Amyloid Assembly Research Articles

Amyloid assembly and disassembly.

ABSTRACTAmyloid fibrils are protein homopolymers that adopt diverse cross-β conformations. Some amyloid fibrils are associated with the pathogenesis of devastating neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease. Conversely, functional amyloids play beneficial roles in melanosome biogenesis, long-term memory formation and release of peptide hormones. Here, we showcase advances in our understanding of amyloid assembly and structure, and how distinct amyloid strains formed by the same protein can cause distinct neurodegenerative diseases. We discuss how mutant steric zippers promote deleterious amyloidogenesis and aberrant liquid-to-gel phase transitions. We also highlight effective strategies to combat amyloidogenesis and related toxicity, including: (1) small-molecule drugs (e.g. tafamidis) to inhibit amyloid formation or (2) stimulate amyloid degradation by the proteasome and autophagy, and (3) protein disaggregases that disassemble toxic amyloid and soluble oligomers. We anticipate that these advances will inspire therapeutics for several fatal neurodegenerative diseases.

Responsibility of lipid compositions for the amyloid ß assembly induced by ganglioside nanoclusters in mouse synaptosomal membranes

The assembly of amyloid s protein (As) on ganglioside-enriched microdomains in presynaptic neuronal membranes is involved in the onset of Alzheimer’s disease. We previously found that a highly enriched ganglioside domain generated in a reconstituted lipid bilayer composed of synaptosomal lipids functioned as an As-sensitive ganglioside nanocluster (ASIGN) that may induce a spherical As assembly. In the present study, the major lipid components of a detergent-resistant membrane microdomain (DRM) fraction of synaptosomes prepared from an aged mouse brain were identified, and we demonstrate that the proportion of As-insensitive gangliosides such as GM3 directly affects the As assembly in synaptosomal membranes. Liquid chromatography coupled to mass spectrometry analyses indicated that ganglio a-series gangliosides (GM1, GM2, GM3, and GD1a) were abundant in DRM fractions. In addition, the GM3/GM1 ratio and the cholesterol content in the synaptosomal fraction differed from those in the non-synaptosomal fraction, in which GM1 and GM3 are sensitive and insensitive gangliosides, respectively. The highly enriched ganglioside nanocluster was identified by the surface topography of ternary mixed lipid bilayers composed of gangliosides, sphingomyelin, and cholesterol. Our results indicate that the composition of gangliosides is responsible for the characteristics of the nanocluster generated in the presynaptic neuronal membranes. Amyloid s-protein (As) is converted to toxic forms through interactions with the ganglioside in neuronal membranes. The highly ganglioside-enriched microdomain (ganglioside cluster) in neuronal membranes plays a key role in As assembly. In the present study, lipid components of synaptosome extracted from mouse aged brain was determined by LC-MS spectroscopy. We demonstrated that ganglioside ratio (GM3 to GM1) and cholesterol content are an important factor for inducing As assembly. These results provides important insight into the mechanism of polypeptide assembly on the neuronal membrane in Alzheimer’s disease patients.

Bacterial Model Membranes Reshape Fibrillation of a Functional Amyloid Protein.

Biofilms are aggregates of cells that form surface-associated communities. The cells in biofilms are interconnected with an extracellular matrix, a network that is made mostly of polysaccharides, proteins, and sometimes nucleic acids. Some extracellular matrix proteins form fibers, termed functional amyloid or amyloid-like, to differentiate their constructive function from disease-related amyloid fibers. Recent functional amyloid assembly studies have neglected their interaction with membranes, despite their native formation in a cellular environment. Here, we use TasA, a major matrix protein in biofilms of the soil bacterium Bacillus subtilis, as a model functional amyloid protein and ask whether the bacterial functional amyloid interacts with membranes. Using biochemical, spectroscopic, and microscopic tools, we show that TasA interacts distinctively with bacterial model membranes and that this interaction mutually influences the morphology and structure of the protein and the membranes. At the protein level, fibers of similar structure and morphology are formed in the absence of membranes and in the presence of eukaryotic model membranes. However, in the presence of bacterial model membranes, TasA forms disordered aggregates with a different β sheet signature. At the membrane level, fluorescence microscopy and anisotropy measurements indicate that bacterial membranes deform more considerably than eukaryotic membranes upon interaction with TasA. Our findings suggest that TasA penetrates bacterial more than eukaryotic model membranes and that this leads to membrane disruption and to reshaping the TasA fiber formation pathway. Considering the important role of TasA in providing integrity to biofilms, our study may direct the design of antibiofilm drugs to the protein-membrane interface.

