Amyloid Structures Research Articles

Molecular pathology of neurodegenerative diseases by cryo-EM of amyloids.

Abnormal assembly of tau, α-synuclein, TDP-43 and amyloid-β proteins into amyloid filaments defines most human neurodegenerative diseases. Genetics provides a direct link between filament formation and the causes of disease. Developments in cryo-electron microscopy (cryo-EM) have made it possible to determine the atomic structures of amyloids from postmortem human brains. Here we review the structures of brain-derived amyloid filaments that have been determined so far and discuss their impact on research into neurodegeneration. Whereas a given protein can adopt many different filament structures, specific amyloid folds define distinct diseases. Amyloid structures thus provide a description of neuropathology at the atomic level and a basis for studying disease. Future research should focus on model systems that replicate the structures observed in disease to better understand the molecular mechanisms of disease and develop improved diagnostics and therapies.

Interfacial Aβ fibril formation is modulated by the disorder-order state of the lipids: The concept of the physical environment as amyloid inductor in biomembranes

The behavior of amphiphilic molecules such as lipids, peptides and their mixtures at the air/water interface allow us to evaluate and visualize the arrangement formed in a confined and controlled surface area. We have studied the surface properties of the zwitterionic DPPC lipid and Aβ(1–40) amyloid peptide in mixed films at different temperatures (from 15 to 40 °C). In this range of temperature the surface properties of pure Aβ(1–40) peptide remained unchanged, whereas DPPC undergoes its characteristic liquid-expanded → liquid-condensed bidimensional phase transition that depends on the temperature and lateral pressure. This particular property of DPPC makes it possible to dynamically study the influence of the lipid phase state on amyloid structure formation at the interface in a continuous, isothermal and abrupt change on the environmental condition. As the mixed film is compressed the fibril-like structure of Aβ(1–40) is triggered specifically in the liquid-expanded region, independently of temperature, and it is selectively excluded from the well-visible liquid condensed domains of DPPC. The Aβ amyloid fibers were visualized by using BAM and AFM and they were Thio T positive. In mixed DPPC/Aβ(1–40) films the condensed domains (in between 11 mN/m to 20 mN/m) become irregular probably due to the fibril-like structures is imposing additional lateral stress sequestering lipid molecules in the surrounding liquid-expanded phase to self-organize into amyloids.

PRIONS AND AMYLOIDS AS SPATIAL TEMPLATES OF THE PROTEOME

Until recently, studies of amyloids were aimed exclusively at revealing their role in the occurrence of dangerous diseases in humans and animals. However, they are widely distributed in nature and are involved in the regulation of essential vital processes in representatives of all three domains of the living world: archaea, bacteria and eukaryotes. The question of the biological significance of the prions – a special class of amyloids, isstill under discussion. The discovery of new functional amyloids became possible due to the development of the bioinformatic and proteomic methods for identification of amyloid-forming proteins. The review describes the way from the study of pathological amyloid structures to the investigation of adaptive amyloids inbacteria, plants, and animals. The importance of the amyloid structure, based on the principle of conformation template copying, as one of the most important forms of supramolecular organization of proteins isshown.

Cross-Linked α-Synuclein as Inhibitor of Amyloid Formation.

The aggregation and amyloid formation of α-synuclein is associated with Parkinson's disease and other synucleinopathies. In its native, monomeric form α-synuclein is an intrinsically disordered protein represented by highly dynamic conformational ensembles. Inhibition of α-synuclein aggregation using small molecules, peptides, or proteins has been at the center of interest in recent years. Our aim was to explore the effects of cross-linking on the structure and aggregation/amyloid formation properties of α-synuclein. Comparative analysis of available high-resolution amyloid structures and representative structural models and MD trajectory of monomeric α-synuclein revealed that potential cross-links in the monomeric protein are mostly incompatible with the amyloid forms and thus might inhibit fibrillation. Monomeric α-synuclein has been intramolecularly chemically cross-linked under various conditions using different cross-linkers. We determined the location of cross-links and their frequency using mass spectrometry and found that most of them cannot be realized in the amyloid structures. The inhibitory potential of cross-linked proteins has been experimentally investigated using various methods, including thioflavin-T fluorescence and transmission electron microscopy. We found that conformational constraints applied by cross-linking fully blocked α-synuclein amyloid formation. Moreover, DTSSP-cross-linked molecules exhibited an inhibitory effect on the aggregation of unmodified α-synuclein as well.

