Accumulation of amyloid fibrils is a key factor in the pathogenesis of progressive diseases, including neurodegenerative disorders such as Alzheimer's and Parkinson's disease, as well as systemic amyloidosis. Although amyloid deposits are known to persist in vivo for years or even decades, it remains unclear how the structure and biological properties of mature fibrils evolve during prolonged post-assembly residence. Here, we addressed this question using a simplified cell-free aqueous model designed to isolate the intrinsic time-dependent behavior of mature amyloid aggregates from the complexity of the biological milieu. Specifically, we investigated the long-term evolution of two polymorphs of lysozyme amyloid fibrils as model systems with distinct clustering propensities that mimic the diversity of amyloid deposits. We challenge the assumption of amyloid stability by demonstrating their spontaneous degradation over 16 months at physiological temperature, a process we define as amyloid "aging". This process is characterized by aggregate declustering, fibril shortening, and progressive depolymerization into monomeric subunits, accompanied by a pronounced reduction in intrinsic toxicity across multiple human cell lines. Notably, "aged" fibrils were disassembled and degraded more efficiently than freshly prepared aggregates by the molecular chaperone α-B-crystallin and immune-associated proteases, including matrix metalloproteinase-9 and cathepsins B and D. However, this enhanced degradation revealed a paradox: accelerated processing of "aged" amyloids did not always reduce cytotoxicity and, in some cases, even exacerbated it, consistent with the generation of biologically active fibril-derived species rather than their complete conversion into non-toxic monomers. We provide the first systematic evidence that mature amyloids are dynamic structures undergoing spontaneous degradation that fundamentally alters their cytotoxicity and susceptibility to biological clearance. Our results introduce amyloid aging as a previously underappreciated dimension of amyloid biology and emphasize the need to account for fibril "age" when evaluating pathogenic potential and developing anti-amyloid therapeutic strategies.Spontaneous "aging" of amyloid fibrils leads to suprastructural remodeling: declustering, fibril shortening, and attenuated cytotoxicity. While "aged" aggregates are degraded more efficiently by immune-associated proteases and α-B-crystallin than "fresh" fibrils, this enhanced degradation does not reduce cytotoxicity and, in some cases, exacerbates it, highlighting the complex interplay between aggregate maturity and biological outcomes.

The pathogenesis of neurodegenerative diseases such as Alzheimer's and Parkinson's, systemic and local amyloidoses is closely associated with amyloid fibril accumulation. Dysregulation of energy metabolism and a decline in the level of adenosine triphosphate (ATP), a key intracellular energy source and extracellular signaling molecule, contribute to the development of these pathologies. Despite the well-established role of ATP as a biological hydrotrope that prevents the initial aggregation of proteins, the impact of this nucleotide on the structure and properties of pre-formed (mature) amyloids is still poorly understood. In this study, we showed the ability of ATP to bind with affinity in the low-to-mid micromolar range to mature amyloids of lysozyme, which associated with hereditary lysozyme amyloidosis, and superfolder GFP, as model objects with unique properties, demonstrating a significant increase in the number of binding sites compared to native proteins. We demonstrated that this interaction induces declustering of both aggregate types, accompanied by fibril disordering specific to certain amyloidogenic proteins. Importantly, ATP-mediated fibril remodeling resulted in a significant reduction in their toxicity to mammalian cell lines, as well as a decrease in the aggregate resistance to chemical and thermal degradation. The obtained results reveal the complex nature of ATP's effects: while acting as an endogenous amyloid detoxification factor, it is also susceptible to sequestration in amyloid deposits due to its affinity in the low-to-mid micromolar range. Amyloid-associated ATP sequestration may lead to nucleotide deficiency and aggravation of amyloidosis.

