various amyloidogenic proteins obtained using their fusion with GFP-like proteins., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Insoluble protein aggregates with fibrillar morphology called amyloids and β-barrel proteins both share a β-sheet-rich structure. Correctly folded β-barrel proteins can not only function in monomeric (dimeric) form, but also tend to interact with one another-followed, in several cases, by formation of higher order oligomers or even aggregates. In recent years, findings proving that β-barrel proteins can adopt cross-β amyloid folds have emerged. Different β-barrel proteins were shown to form amyloid fibrils in vitro. The formation of functional amyloids in vivo by β-barrel proteins for which the amyloid state is native was also discovered. In particular, several prokaryotic and eukaryotic proteins with β-barrel domains were demonstrated to form amyloids in vivo, where they participate in interspecies interactions and nutrient storage, respectively. According to recent observations, despite the variety of primary structures of amyloid-forming proteins, most of them can adopt a conformational state with the β-barrel topology. This state can be intermediate on the pathway of fibrillogenesis ("on-pathway state"), or can be formed as a result of an alternative assembly of partially unfolded monomers ("off-pathway state"). The β-barrel oligomers formed by amyloid proteins possess toxicity, and are likely to be involved in the development of amyloidoses, thus representing promising targets for potential therapy of these incurable diseases. Considering rapidly growing discoveries of the amyloid-forming β-barrels, we may suggest that their real number and diversity of functions are significantly higher than identified to date, and represent only "the tip of the iceberg". Here, we summarize the data on the amyloid-forming β-barrel proteins, their physicochemical properties, and their biological functions, and discuss probable means and consequences of the amyloidogenesis of these proteins, along with structural relationships between these two widespread types of β-folds.

Proteolytic enzymes are known to be involved in the formation and degradation of various monomeric proteins, but the effect of proteases on the ordered protein aggregates, amyloid fibrils, which are considered to be extremely stable, remains poorly understood. In this work we study resistance to proteolytic degradation of lysozyme amyloid fibrils with two different types of morphology and beta-2-microglobulun amyloids. We showed that the proteolytic enzyme of the pancreas, trypsin, induced degradation of amyloid fibrils, and the mechanism of this process was qualitatively the same for all investigated amyloids. At the same time, we found a dependence of efficiency and rate of fibril degradation on the structure of the amyloid-forming protein as well as on the morphology and clustering of amyloid fibrils. It was assumed that the discovered relationship between fibrils structure and the efficiency of their degradation by trypsin can become the basis of a new express method for the analysis of amyloids polymorphism. Unexpectedly lower resistance of both types of lysozyme amyloids to trypsin exposure compared to the native monomeric protein (which is not susceptible to hydrolysis) was attributed to the higher availability of cleavage sites in studied fibrils. Another intriguing result of the work is that the cytotoxicity of amyloids treated with trypsin was not only failing to decline, but even increasing in the case of beta-2-microglobulin fibrils.

Fluorescent probes thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS) are widely used to study amyloid fibrils that accumulate in the body of patients with serious diseases, such as Alzheimer's, Parkinson's, prion diseases, etc. However, the possible effect of these probes on amyloid fibrils is not well understood. In this work, we investigated the photophysical characteristics, structure, and morphology of mature amyloid fibrils formed from two model proteins, insulin and lysozyme, in the presence of ThT and ANS. It turned out that ANS affects the secondary structure of amyloids (shown for fibrils formed from insulin and lysozyme) and their fibers clusterization (valid for lysozyme fibrils), while ThT has no such effects. These results confirm the differences in the mechanisms of these dyes interaction with amyloid fibrils. Observed effect of ANS was explained by the electrostatic interactions between the dye molecule and cationic groups of amyloid-forming proteins (unlike hydrophobic binding of ThT) that induce amyloids conformational changes. This interaction leads to weakening repulsion between positive charges of amyloid fibrils and can promote their clusterization. It was shown that when fibrillogenesis conditions and, consequently, fibrils structure is changing, as well as during defragmentation of amyloids by ultrasonication, the influence of ANS to amyloids does not change, which indicates the universality of the detected effects. Based on the obtained results, it was concluded that ANS should be used cautiously for the study of amyloid fibrils, since this fluorescence probe have a direct effect on the object of study.