Phenol-soluble modulins form amyloids in contact with multiple surface chemistries

Functional amyloids are commonly produced by many microorganisms and their biological functions are numerous. Staphylococcus aureus can secrete a group of peptides named phenol-soluble modulins (PSMs) in their biofilm extracellular matrix. PSMs have been found inside biofilms both in their soluble form and assembled into amyloid structures. Yet, the actual biological function of these amyloids has been highly debated.Here, we assessed the ability of PSMs to form amyloids in contact with different abiotic surfaces to unravel a potential unknown bioadhesive and/or biofilm stabilization function. We combined surface plasmon resonance imaging, fluorescence aggregation kinetics, and FTIR spectroscopy in order to evaluate the PSM adsorption as well as amyloid formation properties in the presence of various surface chemistries.Overall, PSMs adsorb even on low-binding surfaces, making them highly adaptable adsorbants in the context of bioadhesion. Moreover, the PSM aggregation potential to form amyloid aggregates is not impacted by the presence of the surface chemistries tested. This versatility regarding adsorption and amyloid formation may imply a possible role of PSMs in biofilm adhesion and/or structure integrity.

Data-mining unveils structure–property–activity correlation of viral infectivity enhancing self-assembling peptides

Gene therapy via retroviral vectors holds great promise for treating a variety of serious diseases. It requires the use of additives to boost infectivity. Amyloid-like peptide nanofibers (PNFs) were shown to efficiently enhance retroviral gene transfer. However, the underlying mode of action of these peptides remains largely unknown. Data-mining is an efficient method to systematically study structure–function relationship and unveil patterns in a database. This data-mining study elucidates the multi-scale structure–property–activity relationship of transduction enhancing peptides for retroviral gene transfer. In contrast to previous reports, we find that not the amyloid fibrils themselves, but rather µm-sized β-sheet rich aggregates enhance infectivity. Specifically, microscopic aggregation of β-sheet rich amyloid structures with a hydrophobic surface pattern and positive surface charge are identified as key material properties. We validate the reliability of the amphiphilic sequence pattern and the general applicability of the key properties by rationally creating new active sequences and identifying short amyloidal peptides from various pathogenic and functional origin. Data-mining—even for small datasets—enables the development of new efficient retroviral transduction enhancers and provides important insights into the diverse bioactivity of the functional material class of amyloids.

Heterotypic liquid-liquid phase separation of tau and α-synuclein: Implications for overlapping neuropathologies

Tauopathies and synucleinopathies are characterized by the aggregation of Tau and α-synuclein (AS) into amyloid structures, respectively. Individuals with these neuropathies have an elevated risk of developing subsequent neurodegenerative or comorbid disorders. Intriguingly, post-mortem brain examinations have revealed co-localization of Tau and AS aggregates, suggesting a synergistic pathological relationship with an adverse prognosis. The role of liquid-liquid phase separation (LLPS) in the development of neurodegenerative diseases is currently receiving significant attention, as it can contribute to the aggregation and co-deposition of amyloidogenic proteins. In this study, we investigated the phase separation behavior of Tau and AS under various insults, some of which are implicated in disease progression. Our findings demonstrate the formation of heterotypic droplets composed of Tau and AS at physiologically relevant mole ratios that mimic neurons' soma and terminal buttons. Importantly, these heterotypic droplets exhibit increased resistance to electrostatic screening compared to homotypic condensates. Moreover, we observed that biologically relevant biomolecules, known to be dysregulated in disease, exert different effects on these droplets. Additionally, we provide evidence that phase separation itself influences the amyloid aggregation of Tau and AS, underscoring the significance of this process in the development of aggregopathies.