Amyloid Assembly Endows Gad m 1 with Biomineralization Properties.

Acid proteins capable of nucleating Ca2+ and displaying aggregation capacity play key roles in the formation of calcium carbonate biominerals. The helix-loop helix EF-hands are the most common Ca2+-binding motifs in proteins. Calcium is bound by the loop region. These motifs are found in many proteins that are regulated by calcium. Gad m 1, an Atlantic cod β-parvalbumin isoform, is a monomeric EF-hand protein that acts as a Ca2+ buffer in fish muscle; the neutral and acid apo-forms of this protein can form amyloids. Since Ca2+-nucleating proteins have a propensity to form extended β-strand structures, we wondered whether amyloid assemblies of an EF-hand protein were able to influence calcium carbonate crystallization in vitro. Here, we used the Gad m 1 chain as a model to generate monomeric and amyloid assemblies and to analyze their effect on calcite formation in vitro. We found that only amyloid assemblies alter calcite morphology.

Adsorption and Fibrillization of Islet Amyloid Polypeptide at Self-Assembled Monolayers Studied by QCM-D, AFM, and PM-IRRAS.

Aggregation and fibrillization of human islet amyloid polypeptide (hIAPP) plays an important role in the development of type 2 diabetes mellitus. Understanding the interaction of hIAPP with interfaces such as cell membranes at a molecular level therefore represents an important step toward new therapies. Here, we investigate the fibrillization of hIAPP at different self-assembled alkanethiol monolayers (SAMs) by quartz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM), and polarization-modulated infrared reflectionabsorption spectroscopy (PM-IRRAS). We find that hydrophobic interactions with the CH3-terminated SAM tend to retard hIAPP fibrillization compared to the carboxylic acid-terminated SAM where attractive electrostatic interactions lead to the formation of a three-dimensional network of interwoven fibrils. At the hydroxyl- and amino-terminated SAMs, fibrillization appears to be governed by hydrogen bonding between the peptide and the terminating groups which may even overcome electrostatic repulsion. These results thus provide fundamental insights into the molecular mechanisms governing amyloid assembly at interfaces.

The Extracellular Polymeric Substances of Legionella pneumophila Biofilms Contain Amyloid Structures.

Human infection by bacteria of the genus Legionella most often result in the pneumonia known as Legionnaires Disease. Legionella is found as a resident of adherent biofilms in man-made water systems. Disinfection efforts to prevent Legionella infections require a better understanding of the structures that promote Legionella surface attachment and biofilm colonization. Various enzymatic treatments, including multiple carbohydrate-targeting mixtures, failed to disrupt Legionella biofilms, despite the presence of carbohydrates in the biofilms as shown by biochemical methods and concanavalin-A lectin staining. Moreover, Legionella biofilms contained amyloids as detected by three microscopic staining methods (congo red, thioflavin T, and the amyloid-specific antibody WO2). Amyloid structures were seen in biofilms of both L. pneumophila and L. longbeachae, the two Legionella species most associated with human infection. Inhibition of amyloid assembly by congo red and thioflavin T limited both self-aggregation and surface attachment of L. pneumophila, indicating that functional amyloid structures have a key role in initial biofilm formation by these pathogenic bacteria.

Supramolecular Modulation of Structural Polymorphism in Pathogenic α-Synuclein Fibrils Using Copper(II) Coordination.