The accumulation of amyloid fibrils in the brain leads to cognitive and motor impairment in incurable neurodegenerative disorders, particularly in Alzheimer's and Parkinson's diseases. Emerging anti-amyloid immunotherapies rely on recruiting immune proteases to clear these pathological aggregates, yet a stark disconnect remains between the high proteolytic efficiency observed in dilute in vitro assays and the limited efficacy of these treatments in vivo. This discrepancy likely arises because standard models neglect the dense macromolecular crowding characteristic of the cellular milieu. Here, we address this gap by investigating the degradation of alpha-synuclein amyloids by key immune proteases (cathepsin B, cathepsin D, and matrix metalloproteinase-9) in the presence of synthetic crowding agents at physiological densities. We found that macromolecular crowding in a crowding agent- and dose-dependent manner inhibits protease-induced amyloid declusterization, cluster size reduction, fibril fragmentation, and structural disorganization. We propose that the observed inhibition arises from the excluded volume effect, which stabilizes fibril structure and restricts both enzyme mobility and steric access to the fibril surface rather than suppressing catalytic activity. By restricting the extent of enzymatic amyloid degradation, this "crowding shield" modulated the cytotoxic consequences of enzymatic processing in human cell lines, attenuating enzyme-induced shifts in fibril toxicity in a condition-dependent manner. These findings identify the crowded cellular environment as a critical stabilizer of pathogenic protein aggregates, explaining their persistence in vivo.

Tetrapyrrole-binding proteins are promising near-infrared fluorescent markers. We recently showed that the near-infrared fluorescent protein iRFP713, derived from bacteriophytochrome from Rhodopseudomonas palustris (RpBphP2), when mutated (iRFP713/C15S/V254C; hereafter referred to as iRFP) and complexed with phycocyanobilin (PCB) as a chromophore (iRFP-PCB), has a significantly higher fluorescence quantum yield than that of iRFP complexed with biliverdin (iRFP-BV). Here, we show that iRFP-PCB can be used as a fluorescent biomarker in eukaryotic cells (HEK293T) and that the effective brightness of iRFP-PCB is more than four times higher than that of iRFP-BV. We elucidated the structural basis of iRFP interaction with PCB by determining its crystal structure. Interestingly, we observed both parallel and antiparallel arrangements of iRFP protomers in an asymmetric unit cell. We used molecular dynamics simulations to show that the mobility of the protein and chromophore covalently bound to Cys254 is similar in different assembly states and in complexes with BV or PCB. Overall, the results indicate that PCB is a promising chromophore for the development of new fluorescent biomarkers from bacterial phytochromes and provide a basis for the further engineering of biomarkers from iRFP and related proteins.

Growing evidence links gut microbiota to neurodegenerative diseases, yet direct molecular interactions between bacterial and host amyloid proteins remain incompletely understood. Bacterial amyloids represent an understudied yet potentially critical component of gut-brain communication in neurodegeneration. Here, we provide the first investigation of whether amyloids formed by outer membrane proteins (OMPs) of enterobacteria can modulate neurodegeneration-associated protein aggregation. We examined the effects of pre-formed amyloid fibrils from OmpC and OmpF of Escherichia coli and Salmonella enterica on fibrillogenesis of α-synuclein and amyloid-β, whose pathological accumulation in brain is associated with Parkinson's and Alzheimer's diseases, respectively. Using a comprehensive array of physicochemical methods, we discovered that bacterial OMP amyloids altered the structural properties and clustering tendency of mammalian amyloids in a target-specific manner. In particular, for α-synuclein, OMP amyloids modified the irregular "fuzzy coat" surrounding the ordered β-core, increasing fibril clustering without affecting core structure, quantity, or cytotoxicity. In contrast, amyloid-β fibrils showed more extensive structural changes, with modifications to both the "fuzzy coat" and β-sheet core, accompanied by a decreased clustering tendency and significantly reduced toxicity for mammalian neuroblastoma and epithelial adenocarcinoma cell lines. Our findings demonstrate that amyloids formed from OMPs produced by Enterobacteriaceae species represent a previously unrecognized class of amyloid modulators capable of influencing pathological aggregation of mammalian proteins through intermolecular contacts. These results open a discussion on the dual role of bacterial amyloids in neurodegeneration, as they may be capable not only of promoting pathological amyloidogenesis but also of mitigating the toxic effects of host amyloid aggregates.