Putative Pore Structures of Amyloid β 25-35 in Lipid Bilayers.

The amyloid β peptide aggregates to form extracellular plaques in the brains of Alzheimer's disease patients. Certain of its fragments have been found to have similar properties to those of the full-length peptide. The best-studied of these is 25-35, which aggregates into fibrils, is toxic to neurons, and forms ion channels in synthetic lipid bilayers. Here, we investigate possible pore-forming structures of oligomers of this peptide in a POPC/POPG membrane. We consider octameric and decameric β-barrels of different topology, strand orientation, and shear, evaluate their stability in an implicit membrane model, and subject the best models to multimicrosecond all-atom molecular dynamics simulations. We find two decameric structures that are kinetically stable in membranes on this time scale: an imperfectly closed antiparallel β-barrel with K28 in the pore lumen and a short parallel β-barrel with K28 toward the membrane interface. Both structures exhibit dehydrated gaps in the pore lumen, which are larger for the antiparallel barrel. Based on these results, the experimental cation selectivity, the dependence of ion channel activity on voltage direction, and certain mutation data, the parallel model seems more compatible with experimental data.

Fibrils Emerging from Droplets: Molecular Guiding Principles behind Phase Transitions of a Short Peptide-Based Condensate Studied by Solid-State NMR.

Biochemical reactions occurring in highly crowded cellular environments require different means of control to ensure productivity and specificity. Compartmentalization of reagents by liquid-liquid phase separation is one of these means. However, extremely high local protein concentrations of up to 400 mg/ml can result in pathological aggregation into fibrillar amyloid structures, a phenomenon that has been linked to various neurodegenerative diseases. Despite its relevance, the process of liquid-to-solid transition inside condensates is still not well understood at the molecular level. We thus herein use small peptide derivatives that can undergo both liquid-liquid and subsequent liquid-to-solid phase transition as model systems to study both processes. Using solid-state nuclear magnetic resonance (NMR) and transmission electron microscopy (TEM), we compare the structure of condensed states of leucine, tryptophan and phenylalanine containing derivatives, distinguishing between liquid-like condensates, amorphous aggregates and fibrils, respectively. A structural model for the fibrils formed by the phenylalanine derivative was obtained by an NMR-based structure calculation. The fibrils are stabilised by hydrogen bonds and side-chain π-π interactions, which are likely much less pronounced or absent in the liquid and amorphous state. Such noncovalent interactions are equally important for the liquid-to-solid transition of proteins, particularly those related to neurodegenerative diseases.

Improved curcumin bioaccessibility in Pickering emulsion fabricated by rice glutelin fibrils

Pickering emulsion fabricated by food grade-solid colloidal particles as templates for delivering sensitive nutrients has attracted increasing interest in recent years. In this work, rice glutelin fibrils-stabilized Pickering emulsion was successfully fabricated for loading curcumin. In addition, the amyloid and non-amyloid structures were separated from the heated rice glutelin (RG) samples, and the emulsifying and encapsulating performances of these fractions were systematically observed. It was noted that the retentates of glutelin fibrils (RGFs)-stabilized Pickering emulsion exhibited a self-standing behavior, along with the highest curcumin encapsulation efficiency (94.17%) among other samples. Compared to the total heated glutelin fibrils (TGFs), a stronger gel strength was observed in emulsion containing purified fibrils, probably due to the closely arranged polyhedral emulsion structure. However, the filtrates of glutelin fibrils (FGFs) particles-prepared emulsion presented a weak gel strength due to the hydrophilic properties, accompanying by a surprisingly rapid release rate of curcumin during digestion. Nevertheless, TGFs-stabilized emulsion achieved a high curcumin bioavailability (35.06%) and sustained controlled release behavior. These findings proved that the sustainable and biocompatible protein amyloid exhibited excellent performance in encapsulating curcumin and improving curcumin bioavailability.