Structural variation of α-synuclein (αSyn) fibrils has been linked to the diverse etiologies of synucleinopathies. However, little is known about what specific mechanism provides αSyn fibrils with pathologic features. Herein, we demonstrate Cu(II)-based supramolecular approach for unraveling the formation process of pathogenic αSyn fibrils and its application in a neurotoxic mechanism study. The conformation of αSyn monomer was strained by macrochelation with Cu(II), thereby disrupting the fibril elongation while promoting its nucleation. This non-canonical process formed shortened, β-sheet enriched αSyn fibrils (<0.2 μm) that were rapidly transmitted and accumulated to neuronal cells, causing neuronal cell death, in sharp contrast to typical αSyn fibrils (ca. 1 μm). Our approach provided the supramolecular basis for the formation of pathogenic fibrils through physiological factors, such as brain Cu(II).

Differential Modulating Effect of MoS2 on Amyloid Peptide Assemblies.

The abnormal fibrillogenesis of amyloid peptides such as amyloid fibril and senior amyloid plaques, is associated with the pathogenesis of many amyloid diseases. Hence, modulation of amyloid assemblies is related to the possible pathogenesis of some diseases. Some two-dimensional nanomaterials, that is, graphene oxide, tungsten disulfide, exhibit strong modulation effects on the amyloid fibrillogenesis. Herein, the modulation effect of molybdenum disulfide on two amyloid peptide assemblies based on the label-free techniques is presented, including quartz crystal microbalance (QCM), AFM, and CD spectroscopy. MoS2 presents different modulating effects on the assembly of amyloid-β peptide (33-42) [Aβ (33-42)] and amylin (20-29), mainly owing to the distinct affinity between amyloid peptides and MoS2 . This is to our knowledge the first report of MoS2 as a modulator for amyloid aggregation. It enriches the variety of 2D nanomodulators of amyloid fibrillogenesis and explains the mechanism for the self-assembly of amyloid peptides, and expands the applications of MoS2 in biology.

Evidence for a central role of PrP helix 2 in the nucleation of amyloid fibrils.

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

Structural Insights into Aggregation Mechanism of Immunoglobulin Light Chain Variable Domain

Light chain (AL) or systemic amyloidosis is an amyloid disease resulting from an over production of immunoglobulin light chain (IgG) due to abnormal proliferation of monoclonal plasma B cells. These abundant circulating light chains misfold and gets deposited in the form of insoluble fibrils in the extracellular space of various organs leading to their impairment. AL amyloidosis is further complicated by the fact that each patient involving a similar or different organ often tend to present a slightly different protein due to somatic hyper mutation that might aggregate following different kinetics. Little is known about the mechanism of amyloid assembly in AL amyloidosis. Investigation of the aggregation pathways and stages is especially important as many of these involve intermediates structures that could be cytotoxic and play a critical role in the initiation of amyloid assembly. In this work, we discuss the mechanism of in vitro fibril formation in germline (wild type/control) protein κI O18/O8 and its amyloidogenic mutants AL-09 and AL-12. Both amyloidogenic proteins aggregate significantly faster as compared to germline protein with similar rates of aggregation for both AL-09 and AL-12. We also observed that during early stages of aggregation, AL-09 rapidly forms transient low molecular weight oligomers that polymerize into mature fibrils. The single restorative mutation AL-09 H87Y (restoring the amino acid in the germline sequence) alters the aggregation kinetics of AL-09. As compared to AL-09, the rate of aggregation in AL-09 H87Y drastically decreases and the low molecular weight oligomers disappear completely. On the contrary, AL-12 appears to aggregate by polymerization of soluble protein without involving any oligomeric intermediates. The single restorative mutation of AL-12 (AL-12 R65S) to its germline sequence had no effect and appears to have very similar rates of aggregation compared to AL-12.

Preparation of α-Synuclein Amyloid Assemblies for Toxicity Experiments.

