Fibril conformation as the basis of species- and strain-dependent seeding specificity of mammalian prion amyloids

Spongiform encephalopathies are believed to be transmitted by self-perpetuating conformational conversion of the prion protein. It was shown recently that fundamental aspects of mammalian prion propagation can be reproduced in vitro in a seeded fibrillization of the recombinant prion protein variant Y145Stop (PrP23-144). Here we demonstrate that PrP23-144 amyloids from different species adopt distinct secondary structures and morphologies, and that these structural differences are controlled by one or two residues in a critical region. These sequence-specific structural characteristics correlate strictly with the seeding specificity of amyloid fibrils. However, cross-seeding of PrP23-144 from one species with preformed fibrils from another species may overcome natural sequence-based structural preferences, resulting in a new amyloid strain that inherits the secondary structure and morphology of the template. These data provide direct biophysical evidence that protein conformations are transmitted in PrP amyloid strains, establishing a foundation for a structural basis of mammalian prion transmission barriers.     

Chiral bifurcation in aggregating insulin: an induced circular dichroism study

The structural unambiguity of folding is lost when disordered protein molecules convert into β-sheet-rich fibrils. The resulting polymorphism of protein aggregates has been studied in the context of its biomedical consequences. Events underlying the conformational variance of amyloid fibrils, as well as physicochemical boundaries between folding and misfolding pathways, remain obscure. Bifurcation and chiral mesoscopic-scale organization of amyloid fibrils are new aspects of protein misfolding. Here we characterize bifurcation events accompanying insulin fibrillation upon intensive vortexing. Upon agitation, two types of insulin fibrils with opposite chiral senses are formed; however, predominance of either species is only stochastically determined. The uncertainty of fibrils’ chiral sense holds only for fibrils grown within the physiological temperature range, while above 50 °C, the bifurcation is no longer observed—fibrils’ chiral moieties become uniformly biased towards ligand probes, as revealed by the extrinsic Cotton effect of thioflavin T, Congo red, and molecular iodine. According to transmission electron microscopy and scanning electron microscopy data, chiral variants of insulin fibrils consist of fibrous superstructures, distinct from spherulites, formed by the protein in nonagitated solutions. Gradual dissociation of the fibrils in the presence of dimethyl sulfoxide is noncooperative and can be resolved into three distinct phases: decay of the higher-order chiral structures, breakdown of fibrils, and unfolding of intermolecular β-sheet. The chiral aggregates are also destabilized by elution of NaCl implying that Debye screening of charged β-sheets provided by chloride counterions is needed for sustaining their kinetic stability. At elevated temperatures, cross-seeding of agitated insulin samples with preformed fibrils revealed a chiral conflict that prevented the passing of structural features of mother seeds to daughter fibrils in a manner typical of amyloid “strains.”     

Insulin amyloid fibrils form an inclusion complex with molecular iodine: a misfolded protein as a nanoscale scaffold

The study describes formation of an intensely violet inclusion complex of insulin amyloid fibrils and molecular iodine. Resonance Raman spectra of complexes formed by staining mature insulin fibrils with iodine and by seeding fibrils in the presence of iodine imply similar topologies of entrapped iodine and oligoiodide species. Iodine captured by growing fibrils remains accessible to a bulk chemical reagent. In light of its small size and the fact that iodine can partition into polar as well as nonpolar media, the data suggest that intrafibrillar structure of insulin amyloid is densely packed with no appreciable void volumes capable of accommodating iodine atoms. The complex is stable: only drastic perturbation of the beta-pleated fibrous scaffold by dimethyl sulfoxide (rather than of the beta-sheet conformation) leads to the release of iodine atoms from surface moieties. While the reaction between iodine and in vivo amyloid deposits was first described by Virchow in the 19th Century [Virchow, R. (1854) Virchows Arch. 6, 268-271], the underlying molecular mechanism has not been thoroughly explored since then. This work shows how the long-forgotten concept can be utilized as a probe of void volumes in protein fibrils, providing a new tool for structural studies on amyloids, and a model for design of protein-based drug delivery media.     