The Cryo-EM Structure of Renal Amyloid Fibril Suggests Structurally Homogeneous Multiorgan Aggregation in AL Amyloidosis

Immunoglobulin light chain amyloidosis (AL) is caused by the aberrant production of amyloidogenic light chains (LC) that accumulate as amyloid deposits in vital organs. Distinct LC sequences in each patient yield distinct amyloid structures. However different tissue microenvironments may also cause identical protein precursors to adopt distinct amyloid structures. To address the impact of the tissue environment on the structural polymorphism of amyloids, we extracted fibrils from the kidney of an AL patient (AL55) whose cardiac amyloid structure was previously determined by our group. Here we show that the 4.0 Å resolution cryo-EM structure of the renal fibril is virtually identical to that reported for the cardiac fibril. These results provide the first structural evidence that LC amyloids independently deposited in different organs of the same AL patient share a common fold.

The role of tandem repeats in bacterial functional amyloids

Repetitivity and modularity of proteins are two related notions incorporated into multiple evolutionary concepts. We discuss whether they may also be essential for functional amyloids. Amyloids are proteins that create very regular and usually highly insoluble fibrils, which are often associated with neurodegeneration. However, recent discoveries showed that amyloid structure of a protein could also be beneficial and desired, e.g., to promote cell adhesion. Functional amyloids are proteins which differ in their characteristics from pathological amyloids, so that the fibril formation could be more under control of an organism. We propose that repeats in the sequence could regulate the aggregation propensity of these proteins. The inclusion of multiple symmetric interactions, due to the presence of the repeats, could be supporting and strengthening the desirable structural properties of functional amyloids. Our results show that tandem repeats in bacterial functional amyloids have a distinct characteristic. The pattern of repeats supports the appropriate level of fibril formation and better controllability of fibril stability. The repeats tend to be more imperfect, which attenuates excessive aggregation propensity. Their desired structure and function are also reinforced by their amino acid profile. Although in the study we focused on bacterial functional amyloids, due to their importance in biofilm formation, we propose that similar mechanisms could be employed in other functional amyloids which are designed by evolution to aggregate in a desirable manner, but not necessarily in pathological amyloids.

Cryo-EM structure and polymorphic maturation of a viral transduction enhancing amyloid fibril

Amyloid fibrils have emerged as innovative tools to enhance the transduction efficiency of retroviral vectors in gene therapy strategies. In this study, we used cryo-electron microscopy to analyze the structure of a biotechnologically engineered peptide fibril that enhances retroviral infectivity. Our findings show that the peptide undergoes a time-dependent morphological maturation into polymorphic amyloid fibril structures. The fibrils consist of mated cross-β sheets that interact by the hydrophobic residues of the amphipathic fibril-forming peptide. The now available structural data help to explain the mechanism of retroviral infectivity enhancement, provide insights into the molecular plasticity of amyloid structures and illuminate the thermodynamic basis of their morphological maturation.

Secondary structure in polymorphic forms of alpha-synuclein amyloids.

Numerous Alpha-synuclein amyloid structures available in PDB enable their comparative analysis. They are all characterized by a flat structure of each individual chain with an extensive network of inter-chain hydrogen bonds. The identification of such amyloid fibril structures requires determining the special conditions imposed on the torsion angles. Such conditions have already been formulated by the Authors resulting in the model of idealised amyloid. Here, we investigate the fit of this model in the group of A-Syn amyloid fibrils. We identify and describe the characteristic supersecondary structures in amyloids. Generally, the amyloid transformation is suggested to be the 3D to 2D transformation engaging mostly the loops linking Beta-structural fragments. The loop structure introducing the 3D organisation of Beta-sheet change to flat form (2D) introduces the mutual reorientation of Beta-strands enabling the large-scale H-bonds generation with the water molecules. Based on the model of idealised amyloid we postulate the hypothesis for amyloid fibril formation based on the shaking, an experimental procedure producing the amyloids.