Amyloid assemblies of certain proteins, including the Parkinson disease-related protein α-synuclein, are commonly associated with the development and spreading of neurodegenerative diseases, although the nature of the most toxic forms and the mechanisms by which they trigger neurodegeneration remain largely unknown. This is at least in part due to the inherent challenges involved in the preparation of stable and structurally homogeneous samples of amyloid assemblies that could be used in toxicity experiments. Here, we describe the preparation of two different types of stable α-synuclein amyloid assemblies, namely a kinetically trapped oligomeric species and a propagating-competent fibrillar polymorph. The degree of heterogeneity in the samples has been defined and carefully minimized, thus allowing for meaningful structure-toxicity relationships in different α-synuclein amyloid assemblies to be established.

An Aggregate Weight-Normalized Thioflavin-T Measurement Scale for Characterizing Polymorphic Amyloids and Assembly Intermediates.

The red shift in the fluorescence excitation spectra of thioflavin dyes upon binding to fibrils has been a boon to the amyloid field, offering simple and effective methods for the qualitative detection of amyloid in tissue samples and for quantitation of particular fibril preparations with gravimetric linearity. The quantitative aspect of the thioflavin T (ThT) response, however, comes with an important caveat that bestows both significant limitations and great untapped power. It is now well established that amyloid fibrils of different proteins, as well as polymorphic fibrils of the same protein, can exhibit vastly different ThT fluorescence intensities for the same weight concentration of aggregates. Furthermore, the aggregated intermediates commonly observed in amyloid assembly reactions can exhibit aggregate weight-normalized (AWN) ThT fluorescence intensities that vary from essentially zero through a wide range of intermediate values before reaching the intensity of homogeneous, mature amyloid. These features make it very difficult to quantitatively interpret, without additional data, the time-dependent development of ThT fluorescence intensity in an assembly reaction. In this chapter, we describe a method for coupling ex situ ThT fluorescence determinations with an analytical HPLC supported sedimentation assay (also described in detail) that can provide significant new insights into amyloid assembly reactions. The time dependent aggregation data provided by the sedimentation assay reveals a time course of aggregation that is largely independent of aggregate properties. In addition, the combination of these data with ThT measurements of the same reaction time points reveals important aspects of average aggregate structure at each time point. Examples of the use and potential value of AWN-ThT measurements during amyloid assembly Aβ and polyglutamine peptides are provided.

Solid-State NMR Studies of Amyloid Materials: A Protocol to Define an Atomic Model of Aβ(1-42) in Amyloid Fibrils.

Intense efforts have been made to understand the molecular structures of misfolded amyloid β (Aβ) in order to gain insight into the pathological mechanism of Alzheimer's disease. Solid-state NMR spectroscopy (SSNMR) is considered a primary tool for elucidating the structures of insoluble and noncrystalline amyloid fibrils and other amyloid assemblies. In this chapter, we describe a detailed protocol to obtain the first atomic model of the 42-residue human Aβ peptide Aβ(1-42) in structurally homogeneous amyloid fibrils from our recent SSNMR study (Nat Struct Mol Biol 22:499-505, 2015). Despite great biological and clinical interest in Aβ(1-42) fibrils, their structural details have been long-elusive until this study. The protocol is divided into four sections. First, the solid-phase peptide synthesis (SPPS) and purification of monomeric Aβ(1-42) is described. We illustrate a controlled incubation method to prompt misfolding of Aβ(1-42) into homogeneous amyloid fibrils in an aqueous solution with fragmented Aβ(1-42) fibrils as seeds. Next, we detail analysis of Aβ(1-42) fibrils by SSNMR to obtain structural restraints. Finally, we describe methods to construct atomic models of Aβ(1-42) fibrils based on SSNMR results through two-stage molecular dynamics calculations.

Methods to Characterize the Nanostructure and Molecular Organization of Amphiphilic Peptide Assemblies.