Independent heterologous fibrillation of insulin and its B-chain peptide

Insulin is very prone to form amyloid fibrils under slightly destabilizing conditions, and the B-chain region plays a critical role in the fibrillation. We show here that the isolated B-chain peptide of bovine insulin also forms fibrils at both acidic and neutral pH. When a mixture of insulin and the B-chain peptide was incubated at either acidic or neutral pH, the formation of fibrils was clearly separated into two phases, with the faster phase corresponding to the formation of homogeneous fibrils from the B-chain and the slower phase corresponding to homogeneous fibrillation of insulin. To further investigate the interaction (or lack thereof) between the two polypeptides, we examined the effects of cross-seeding. The results indicate that seeds of B-chain fibrils accelerate the fibrillation of insulin at pH 1.6 and inhibit the fibrillation at pH 7.5, but seeds of insulin fibrils have little effect on the fibrillation of the B-chain. We conclude that at pH 7.5 simultaneous independent homologous fibrillation occurs, but at low pH, heterologous fibrillation takes place, and with B-chain seeding of insulin, a unique conformation of fibrils is formed. Our results demonstrate that in the co-aggregation of closely related peptides each peptide species may undergo concurrent homogeneous or heterologous polymerization and that fibrils of one species may or may not seed fibrillation of the other. The results demonstrate the significant "species" barrier in amyloid fibril formation between fibrillation induced by different fibrils. A model for the fibrillation of the heterogeneous system of insulin and B-chain insulin is proposed.     

Role of N-terminal familial mutations in prion protein fibrillization and prion amyloid propagation in vitro

A self-perpetuating conformational conversion of the prion protein (PrP) is believed to underlie pathology and transmission of prion diseases. Here we explore the effects of N-terminal pathogenic mutations (P102L, P105L, A117V) and the residue 129 polymorphism on amyloid fibril formation by the human PrP fragment 23-144, an in vitro conversion model that can reproduce certain characteristics of prion replication such as strains and species barriers. We find that these amino acid substitutions neither affect PrP23-144 amyloidogenicity nor introduce barriers to cross-seeding of soluble protein. However, the polymorphism strongly influences the conformation of the amyloid fibrils, as determined by infrared spectroscopy. Intriguingly, unlike conformational features governed by the critical amyloidogenic region of PrP23-144 (residues 138-139), the structural features distinguishing Met-129 and Val-129 PrP23-144 amyloid fibrils are not transmissible by cross-seeding. While based only on in vitro data, these findings provide fundamental insight into the mechanism of prion-based conformational transmission, indicating that only conformational features controlling seeding specificity (e.g. those in critical intermolecular contact sites of amyloid fibrils) are necessarily transmissible by cross-seeding; conformational traits in other parts of the PrP molecule may not be "heritable" from the amyloid template.     

Amyloid fibril formation of alpha-synuclein is accelerated by preformed amyloid seeds of other proteins: implications for the mechanism of transmissible conformational diseases

Alpha-synuclein is one of the causative proteins of familial Parkinson disease, which is characterized by neuronal inclusions named Lewy bodies. Lewy bodies include not only alpha-synuclein but also aggregates of other proteins. This fact raises a question as to whether the formation of alpha-synuclein amyloid fibrils in Lewy bodies may occur via interaction with fibrils derived from different proteins. To probe this hypothesis, we investigated in vitro fibril formation of human alpha-synuclein in the presence of preformed fibril seeds of various different proteins. We used three proteins, Escherichia coli chaperonin GroES, hen lysozyme, and bovine insulin, all of which have been shown to form amyloid fibrils. Very surprisingly, the formation of alpha-synuclein amyloid fibril was accelerated markedly in the presence of preformed seeds of GroES, lysozyme, and insulin fibrils. The structural characteristics of the natively unfolded state of alpha-synuclein may allow binding to various protein particles, which in turn triggers the formation (extension) of alpha-synuclein amyloid fibrils. This finding is very important for understanding the molecular mechanism of Parkinson disease and also provides interesting implications into the mechanism of transmissible conformational diseases.     

Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy

Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and electron microscopy (EM) have been used simultaneously to follow the temperature-induced formation of amyloid fibrils by bovine insulin at acidic pH. The FTIR and CD data confirm that, before heating, insulin molecules in solution at pH 2.3 have a predominantly native-like alpha-helical structure. On heating to 70 degrees C, partial unfolding occurs and results initially in aggregates that are shown by CD and FT-IR spectra to retain a predominantly helical structure. Following this step, changes in the CD and FTIR spectra occur that are indicative of the extensive conversion of the molecular conformation from alpha-helical to beta-sheet structure. At later stages, EM shows the development of fibrils with well-defined repetitive morphologies including structures with a periodic helical twist of approximately 450 A. The results indicate that formation of fibrils by insulin requires substantial unfolding of the native protein, and that the most highly ordered structures result from a slow evolution of the morphology of the initially formed fibrillar species.     

Bovine Insulin Filaments Induced by Reducing Disulfide Bonds Show a Different Morphology, Secondary Structure, and Cell Toxicity from Intact Insulin Amyloid Fibrils

Amyloid fibrils are associated with more than 20 diseases, including Alzheimer's disease and type II diabetes. Insulin is a 51-residue polypeptide hormone, with its two polypeptide chains linked by one intrachain and two interchain disulfide bonds, and has long been known to self-assemble in vitro into amyloid fibrils. We demonstrate here that bovine insulin forms flexible filaments in the presence of a reducing agent, Tris (2-carboxyethyl) phosphine. The insulin filaments, possibly formed due to partial reduction of S-S bonds in insulin molecules, differ from intact insulin fibrils in terms of their secondary structure. The insulin filaments were determined to have an antiparallel β-sheet structure, whereas the insulin fibrils have a parallel β-sheet structure. Of importance, the cell toxicity of the insulin filaments was remarkably lower than that of the insulin fibrils. This finding supports the idea that cell toxicity of amyloids correlates with their morphology. The remarkably low toxicity of the filamentous structure should shed new light on possible pharmacological approaches to the various diseases caused by amyloid fibrils.     

Controlling the dimensions of amyloid fibrils: toward homogenous components for bionanotechnology

Amyloid fibrils have been recognized as having potential in a variety of bionanotechnological applications. However, realization of these applications is constrained by a lack of control over morphology and alignment, both crucial for potential end uses. This article focuses on the use of growth and storage conditions to control the length of amyloid fibrils formed from bovine insulin, with length distributions constructed from transmission electron microscopy (TEM) images. Growth temperature, pH, protein concentration, and storage conditions were examined and were seen to offer a range of conditions that favor different length distribution. The use of amyloid fibrils as nanowires is one area where control of fibril dimensions is desirable, for experimental setup and endpoint applications. The conductive properties of fibrils formed from bovine insulin are presented, with these insulin fibrils being shown to have high resistivity in their unmodified state, with current values in the nanoamp range. These low current values can be increased via modification, or the fibrils used in their native state in applications where low current values are desirable. These findings, coupled with the ability to predict and select for various insulin amyloid fibril dimensions, enhances their utility as nanomaterials.     

Amyloid fibrils as functionalizable components of nanocomposite materials

Amyloid fibrils are a form of protein nanofiber that show promise as components of multifunctional bionanomaterials. In this work, native bovine insulin and bovine insulin that had been previously converted into amyloid fibrils were combined with poly(vinyl alcohol) (PVOH) via solution casting to determine the effect of fibrillization on the thermomechanical properties of the resulting composite. The synthesis method was found to preserve the amyloid fibril structure and properties of the resulting fibril-PVOH composite were investigated. At a filling level of 0.6 wt %, the fibril-reinforced PVOH was 15% stiffer than the PVOH control. Various properties of the films, including the glass transition temperature, degradation temperature, microstructure, and film morphology were characterized. Although more work is required to optimize the properties of the composites, this study provides proof of principle that incorporation of amyloid fibrils into a polymeric material can impart useful changes to the mechanical and morphological properties of the films.     