Amyloid fibrils cause organ and tissue dysfunction in numerous severe diseases. Despite the prevalence and severity of amyloidoses, there is still no effective and safe anti-amyloid therapy. This study investigates the impact of cysteine protease cathepsin B (CTSB) on amyloids associated with Alzheimer’s and Parkinson’s diseases, hemodialysis, and lysozyme amyloidosis. We analyzed the effect of CTSB on the size, structure, and proteotoxicity of amyloid fibrils formed from alpha-synuclein, abeta peptide (1-42), insulin, and lysozyme using a combination of spectroscopic, microscopic, electrophoretic, and colorimetric methods. Our comprehensive research revealed a dual effect of CTSB on amyloid fibrils. Firstly, CTSB induced amyloid fragmentation while preserving their ordered morphology, and, secondly, it “loosened” the tertiary structure of amyloids and reduced the regularity of the secondary structure. This dual mechanism of action was universal across fibrils associated with different pathologies, although the disruption efficacy and predominant type of degradation products depended on the amyloids’ structure, size, and clustering. Notably, CTSB-induced irreversible degradation significantly reduced the toxicity for immortalized and primary cell lines of low-clustered fibrils, such as alpha-synuclein amyloids associated with Parkinson’s disease. These findings enhance our understanding of how endogenous CTSB may regulate amyloid content at the molecular level in different neuropathologies. In addition, our results suggest the potential of CTSB as a component of anti-amyloid drugs in combination with agents that enhance the accessibility of proteolytic sites within amyloid clots and reduce these clusters stability.

Amyloid fibrils, linked to severe pathologies such as neurodegenerative diseases, pose a significant challenge to modern medicine. Lysosomal proteases, particularly cathepsins, have attracted attention for their potential role in modulating amyloid pathologies, especially in the context of immunotherapy. However, the impact of these proteases on mature amyloids remains poorly understood. This study investigates the effects of cathepsin D (CTSD), a lysosomal aspartyl protease, on mature amyloid fibrils associated with local insulin and systemic lysozyme amyloidoses, as well as neurodegenerative Alzheimer's and Parkinson's diseases. Our results demonstrate that CTSD induces fragmentation of all examined fibril types, presumably by disrupting hydrogen bonds between the beta-strands forming the fibril backbone. This fragmentation occurs without depolymerizing or destructuring the amyloids and does not reduce their toxic effects on immortalized and primary cell lines. Furthermore, the size, structure, and properties of CTSD-induced amyloid degradation products suggest that the enzyme may contribute to the rapid accumulation and propagation of pathological amyloids at both intercellular and tissue levels in mammals. This finding is valuable for understanding physiological processes and developing immunotherapeutic strategies, as artificially stimulating the immune response may exacerbate pathological conditions.

Protein dysfunction can be caused by its fibrillogenesis, which is often initiated by rather subtle structural changes. In the case of odorant-binding proteins (OBPs), fibrillogenesis triggering is mediated by local melting of the peripheral C-terminal domain while maintaining the integrity of the bulk of the molecule, the β-barrel. This work is focused on establishing the sequence and duration of structural transformations of OBPs' β-barrel during fibrillogenesis. We found that β-barrel transformation requires oligomerization of OBPs monomers with unlocked C-terminus, whose formation precedes the fibrillogenesis initiation. The fibrillogenesis lag phase involves the gradual bond weakening within the β-barrel without its destruction. During this phase, oligomeric molecules first experience partial disruption of contacts near the β1-strand, followed by its disorganization and the opening of the internal protein cavity. In the exponential phase, complete β-barrel reorganization in aggregates lasts as long as the lag phase, accompanied by the sequential appearance of prefibrillar forms with cytotoxicity and mature amyloid fibrils. Our findings suggest similarities in the intermediate states accumulated during fibrillogenesis as well as common mechanisms and sequence of structural transitions for proteins of β-barrel topology. This contributes to the identification of relevant targets and possible ways to inhibit amyloidogenesis of these proteins.