Methods to characterize the nanostructure and molecular organization of aggregates of peptides such as amyloid or amphiphilic peptide assemblies are reviewed. We discuss techniques to characterize conformation and secondary structure including circular and linear dichroism and FTIR and Raman spectroscopies, as well as fluorescence methods to detect aggregation. NMR spectroscopy methods, especially solid-state NMR measurements to probe beta-sheet packing motifs, are also briefly outlined. Also discussed are scattering methods including X-ray diffraction and small-angle scattering techniques including SAXS (small-angle X-ray scattering) and SANS (small-angle neutron scattering) and dynamic light scattering. Imaging methods are direct methods to uncover features of peptide nanostructures, and we provide a summary of electron microscopy and atomic force microscopy techniques. Selected examples are highlighted showing data obtained using these techniques, which provide a powerful suite of methods to probe ordering from the molecular scale to the aggregate superstructure.

Microfluidic approaches for probing amyloid assembly and behaviour.

The self-assembly of proteins into supramolecular structures and machinery underpins biological activity in living systems. Misassembled, misfolded and aggregated protein structures can, by contrast, have deleterious activity and such species are at the origin of a number of disease states ranging from cancer to neurodegenerative disorders. In particular, the formation of highly ordered protein aggregates, amyloid fibrils, from normally soluble peptides and proteins, is the common pathological hallmark of a range a group of over fifty protein misfolding disorders. Because of the critical role of the process in the aetiology of such disorders, as well as the quest to understand the basic principles of protein folding and misfolding, the amyloid phenomenon has become a central area of modern biomedical research. Advances in our knowledge of the physical properties of amyloid systems have, however, also highlighted the potential of amyloid structures in the context of materials science. In this review, we explore how microfluidic approaches can be used to study aspects of amyloid assembly and behaviour that are challenging to probe under bulk solution conditions. We discuss the use of volume confinement to probe very early events in the amyloid formation process. In addition, the well-defined fluid flow properties within channels with dimensions on the micron scale can be exploited to measure the physical properties of protein aggregates, such as their sizes and charges, to shed light on the physical and chemical parameters defining amyloid species. Moreover, the molecular species formed during aggregation reactions have physical dimensions spanning at least three orders of magnitude, and microfluidic techniques are well suited to work with analytes of such disparate dimensions. Furthermore, the flexibility of the design of microfluidic devices lends itself to adaptable experimental setups, including the study of protein self-assembly within living cells. Finally, we highlight the salient features of microfluidic experiments that facilitate probing complex biological systems, and discuss their use in the exploration of amyloids as a class of functional material.

Engineering and evaluation of amyloid assemblies as a nanovaccine against the Chikungunya virus.

The design of nanoparticles exposing a high density of antigens constitutes a promising strategy to address safety concerns of conventional life-attenuated vaccines as well as to increase the immunogenicity of subunit vaccines. In this study, we developed a fully synthetic nanovaccine based on an amyloid peptide sequence with high self-assembling properties. The immunogenic epitope E2EP3 from the E2 glycoprotein of the Chikungunya virus was used to evaluate the potential of a 10-mer peptide derived from an endogenous amyloidogenic polypeptide as a novel vaccine platform. Chimeric peptides, comprising the peptide antigen attached to the amyloid core by a short flexible linker, were prepared by solid phase synthesis. As observed using atomic force microscopy, these polypeptides self-assembled into linear and unbranched fibrils with a diameter ranging from 6 to 8 nm. A quaternary conformation rich in cross-β-sheets characterized these assemblies, as demonstrated by circular dichroism spectroscopy and thioflavin T fluorescence. ELISA assays and transmission electronic microscopy of immunogold labeled-fibrils revealed a high density of the Chikungunya virus E2 glycoprotein derived epitope exposed on the fibril surface. These amyloid fibrils were cytocompatible and were efficiently uptaken by macrophages. Mice immunization revealed a robust IgG response against the E2EP3 epitope, which was dependent on self-assembly and did not require co-injection of the Alhydrogel adjuvant. These results indicate that cross-β-sheet amyloid assemblies constitute suitable synthetic self-adjuvanted assemblies to anchor antigenic determinants and to increase the immunogenicity of peptide epitopes.