Given the ability of molecular chaperones and chaperone-like proteins to inhibit the formation of pathological amyloid fibrils, the chaperone-based therapy of amyloidosis has recently been proposed. However, since these diseases are often diagnosed at the stages when a large amount of amyloids is already accumulated in the patient's body, in this work we pay attention to the undeservedly poorly studied problem of chaperone and chaperone-like proteins' effect on mature amyloid fibrils. We showed that a heat shock protein alpha-B-crystallin, which is capable of inhibiting fibrillogenesis and is found in large quantities as a part of amyloid plaques, can induce degradation of mature amyloids by two different mechanisms. Under physiological conditions, alpha-B-crystallin induces fluffing and unweaving of amyloid fibrils, which leads to a partial decrease in their structural ordering without lowering their stability and can increase their cytotoxicity. We found a higher correlation between the rate and effectiveness of amyloids degradation with the size of fibrils clusters rather than with amino acid sequence of amyloidogenic protein. Some external effects (such as an increase in medium acidity) can lead to a change in the mechanism of fibrils degradation induced by alpha-B-crystallin: amyloid fibers are fragmented without changing their secondary structure and properties. According to recent data, fibrils cutting can lead to the generation of seeds for new bona fide amyloid fibrils and accelerate the accumulation of amyloids, as well as enhance the ability of fibrils to disrupt membranes and to reduce cell viability. Our results emphasize the need to test the chaperone effect not only on fibrillogenesis, but also on the mature amyloid fibrils, including stress conditions, in order to avoid undesirable disease progression during chaperone-based therapy.

Amyloids are protein aggregates with a highly ordered spatial structure giving them unique physicochemical properties. Different amyloids not only participate in the development of numerous incurable diseases but control vital functions in archaea, bacteria and eukarya. Plants are a poorly studied systematic group in the field of amyloid biology. Amyloid properties have not yet been demonstrated for plant proteins under native conditions in vivo. Here we show that seeds of garden pea Pisum sativum L. contain amyloid-like aggregates of storage proteins, the most abundant one, 7S globulin Vicilin, forms bona fide amyloids in vivo and in vitro. Full-length Vicilin contains 2 evolutionary conserved β-barrel domains, Cupin-1.1 and Cupin-1.2, that self-assemble in vitro into amyloid fibrils with similar physicochemical properties. However, Cupin-1.2 fibrils unlike Cupin-1.1 can seed Vicilin fibrillation. In vivo, Vicilin forms amyloids in the cotyledon cells that bind amyloid-specific dyes and possess resistance to detergents and proteases. The Vicilin amyloid accumulation increases during seed maturation and wanes at germination. Amyloids of Vicilin resist digestion by gastrointestinal enzymes, persist in canned peas, and exhibit toxicity for yeast and mammalian cells. Our finding for the first time reveals involvement of amyloid formation in the accumulation of storage proteins in plant seeds., Competing Interests: The authors have declared that no competing interests exist.