Olfactory dysfunction is a common complication of serious pathologies, including neurodegenerative disorders, bacterial and viral infections, including COVID-19, and others. Despite the widespread prevalence of olfactory disorders, the pathophysiological mechanisms of their development, as well as the molecular basis of their association with the underlying disease, remain incompletely understood. The current work formulates a new concept of the origin of olfactory disorders, linking a decrease in the activation of olfactory neurons and their death to the fibrillogenesis of odorant-binding proteins (OBPs), which are the primary participants of olfactory perception. The potential triggers of OBPs’ amyloidogenesis in vivo are discussed, such as molecular crowding, components of nasal medications, environmental factors, and cross-seeding with viral and bacterial amyloids. Several ways of impairment of olfactory signaling as a result of fibrillogenesis of OBPs are formulated: complete loss of OBPs functionality following amyloid formation; mechanical blockage of the membranes of sensory neurons and damage to chemoreceptors on their surface, preventing olfactory signaling; cytotoxic effect of OBPs’ amyloid on sensory neurons and other cells of the olfactory epithelium. The proposed concept offers a novel perspective on the pathogenesis of olfactory dysfunction, as well as its possible association with amyloidoses, including in neurodegenerations, and infectious diseases. It opens prospects for the development of new therapeutic approaches to the treatment of olfactory disorders.

ABSTRACT More than a century ago, it was known that the accumulation of ordered protein aggregates, amyloid fibrils, accompanies several serious and still largely incurable pathologies, including Alzheimer's and Parkinson's diseases. The striking gap between decades of research identifying amyloids as one of the key drivers of neurodegeneration and the persistent lack of effective anti‐amyloid therapies reveals a perplexing contradiction, which we define as the “amyloid paradox.” To address this paradox, here we summarize and analyze current perspectives on the unique properties and pathogenic mechanisms of amyloids, highlighting the variability and complexity of their biological consequences and uncovering the risks and limitations encountered in combating these aggregates. We conceptualize amyloid fibril pathogenicity as a complex cascade extending well beyond direct cytotoxicity, such as that arising from disruption of membranes and other cellular organelles. This review encompasses amyloids' disruptive effects on cellular processes and ability to trigger inflammatory responses, their resistance to degradation, capacity to regenerate after apparent destruction, tendency to propagate throughout the organism, propensity to cytotoxicity‐increasing transformation, and ability to sequester and pathologically modify essential biomolecules. This integrated analysis reveals why single‐target therapeutic approaches often fail and suggests that effective anti‐amyloid strategies must address multiple aspects of amyloid pathogenicity simultaneously. The conceptual reframing of the threats of amyloid fibrils helps explain the origins of the amyloid paradox, enhances our understanding of these complex pathogenic agents, and provides a foundation for developing more effective and safe therapeutic strategies for neurodegenerative diseases. These strategies should address the complex and interconnected nature of amyloid pathogenicity rather than its targeting isolated aspects.

>Currently,our understanding of the pathogenesis of major neurodegenerative disorders,such as Alzheimer's,Parkinson's,and Huntington's diseases,is largely shaped by the amyloid cascade hypothesis.Pa rticularly,this hypothesis posits that in Alzheimer's disease,the aggregation of amyloid-beta peptide initiates a series of pathological processes leading to neuronal dysfunction and death (Zhang et al.,2024).

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.

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.

Diseases associated with the accumulation of ordered protein aggregates, amyloid fibrils, once thought to be rare, are predicted to soon become epidemics [...]

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.

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.

BackgroundThe 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 ReviewThe 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 ReviewOur 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.