A structural model of the hierarchical assembly of an amyloid nanosheet by an infrared probe technique.

Rational design of amyloid-based materials requires structural insight into such materials. Here, we explore the use of a side-chain-based infrared (IR) probe technique combined with atomic force microscopy, Raman spectroscopy, UV-Vis spectroscopy, and thermogravimetric analysis coupled with mass spectrometry to elucidate the structural details of an amyloid nanosheet formed by an Aβ(16-22) variant, KLVXFAK, where X is p-cyanophenylalanine with its side-chain cyano group being an IR probe. Through the structural constraints obtained with the combined tools, we are able to propose a novel structural model for the amyloid nanosheet. The nanosheet can be viewed as a stack of class 7-type steric-zipper-like amyloid structures with a unique sheared intersheet arrangement: the β-sheets are stacked along a zippering axis with each individual β-sheet sheared relative to its adjacent β-sheets by two residues through the cyano-lysine intersheet hydrogen bond. With such a configuration, the side view of the nanosheet is similar to that of a stack of tilted-aligned roof tiles with each individual β-sheet being each tile. In addition, this work provides a nice example of how to utilize the side-chain-based IR probe technique combined with other supplemental tools to build a hierarchical structural model for a complex amyloid assembly.

Preparation and Screening of Catalytic Amyloid Assemblies.

Aggregation of proteins into amyloids has long been recognized as one of the major contributors to disease and aging. Amyloids are known to catalyze their own formation but they have been considered the rock-bottom thermodynamic minimum of the protein fold without much functionality. We have recently demonstrated that aggregation of short peptides in the presence of metal ions gives rise to efficient catalytic activity. Here we present a detailed protocol for the synthesis and purification of these peptides and the preparation of amyloid-like fibrils. Then we describe an easy-to-perform, high-throughput assay to measure their hydrolytic activity.

Multistep Conformation Selection in Amyloid Assembly.

Defining pathways for amyloid assembly could impact therapeutic strategies for as many as 50 disease states. Here we show that amyloid assembly is subject to different forces regulating nucleation and propagation steps and provide evidence that the more global β-sheet/β-sheet facial complementarity is a critical determinant for amyloid nucleation and structural selection.

Hypothesis for the cause and therapy of neurodegenerative diseases

The cause and therapy of neurodegenerative diseases remain unsolved puzzles. These diseases are correlated with presence of beta sheet-rich amyloid assemblies. Here, I derive and assemble puzzle pieces to obtain a loose end-tying hypothesis for cause with direct implications for therapy. I use the following extrapolations to find connectable puzzle pieces: (a) the traditional extrapolation that amyloid/amyloid precursors cause disease, (b) a recent extrapolation that amyloid-forming proteins, some of which are virus protein homologs, are components of an empirically obscure innate immune system that counters insults, including those by both viruses and bacteria, (c) a new extrapolation that various insults produce assemblies with structural features in common and that amyloid-forming, innate immune system proteins recognize these features and, then, counter insults by co-assembly, (d, 1) a second new extrapolation that beta sheet is a common structural feature and is extended during insult-neutralizing co-assembly and (d, 2) an appendix, derived from studies of phages T3 and T4, that most insult-produced assemblies are obscure to current biochemical analysis. The hypothesis is the following. One function of amyloid-forming proteins is non-classical innate immunity to biological insults. This immunity works via beta sheet-extending co-assembly of amyloid-forming proteins with beta sheet-containing insult products. For example, co-assembly with beta sheet-containing viral assembly intermediates inhibits virus production. Amyloid-forming proteins cause neurodegenerative disease when errant, typically overproduced. Other innate immunity systems sometimes exacerbate symptoms. This hypothesis suggests the following therapy, based on manipulating Nature’s chemistry. First, conduct directed evolution to obtain low-pathogenicity, chronic symptom-producing viruses with assembly intermediates that co-assemble with and destabilize both amyloid and amyloid sub-assemblies. Then, infect patients with these viruses.