Natural protective amyloids

Amyloidoses are a group of diseases including neurodegenerative diseases like Alzheimer's disease and also type II diabetes, spongiform encephalopathies and many others, believed to be caused by protein aggregation and subsequent amyloid fibril formation. However, occasionally, living organisms exploit amyloid fibril formation, a property inherent into amino acid sequences, and perform specific physiological functions from amyloids, in differing biological contexts. Some of these functional amyloids are natural protective amyloids. Here, we review recent evidence on silkmoth chorion protein synthetic peptide-analogues that documents the function of silkmoth chorion, the major component of the eggshell, a structure with extraordinary physiological and mechanical properties, as a natural protective amyloid. Also, we briefly discuss the reported function of other natural, protective amyloids like fish chorion, the protein Pmel17 which forms amyloid fibrils that act as templates and accelerate the covalent polymerization of reactive small molecules into melanin, the hydrophobins and the antifreeze protein from winter flounder. Molecular self-assembly is becoming an increasingly popular route to new supramolecular structures and molecular materials and the inspiration for such structures is commonly derived from self-assembling systems in biology. Therefore, a careful examination of these studies may set the basis for the exploration of new routes for the formation of novel biocompatible polymeric structures with exceptional physico-chemical properties, for potentially new biomedical and industrial applications.     

Multifaceted Influences of Melanin‐Like Particles on Amyloid‐beta Aggregation

The properties of eumelanin‐like particles (EMPs) and pheomelanin‐like particles (PMPs) in regulating the process of amyloid formation of amyloid‐beta 42 (Aβ42) were examined. EMPs and PMPs are effective both in interfering with amyloid aggregation of Aβ42 and in remodeling matured Αβ42 fibers. The results suggest that some (but not all) molecular species consisting of melanin‐like particles (MPs) are responsible for their inhibiting property toward amyloid formation, and the influence is likely manifested by long‐range interactions. Incubating preformed Aβ42 fibers with catechols or MPs leads to the formation of mesh‐like, interconnected Aβ42 fibers encapsulated with melanin‐like material. MPs are kinetically more effective than catechol monomers in this process, and a detailed investigation reveals that 4,5‐dihydroxyindole, a major intermediate in the formation of melanin‐like species, and its derivatives are mainly responsible for remodeling amyloid fibers.     

Mechanisms of protein oligomerization: inhibitor of functional amyloids templates α-synuclein fibrillation

Small organic molecules that inhibit functional bacterial amyloid fibers, curli, are promising new antibiotics. Here we investigated the mechanism by which the ring-fused 2-pyridone FN075 inhibits fibrillation of the curli protein CsgA. Using a variety of biophysical techniques, we found that FN075 promotes CsgA to form off-pathway, non-amyloidogenic oligomeric species. In light of the generic properties of amyloids, we tested whether FN075 would also affect the fibrillation reaction of human α-synuclein, an amyloid-forming protein involved in Parkinson's disease. Surprisingly, FN075 stimulates α-synuclein amyloid fiber formation as measured by thioflavin T emission, electron microscopy (EM), and atomic force microscopy (AFM). NMR data on (15)N-labeled α-synuclein show that upon FN075 addition, α-synuclein oligomers with 7 nm radius form in which the C-terminal 40 residues remain disordered and solvent exposed. The polypeptides in these oligomers contain β-like secondary structure, and the oligomers are detectable by AFM, EM, and size-exclusion chromatography (SEC). Taken together, FN075 triggers oligomer formation of both proteins: in the case of CsgA, the oligomers do not proceed to fibers, whereas for α-synuclein, the oligomers are poised to rapidly form fibers. We conclude that there is a fine balance between small-molecule inhibition and templation that depends on protein chemistry.     