Role of complementarity-determining regions 1 and 3 in pathologic amyloid formation by human immunoglobulin κ1 light chains

Background Immunoglobulin light chain (LC) amyloidosis is a life-threatening disease complicated by vast numbers of patient-specific mutations. We explored 14 patient-derived and engineered proteins related to κ1-family germline genes IGKVLD-33*01 and IGKVLD-39*01. Methods Hydrogen-deuterium exchange mass spectrometry analysis of conformational dynamics in recombinant LCs and their fragments was integrated with studies of thermal stability, proteolytic susceptibility, amyloid formation and amyloidogenic sequence propensity. The results were mapped on the structures of native and fibrillary proteins. Results Proteins from two κ1 subfamilies showed unexpected differences. Compared to their germline counterparts, amyloid LC related to IGKVLD-33*01 was less stable and formed amyloid faster, whereas amyloid LC related to IGKVLD-39*01 had similar stability and formed amyloid slower, suggesting different major factors influencing amyloidogenesis. In 33*01-related amyloid LC, these factors involved destabilization of the native structure and probable stabilization of amyloid. The atypical behavior of 39*01-related amyloid LC stemmed from increased dynamics/exposure of amyloidogenic segments in βC′V and βEV that could initiate aggregation and decreased dynamics/exposure near the Cys23–Cys88 disulfide. Conclusions The results suggest distinct amyloidogenic pathways for closely related LCs and point to the complementarity-defining regions CDR1 and CDR3, linked via the conserved internal disulfide, as key factors in amyloid formation.

Structural Specificity of Polymorphic Forms of α-Synuclein Amyloid.

The structural transformation producing amyloids is a phenomenon that sheds new light on the protein folding problem. The analysis of the polymorphic structures of the α-synuclein amyloid available in the PDB database allows analysis of the amyloid-oriented structural transformation itself, but also the protein folding process as such. The polymorphic amyloid structures of α-synuclein analyzed employing the hydrophobicity distribution (fuzzy oil drop model) reveal a differentiation with a dominant distribution consistent with the micelle-like system (hydrophobic core with polar shell). This type of ordering of the hydrophobicity distribution covers the entire spectrum from the example with all three structural units (single chain, proto-fibril, super-fibril) exhibiting micelle-like form, through gradually emerging examples of local disorder, to structures with an extremely different structuring pattern. The water environment directing protein structures towards the generation of ribbon micelle-like structures (concentration of hydrophobic residues in the center of the molecule forming a hydrophobic core with the exposure of polar residues on the surface) also plays a role in the amyloid forms of α-synuclein. The polymorphic forms of α-synuclein reveal local structural differentiation with a common tendency to accept the micelle-like structuralization in certain common fragments of the polypeptide chain of this protein.

Amyloid fibril formation, structure and domain swapping of acyl-coenzyme A thioesterase-7.

Acyl-coenzyme A thioesterase (Acot) enzymes are involved in a broad range of essential intracellular roles including cell signalling, lipid metabolism, inflammation and the opening of ion channels. Dysregulation in lipid metabolism has been linked to neuroinflammatory and neurological disorders such as Alzheimer's and Parkinson's diseases. Structurally, Acot enzymes adopt a circularised trimeric arrangement with each monomer containing an N- and a C-terminal hotdog domain. Acot7 spontaneously forms amyloid fibrils in vitro under physiological conditions. The resultant amyloid fibrillar structures were characterised by dye-binding fluorescence assays, far-UV circular dichroism spectroscopy, transmission electron microscopy and X-ray fibre diffraction. Acot7 has an unusual mechanism of aggregation with no lag phase. The initial phase (~ 18 h) of aggregation involves conformational rearrangement within the oligomers to form species of enhanced β-sheet character. The subsequent loss of α-helical structure is accompanied by large-scale amyloid fibril formation. The crystal structure of Acot7 revealed an unexpected arrangement of the two domains within the circularised trimeric structure, which is the basis for a proposed mechanism of amyloid fibril formation involving domain swapping during the initial phase of aggregation. Acot7 formed fibrils in the presence of its substrate arachidonoyl-CoA and its inhibitors and maintained its enzyme activity during fibril assembly. It is proposed that the Acot7 fibrillar form acts as functional amyloid.