Accumulation of amyloid fibrils in organism accompanies many serious diseases, such as Alzheimer's and Parkinson's diseases, diabetes, prion diseases, etc. It is generally accepted that amyloids are highly resistant to degradation, which complicates their elimination in vivo and is one of the reasons for their pathogenicity. However, using a wide range of physicochemical approaches and specially elaborated method for the tested samples preparation by equilibrium microdialysis technique, it is proved that the stability of amyloids is greatly exaggerated. It turned out that amyloid fibrils formed from at least two amyloidogenic proteins, one of which is a model object for fibrils studying and the second is the cause of hemodialysis amyloidosis in an acute renal failure, are less stable than monomeric proteins. A mechanism of the degradation/reassembly of amyloid fibrils was proposed. It was shown that amyloid «seed» is a factor affecting not only the rate of the fibrils formation, but also their structure. Obtained results are a step towards identifying effects that can lead to degradation of amyloids and their clearance without adverse influence on the functionally active state of the protein or to change the structure and, as a result, the pathogenicity of these protein aggregates., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Amyloids represent protein fibrils with a highly ordered spatial structure, which not only cause dozens of incurable human and animal diseases but also play vital biological roles in Archaea, Bacteria, and Eukarya. Despite the fact that association of bacterial amyloids with microbial pathogenesis and infectious diseases is well known, there is a lack of information concerning the amyloids of symbiotic bacteria. In this study, using the previously developed proteomic method for screening and identification of amyloids (PSIA), we identified amyloidogenic proteins in the proteome of the root nodule bacterium Rhizobium leguminosarum. Among 54 proteins identified, we selected two proteins, RopA and RopB, which are predicted to have β-barrel structure and are likely to be involved in the control of plant-microbial symbiosis. We demonstrated that the full-length RopA and RopB form bona fide amyloid fibrils in vitro. In particular, these fibrils are β-sheet-rich, bind Thioflavin T (ThT), exhibit green birefringence upon staining with Congo Red (CR), and resist treatment with ionic detergents and proteases. The heterologously expressed RopA and RopB intracellularly aggregate in yeast and assemble into amyloid fibrils at the surface of Escherichia coli . The capsules of the R. leguminosarum cells bind CR, exhibit green birefringence, and contain fibrils of RopA and RopB in vivo., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Fluorescent dye trans-2-[4-(dimethylamino)styryl]-3-ethyl-1,3-benzothiazolium perchlorate (DMASEBT) is a relatively recently synthesized probe for detection of amyloid fibrils accumulating in the organs and tissues of patients with a wide range of serious incurable diseases. DMASEBT was developed as an alternative of its widely used analogue thioflavin T (ThT), which is the "gold standard" for the amyloid fibrils study. Our results show the similarity of both dyes binding to amyloid fibrils and allow one to propose a mechanism of such probes interaction with some types of the fibrils. At the same time, DMASEBT has a significant advantage, namely, improved photophysical properties compared with ThT, which allows for the detection of DMASEBT-stained amyloid fibrils in the spectral region of the "transparency window of biological tissues". The ability of the dye to penetrate into the cells was shown to open the prospect of this dye's use for amyloid fibrils bioimaging and biosensing in vivo. Furthermore, it was proven that DMASEBT can be used not only as a test for amyloid fibrils formation but also for the comparative study of the fibrils structure (both their fibers and bunches), which in turn may underlie the variability of amyloidosis and affect the cytotoxicity of these protein aggregates.

The persistence of high concentrations of beta-2-microglobulin (β2M) in the blood of patients with acute renal failure leads to the development of the dialysis-related amyloidosis. This disease manifests in the deposition of amyloid fibrils formed from the various forms of β2M in the tissues and biological fluids of patients. In this paper, the amyloid fibrils formed from the full-length β2M (β2m) and its variants that lack the 6 and 10 N-terminal amino acids of the protein polypeptide chain (ΔN6β2m and ΔN10β2m, respectively) were probed by using the fluorescent dye thioflavin T (ThT). For this aim, the tested solutions were prepared via the equilibrium microdialysis approach. Spectroscopic analysis of the obtained samples allowed us to detect one binding mode (type) of ThT interaction with all the studied variants of β2M amyloid fibrils with affinity ~10⁴ M -1 . This interaction can be explained by the dye molecules incorporation into the grooves that were formed by the amino acids side chains of amyloid protofibrils along the long axis of the fibrils. The decrease in the affinity and stoichiometry of the dye interaction with β2M fibrils, as well as in the fluorescence quantum yield and lifetime of the bound dye upon the shortening of the protein amino acid sequence were shown. The observed differences in the ThT-β2M fibrils binding parameters and characteristics of the bound dye allowed to prove not only the difference of the ΔN10β2m fibrils from other β2M fibrils (that can be detected visually, for example, by transmission electron microscopy (TEM), but also the differences between β2m and ΔN6β2m fibrils (that can not be unequivocally confirmed by other approaches). These results prove an essential role of N-terminal amino acids of the protein in the formation of the β2M amyloid fibrils. Information about amyloidogenic protein sequences can be claimed in the development of ways to inhibit β2M fibrillogenesis for the treatment of dialysis-related amyloidosis.