Chirality of amyloid suprastructures

Amyloids are pathological fibrillar aggregates of proteins related to more than 20 different diseases. Amyloid fibers have a characteristic cross-beta structure consisting of a series of beta-strands extended perpendicular to the fiber axis and joined by hydrogen bonds parallel to the fiber direction. Fibril aggregation results in helical suprastructures. Here we used high-resolution SEM and cryo-SEM for the study of chirality in the amyloid suprastructure. We found that amyloids of Abeta1-40 and hen lysozyme form at all hierarchical levels always and only left-handed helices. In contrast, amyloid fibers formed by the N-terminal sequence of serum amyloid A (SAA1-12) consist of right-handed helices exclusively. Consistently, the peptide enantiomer, formed of (R)-aminoacids, aggregates exclusively in left-handed helices. We conclude that the opposite handedness of the SAA1-12 amyloids is an intrinsic feature of the peptide structure. The left-handed chirality observed for the Abeta1-40 and hen lysozyme amyloid suprastructures is consistent with the conventional beta-sheet structural model. In contrast, the right-handedness observed in (all-S) SAA1-12 fibers indicates that the cross-beta structure of SAA1-12 fibers is probably not formed of beta-sheets. Whatever the answer to the dilemma of the right-handed helicity of SAA1-12 amyloid fibers is, its existence shows that the supramolecular chirality of amyloid fibers is not only dictated by the molecular chirality of the component molecules but also by their structural organization.     

Functional amyloids from bacterial biofilms – structural properties and interaction partners

Protein aggregation and amyloid formation have historically been linked with various diseases such as Alzheimer''s and Parkinson''s disease, but recently functional amyloids have gained a great deal of interest in not causing a disease and having a distinct function in vivo. Functional bacterial amyloids form the structural scaffold in bacterial biofilms and provide a survival strategy for the bacteria along with antibiotic resistance. The formation of functional amyloids happens extracellularly which differs from most disease related amyloids. Studies of functional amyloids have revealed several distinctions compared to disease related amyloids including primary structures designed to optimize amyloid formation while still retaining a controlled assembly of the individual subunits into classical cross-β-sheet structures, along with a unique cross-α-sheet amyloid fold. Studies have revealed that functional amyloids interact with components found in the extracellular matrix space such as lipids from membranes and polymers from the biofilm. Intriguingly, a level of complexity is added as functional amyloids also interact with several disease related amyloids and a causative link has even been established between functional amyloids and neurodegenerative diseases. It is hence becoming increasingly clear that functional amyloids are not inert protein structures found in bacterial biofilms but interact with many different components including human proteins related to pathology. Gaining a clear understanding of the factors governing the interactions will lead to improved strategies to combat biofilm associated infections and the correlated antibiotic resistance. In the current review we summarize the current state of the art knowledge on this exciting and fast growing research field of biofilm forming bacterial functional amyloids, their structural features and interaction partners.

Functional bacterial amyloids forming biofilms have unique structural characteristics while still being similar to pathological ones. Through many identified interaction partners, they emerge as complex and essential components of biofilms.     

Identification of Regions in Apomyoglobin that Form Intermolecular Interactions in Amyloid Aggregates Using High-Performance Mass Spectrometry

The formation of amyloid aggregates in human organs and tissues causes the development of incurable diseases. However, experimental studies of the mechanism of amyloid formation by proteins and the structural characteristics of amyloids are complicated because of the heterogeneity and high molecular weight of the aggregates. We used limited proteolysis and mass spectrometry for the identification of regions in the apomyoglobin polypeptide chain, which give rise to intermolecular interactions in amyloid structures. Tandem mass spectroscopy enabled the identification of regions in the myoglobin polypeptide chain, which form the core of amyloid structures. It was shown that the main structural elements for the formation of the core of amyloid fibrils in myoglobin were regions from 60 through 90 and from 97 through 124 amino acid residues. These regions coincide well with those theoretically predicted. This approach yielded important data on the structure of protein molecules in aggregates and on conformational rearrangements of apomyoglobin upon amyloid formation.     