Controversial Properties of Amyloidogenic Proteins and Peptides: New Data in the COVID Era.

For a long time, studies of amyloidogenic proteins and peptides (amyloidogenic PPs) have been focused basically on their harmful properties and association with diseases. A vast amount of research has investigated the structure of pathogenic amyloids forming fibrous deposits within or around cells and the mechanisms of their detrimental actions. Much less has been known about the physiologic functions and beneficial properties of amyloidogenic PPs. At the same time, amyloidogenic PPs have various useful properties. For example, they may render neurons resistant to viral infection and propagation and stimulate autophagy. We discuss here some of amyloidogenic PPs' detrimental and beneficial properties using as examples beta-amyloid (β-amyloid), implicated in the pathogenesis of Alzheimer's disease (AD), and α-synuclein-one of the hallmarks of Parkinson's disease (PD). Recently amyloidogenic PPs' antiviral and antimicrobial properties have attracted attention because of the COVID-19 pandemic and the growing threat of other viral and bacterial-induced diseases. Importantly, several COVID-19 viral proteins, e.g., spike, nucleocapsid, and envelope proteins, may become amyloidogenic after infection and combine their harmful action with the effect of endogenous APPs. A central area of current investigations is the study of the structural properties of amyloidogenic PPs, defining their beneficial and harmful properties, and identifying triggers that transform physiologically important amyloidogenic PPs into vicious substances. These directions are of paramount importance during the current SARS-CoV-2 global health crisis.

Dissolution of Lysozyme Amyloid Fibrils Using Magnetic Nanoparticles in an Alternating Magnetic Field: Design of an Effective Treatment for Cutaneous Amyloidosis

The purpose of this study was to apply functionalized magnetic nanoparticles for the treatment of amyloidosis, a disease characterized by the accumulation of aberrant protein forms with an insoluble amyloid structure. The dissolution and clearance of these extremely stable fibrils from lesions is very complicated. For this purpose, we examined the possibility of using magnetic nanoparticles that generate heat in an external alternating magnetic field with a frequency of 3.5 MHz. As a convenient model system, we used lysozyme fibrils. For the quantification of fibrillar status, we used Thioflavin T and Congo red, specific dyes which change their spectroscopic properties upon binding with the cross-beta structure of fibrils. We found that by using fluorescence, and polarization microscopy, as well as absorption spectrophotometry, the amyloid-like fibrils can be almost completely dissolved. The obtained results suggest that the application of magnetic nanoparticles could be a possible therapeutic intervention in cutaneous amyloidosis.

EMBER multidimensional spectral microscopy enables quantitative determination of disease- and cell-specific amyloid strains

In neurodegenerative diseases, proteins fold into amyloid structures with distinct conformations (strains) that are characteristic of different diseases. However, there is a need to rapidly identify amyloid conformations insitu. Here, we use machine learning on the full information available in fluorescent excitation/emission spectra of amyloid-binding dyes to identify six distinct different conformational strains invitro, as well as amyloid-β (Aβ) deposits in different transgenic mouse models. Our EMBER (excitation multiplexed bright emission recording) imaging method rapidly identifies conformational differences in Aβ and tau deposits from Down syndrome, sporadic and familial Alzheimer's disease human brain slices. EMBER has insitu identified distinct conformational strains of tau inclusions in astrocytes, oligodendrocytes, and neurons from Pick's disease. In future studies, EMBER should enable high-throughput measurements of the fidelity of strain transmission in cellular and animal neurodegenerative diseases models, time course of amyloid strain propagation, and identification of pathogenic versus benign strains.