In this work, α-synuclein amyloid fibrils-the formation of which is a biomarker of Parkinson's disease-were investigated using the fluorescent probe thioflavin T (ThT). The experimental conditions of protein fibrillogenesis were chosen so that a sufficient number of continuous measurements could be performed to characterize and analyze all stages of this process. The reproducibility of fibrillogenesis and the structure of the obtained aggregates (which is a critical point for further investigation) were proven using a wide range of physical-chemical methods. For the determination of ThT-α-synuclein amyloid fibril binding parameters, the sample and reference solutions were prepared using equilibrium microdialysis. By utilizing absorption spectroscopy of these solutions, the ThT-fibrils binding mode with a binding constant of about 10⁴ M -1 and stoichiometry of ThT per protein molecule of about 1:8 was observed. Fluorescence spectroscopy of the same solutions with the subsequent correction of the recorded fluorescence intensity on the primary inner filter effect allowed us to determine another mode of ThT binding to fibrils, with a binding constant of about 10⁶ M -1 and stoichiometry of about 1:2500. Analysis of the photophysical characteristics of the dye molecules bound to the sites of different binding modes allowed us to assume the possible localization of these sites. The obtained differences in the ThT binding parameters to the amyloid fibrils formed from α-synuclein and other amyloidogenic proteins, as well as in the photophysical characteristics of the bound dye, confirmed the hypothesis of amyloid fibril polymorphism.

The aim of the present work was investigation of the fluorescent dye thioflavin T (ThT) binding to acetylcholinesterase (AChE). ThT is an effective test for protease activity, as well as a probe for amyloid fibril formation. Despite the extended and active investigation of ThT-AChE binding, there is still no common view on the stoichiometry of this interaction. In particular, there is a hypothesis explaining the spectral properties of bound to AChE dye and high quantum yield of its fluorescence by formation of dimers or excimers of ThT. In order to confirm or deny this hypothesis, we proposed a new experimental approach for examination of ThT-AChE interaction based on spectroscopic investigation of samples prepared by equilibrium microdialysis. This approach allowed us to prove 1/1 ThT/AChE binding stoichiometry. The increase of ThT fluorescence quantum yield and lifetime accompanying its binding to AChE can be explained by the molecular rotor nature of this dye. Together with the coincidence of the positions of free and AChE-bound ThT fluorescence spectra, the obtained results prove the groundlessness of the hypotheses about ThT aggregation while binding to AChE. The model of ThT localization in the active site of AChE was proposed by using molecular docking simulations. These results also allowed us to suggest the key role of aromatic residues in ThT-AChE interaction, as observed for some amyloid fibrils.

Amyloids are protein fibrils with a characteristic spatial structure. Amyloids were long perceived as the pathogens involved in a set of lethal diseases in humans and animals. In recent decades, it has become clear that amyloids represent a quaternary protein structure that is not only pathological but also functionally important and is widely used by different organisms, ranging from archaea to animals, to implement diverse biological functions. The greatest biological variety of amyloids is found in prokaryotes, where they control the formation of biofilms and cell wall sheaths, facilitate the overcoming of surface tension, and regulate the metabolism of toxins. Several amyloid proteins were identified in the important model, biotechnological and pathogenic bacterium Escherichia coli. In previous studies, using a method for the proteomic screening and identification of amyloids, we identified 61 potentially amyloidogenic proteins in the proteome of E. coli. Among these proteins, YghJ was the most enriched with bioinformatically predicted amyloidogenic regions. YghJ is a lipoprotein with a zinc metalloprotease M60-like domain that is involved in mucin degradation in the intestine as well as in proinflammatory responses. In this study, we analyzed the amyloid properties of the YghJ M60-like domain and demonstrated that it forms amyloid-like fibrils in vitro and in vivo.