A Single Spherical Assembly of Protein Amyloid Fibrils Formed by Laser Trapping

Protein amyloids have received much attention owing to their correlation with serious diseases and to their promising mechanical and optical properties as future materials. Amyloid formation has been conducted by tuning temperature and chemical conditions, so that its nucleation and the following growth are analyzed as ensemble dynamics. A single spherical assembly of amyloid fibrils of cytochrome c domain‐swapped dimer was successfully generated upon laser trapping. The amyloid fibrillar structure was confirmed by fluorescence characterization and electron microscopy. The prepared spheres were further manipulated individually in solution to fabricate a three‐dimensional microstructure and a line pattern. Amyloid formation dynamics and amyloid‐based microstructure fabrication are demonstrated based on direct observation of a single spherical assembly, which foresees a new approach in amyloid studies.     

Self‐Assembled Cyclic d,l‐α‐Peptides as Generic Conformational Inhibitors of the α‐Synuclein Aggregation and Toxicity: In Vitro and Mechanistic Studies

Many peptides and proteins with large sequences and structural differences self‐assemble into disease‐causing amyloids that share very similar biochemical and biophysical characteristics, which may contribute to their cross‐interaction. Here, we demonstrate how the self‐assembled, cyclic d,l ‐α‐peptide CP‐2 , which has similar structural and functional properties to those of amyloids, acts as a generic inhibitor of the Parkinson′s disease associated α‐synuclein (α‐syn) aggregation to toxic oligomers by an ?off‐pathway“ mechanism. We show that CP‐2 interacts with the N‐terminal and the non‐amyloid‐β component region of α‐syn, which are responsible for α‐syn′s membrane intercalation and self‐assembly, thus changing the overall conformation of α‐syn. CP‐2 also remodels α‐syn fibrils to nontoxic amorphous species and permeates cells through endosomes/lysosomes to reduce the accumulation and toxicity of intracellular α‐syn in neuronal cells overexpressing α‐syn. Our studies suggest that targeting the common structural conformation of amyloids may be a promising approach for developing new therapeutics for amyloidogenic diseases.     

Dynamics of Zn(II) binding as a key feature in the formation of amyloid fibrils by Aβ11-28

Supramolecular assembly of peptides and proteins into amyloid fibrils is of multifold interest, going from materials science to physiopathology. The binding of metal ions to amyloidogenic peptides is associated with several amyloid diseases, and amyloids with incorporated metal ions are of interest in nanotechnology. Understanding the mechanisms of amyloid formation and the role of metal ions can improve strategies toward the prevention of this process and enable potential applications in nanotechnology. Here, studies on Zn(II) binding to the amyloidogenic peptide Aβ11-28 are reported. Zn(II) modulates the Aβ11-28 aggregation, in terms of kinetics and fibril structures. Structural studies suggest that Aβ11-28 binds Zn(II) by amino acid residues Glu11 and His14 and that Zn(II) is rapidly exchanged between peptides. Structural and aggregation data indicate that Zn(II) binding induces the formation of the dimeric Zn(II)(1)(Aβ11-28)(2) species, which is the building block of fibrillar aggregates and explains why Zn(II) binding accelerates Aβ11-28 aggregation. Moreover, transient Zn(II) binding, even briefly, was enough to promote fibril formation, but the final structure resembled that of apo-Aβ11-28 amyloids. Also, seeding experiments, i.e., the addition of fibrillar Zn(II)(1)(Aβ11-28)(2) to the apo-Aβ11-28 peptide, induced aggregation but not propagation of the Zn(II)(1)(Aβ11-28)(2)-type fibrils. This can be explained by the dynamic Zn(II) binding between soluble and aggregated Aβ11-28. As a consequence, dynamic Zn(II) binding has a strong impact on the aggregation behavior of the Aβ11-28 peptide and might be a relevant and so far little regarded parameter in other systems of metal ions and amyloidogenic peptides.     

Inhibitory effect of safranal and crocin,two principle compounds of <Emphasis Type="Italic">Crocus sativus</Emphasis>, on fibrillation of lysozyme

A key feature in more than twenty amyloid-related diseases is the aggregation of intra-and/or extracellular misfolded proteins as amyloid fibrils. Therefore, preventing or reversing amyloid aggregation by using of small molecules is considered as useful approaches to the treatment of these diseases. We have evaluated the ability of safranal and crocin, to inhibit amyloid self-assembly of hen egg white lysozyme (HEWL), as an in vitro model system. Structural properties of HEWL in the presence of these compounds were investigated individually using thioflavin T, anilinonaphthalene-8-sulfonic acid fluorescence assays, far-UV circular dichroism and scanning electron microscopy as well as docking method. Our results showed that incubation of HEWL with either crocin or safranal at various concentrations leads a significant inhibition in the rate of amyloid formation. Docking analysis revealed crocin and safranal interact with the central hydrophobic region of lysozyme through van der Waals interaction. Hydroxyl group in crocin through hydrogen bonds connected to the several hydrophilic amino acids of lysozyme, while in safranal there are just one aldehyde group that through hydrogen bonds connected to aspartic acid in lysozyme. It can be concluded that both hydrophobic and hydrophilic groups contribute to lower lysozyme fibril accumulation.     

Ubiquitous Amyloids

The common view of amyloids and prion proteins is that they are associated with many currently incurable diseases and present a great danger to an organism. This danger comes from the fact that not only prion proteins, but also the infectious form(s) of amyloids, as it has been shown recently, are able to transmit the disease. On the other hand, organisms take advantage of the strength and durability of specific forms of amyloids. Such forms do not spread any disease. Also, in nanotechnology there is a constantly growing need to employ amyloid fibrils in many industrial applications. With increasing knowledge about amyloids and prion proteins we are aware that the amyloidal state is inherent to any protein, making the problem of amyloid formation a central one in aging-related diseases. However, the “good” amyloids can be beneficial and even necessary for our health. Furthermore, because of their mechanical properties, the amyloids are of great interest to engineers.     

“What Doesn’t Kill You Makes You Stronger”: Future Applications of Amyloid Aggregates in Biomedicine

Amyloid proteins are linked to the pathogenesis of several diseases including Alzheimer’s disease, but at the same time a range of functional amyloids are physiologically important in humans. Although the disease pathogenies have been associated with protein aggregation, the mechanisms and factors that lead to protein aggregation are not completely understood. Paradoxically, unique characteristics of amyloids provide new opportunities for engineering innovative materials with biomedical applications. In this review, we discuss not only outstanding advances in biomedical applications of amyloid peptides, but also the mechanism of amyloid aggregation, factors affecting the process, and core sequences driving the aggregation. We aim with this review to provide a useful manual for those who engineer amyloids for innovative medicine solutions.     

The formation of amyloid fibrils from proteins in the lysozyme family

Amyloid fibrils are highly ordered protein assemblies known to contribute to the pathology of a variety of genetic and aging-associated diseases. More recently, these fibrils have been shown to be useful as structural scaffolds in both natural biological systems and nanotechnology applications. The intense interest in amyloid fibrils has led to the investigation of well-characterized proteins, such as hen egg white lysozyme (HEWL), as model systems to examine structural and mechanistic principles that may be generally applicable to all amyloid fibrils. The purpose of this review is to critically examine the fibril-formation literature of proteins in the lysozyme family with respect to the known structure and folding properties of these proteins. The goal is to identify similarities and differences within the family, examine general misfolding / aggregation principles, and identify key areas of importance for future work on the fibril formation of these proteins.     

On the Binding of Congo Red to Amyloid Fibrils

Amyloids are characterized by their capacity to bind Congo red (CR), one of the most used amyloid‐specific dyes. The structural features of CR binding were unknown for years, mainly because of the lack of amyloid structures solved at high resolution. In the last few years, solid‐state NMR spectroscopy enabled the determination of the structural features of amyloids, such as the HET‐s prion forming domain (HET‐s PFD), which also has recently been used to determine the amyloid–CR interface at atomic resolution. Herein, we combine spectroscopic data with molecular docking, molecular dynamics, and excitonic quantum/molecular mechanics calculations to examine and rationalize CR binding to amyloids. In contrast to a previous assumption on the binding mode, our results suggest that CR binding to the HET‐s PFD involves a cooperative process entailing the formation of a complex with 1:1 stoichiometry. This provides a molecular basis to explain the bathochromic shift in the maximal absorbance wavelength when CR is bound to amyloids.     

Directing the secondary structure of polypeptides at will: from helices to amyloids and back again?

An ageing society faces an increasing number of neurodegenerative diseases such as Alzheimer's, Parkinson's, and Creutzfeld-Jacob disease. The deposition of amyloid fibrils is a pathogenic factor causing the destruction of neuronal tissue. Amyloid-forming proteins are mainly alpha-helical in their native conformation, but undergo an alpha-helix to beta-strand conversion before or during fibril formation. Partially unfolded or misfolded beta-sheet fragments are discussed as direct precursors of amyloids. To potentially cure neurodegenerative diseases we need to understand the complex folding mechanisms that shift the equilibrium from the functional to the pathological isoform of the proteins involved. This paper describes a novel approach that allows us to study the interplay between peptide primary structure and environmental conditions for peptide and protein folding in its whole complexity on a molecular level. This de novo designed peptide system may achieve selective inhibition of fibril formation.     

Inhibiting and Reversing Amyloid‐β Peptide (1–40) Fibril Formation with Gramicidin S and Engineered Analogues

In Alzheimer’s disease, amyloid‐β (Aβ) peptides aggregate into extracellular fibrillar deposits. Although these deposits may not be the prime cause of the neurodegeneration that characterizes this disease, inhibition or dissolution of amyloid fibril formation by Aβ peptides is likely to affect its development. ThT fluorescence measurements and AFM images showed that the natural antibiotic gramicidin S significantly inhibited Aβ amyloid formation in vitro and could dissolve amyloids that had formed in the absence of the antibiotic. In silico docking suggested that gramicidin S, a cyclic decapeptide that adopts a β‐sheet conformation, binds to the Aβ peptide hairpin‐stacked fibril through β‐sheet interactions. This may explain why gramicidin S reduces fibril formation. Analogues of gramicidin S were also tested. An analogue with a potency that was four‐times higher than that of the natural product was identified.     

Polymer physics inspired approaches for the study of the mechanical properties of amyloid fibrils

Amyloid structures constitute a class of highly ordered nanomaterials formed by insoluble protein aggregates. These aggregates are characterized by a cross‐β structural motif in which β‐sheets are oriented perpendicular to the fibril axis and bound together by a dense hydrogen bonding network. Although they have been associated with several neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases, amyloid fibrils have also been found in many physiologically beneficial roles, for instance in adhesives and hormone storage. Inspired by this natural occurrence of functional amyloid, the hierarchal self‐assembly of these structures has recently been used to develop artificial biomaterials for applications in medicine and nanotechnology. In order to realize the full potential of amyloids as functional materials, it is important to understand their fundamental mechanical properties. This review explores a range of experimental strategies to determine the mechanical properties of amyloid fibrils and discusses the results in the context of polymer physics concepts. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2014 , 52, 281–292     

Deamidation accelerates amyloid formation and alters amylin fiber structure

Deamidation of asparagine and glutamine is the most common nonenzymatic, post-translational modification. Deamidation can influence the structure, stability, folding, and aggregation of proteins and has been proposed to play a role in amyloid formation. However there are no structural studies of the consequences of deamidation on amyloid fibers, in large part because of the difficulty of studying these materials using conventional methods. Here we examine the effects of deamidation on the kinetics of amyloid formation by amylin, the causative agent of type 2 diabetes. We find that deamidation accelerates amyloid formation and the deamidated material is able to seed amyloid formation by unmodified amylin. Using site-specific isotope labeling and two-dimensional infrared (2D IR) spectroscopy, we show that fibers formed by samples that contain deamidated polypeptide contain reduced amounts of β-sheet. Deamidation leads to disruption of the N-terminal β-sheet between Ala-8 and Ala-13, but β-sheet is still retained near Leu-16. The C-terminal sheet is disrupted near Leu-27. Analysis of potential sites of deamidation together with structural models of amylin fibers reveals that deamidation in the N-terminal β-sheet region may be the cause for the disruption of the fiber structure at both the N- and C-terminal β-sheet. Thus, deamidation is a post-translational modification that creates fibers that have an altered structure but can still act as a template for amylin aggregation. Deamidation is very difficult to detect with standard methods used to follow amyloid formation, but isotope-labeled IR spectroscopy provides a means for monitoring sample degradation and investigating the structural consequences of deamidation.     

Ex situ atomic force microscopy analysis of beta-amyloid self-assembly and deposition on a synthetic template

The beta-amyloid (Abeta) deposition, which is the conversion of soluble Abeta peptides to insoluble plaques on a surface, is an essential pathological process in Alzheimer's disease (AD). The identification and characterization of possible environmental factors that may influence amyloid deposition in vivo are important to unveil the underlying etiology of AD. According to the amyloid cascade hypothesis, diffuse plaques are initial and visual deposits in the early event of AD, leading to amyloid plaques. To study amyloid deposition and growth in vitro, we prepared a synthetic template by immobilizing Abeta seeds on an N-hydroxysuccinimide ester-activated solid surface. According to our analysis with an ex situ atomic force microscope, the formation of amyloid plaque-like aggregates was mediated by the interaction between Abeta in a solution and on a synthetic template, suggesting that Abeta oligomers function well as seeds for amyloid deposition. It was observed that insoluble amyloid aggregates formed on the template surface serve as a sink of soluble Abeta in a solution as well as mediate the formation of intermediates in the pathway of amyloid fibrillization in a solution. Relative seeding efficiencies of fresh monomers, oligomers, and fully grown fibrils were analyzed by measuring the deposited plaque volume and its height distribution through atomic force microscopy. The result revealed that oligomeric forms of Abeta act more efficiently as seeds than monomers or fibrils do. Fluorescence spectroscopy with thioflavin T confirmed that amyloid aggregate formation proceeds in a concentration-dependent manner. Analysis with Fourier transform infrared spectroscopy indicated a progressive transition of soluble Abeta42 monomer to amyloid fibrils having antiparallel beta-sheet structure on the template. Furthermore, studies on the interaction between Abeta40 and 42, two major variants of Abeta derived from the amyloid precursor protein, showed that amyloid aggregate formation on the surface was accelerated further by the homogeneous association of soluble Abeta42 onto Abeta42 seeds than by other combinations. A slightly acidic condition was found to be unfavorable for amyloid formation. This study gives insight into understanding the effects of environmental factors on amyloid formation via the use of a synthetic template system.     

X-ray scattering study of the effect of hydration on the cross-beta structure of amyloid fibrils

We have investigated the effect of sample hydration on the wide-angle X-ray scattering patterns of amyloid fibrils from two different sources, hen egg white lysozyme (HEWL) and an 11-residue peptide taken from the sequence of transthyretin (TTR105-115). Both samples show an inter-strand reflection at 4.7 A and an inter-sheet reflection which occurs at 8.8 and approximately 10 A for TTR105-115 and HEWL fibrils, respectively. The positions, widths, and relative intensities of these reflections are conserved in patterns obtained from dried stalks and hydrated samples over a range of fibril concentrations. In 2D scattering patterns obtained from flow-aligned hydrated samples, the inter-strand and inter-sheet reflections showed, respectively, axial and equatorial alignment relative to the fibril axis, characteristic of the cross-beta structure. Our results show that the cross-beta structure of the fibrils is not a product of the dehydrating conditions typically employed to produce aligned samples, but is conserved in individual fibrils in hydrated samples under dilute conditions comparable to those associated with other biophysical and spectroscopic techniques. This suggests a structure consisting of a stack of two or more sheets whose interfaces are inaccessible to bulk water.