Quercetin inhibits amyloid fibrillation of bovine insulin and destabilizes preformed fibrils

Growing interest and research efforts have recently been focused on elucidating the molecular mechanism of amyloid formation and the screening of effective inhibitors to interrupt amyloid structures. In the present study, the anti-amyloidogenic effects of quercetin were investigated in vitro using bovine insulin as a model protein. The results demonstrated that quercetin dose-dependently inhibited amyloid formation of insulin. Moreover, quercetin destabilized the preformed insulin fibrils and transformed the fibrils into amorphous aggregates. Hemolysis was observed when human erythrocytes were co-incubated with insulin fibrils. Quercetin inhibited fibril-induced hemolysis in a dose-dependent manner. SDS–PAGE showed that insulin fibrils induced the aggregation of cytoskeletal proteins of erythrocyte membranes and that quercetin attenuated this fibril-induced cytoskeletal aggregation. The results of the present work suggest that quercetin may serve as a lead structure for the design of novel anti-amyloidogenic drugs.     

Structural polymorphism and possible pathways of amyloid fibril formation on the example of insulin protein

In this review we analyze the main works on amyloid formation of insulin. There are many environmental factors affecting the formation of insulin amyloid fibrils (and other amyloidogenic proteins) such as: protein concentration, pH, ionic strength of solution, medium composition (anions, cations), presence of denaturants (urea, guanidine chloride) or stabilizers (saccharose), temperature regime, agitation. Since polymorphism is potentially crucial for human diseases and may underlie the natural variability of some amyloid diseases, in this review we focus attention on polymorphism that is an important biophysical difference between native protein folding suggesting correspondence between the amino acid sequence and unique folding state, and formation of amyloid fibrils, when the same amino acid sequence can form amyloid fibrils of different morphology. At present, according to the literature data, we can choose three ways of polymerization of insulin molecules depending on the nucleus size. The first suggests that fibrillogenesis can occur through assembly of insulin monomers. The second suggests that precursors of fibrils are dimers, and the third assumes that precursors of fibrils are oligomers. Additional experimental works and new methods of investigation and assessment of results are needed to clarify the general picture of insulin amyloid formation.     

Oligomeric forms of insulin amyloid aggregation disrupt outgrowth and complexity of neuron-like PC12 cells

Formation of protein amyloid fibrils consists of a series of intermediates including oligomeric aggregates, proto-fibrillar structures, and finally mature fibrils. Recent studies show higher toxicity for oligomeric and proto-fibrillar intermediates of protein relative to their mature fibrils. Here the kinetic of the insulin amyloid fibrillation was evaluated using a variety of techniques including ThT fluorescence, Congo red absorbance, circular dichroism, and atomic force microscopy (AFM). The solution surface tension changes were attributed to hydrophobic changes in insulin structure and were detected by Du Noüy Ring method. Determination of the surface tension of insulin oligomeric, proto-fibrillar and fibrillar forms indicated that the hydrophobicity of solution is enhanced by the formation of the oligomeric forms of insulin compared to other forms. In order to investigate the toxicity of the different forms of insulin we monitored morphological alterations of the differentiated neuron-like PC12 cells following incubation with native, oligomeric aggregates, proto-fibrillar, and fibrillar forms of insulin. The cell body area, average neurite length, neurite width, number of primary neurites, and percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different forms of insulin. We observed that the oligomeric form of insulin impaired the growth and complexity of PC12 cells compared to other forms. Together our data suggest that the lower surface tension of oligomers and their perturbation affects the morphology of PC12 cells, mainly due to their enhanced hydrophobicity and detergent-like structures.     

Ultrastructural organization of amyloid fibrils by atomic force microscopy

Atomic force microscopy has been employed to investigate the structural organization of amyloid fibrils produced in vitro from three very different polypeptide sequences. The systems investigated are a 10-residue peptide derived from the sequence of transthyretin, the 90-residue SH3 domain of bovine phosphatidylinositol-3'-kinase, and human wild-type lysozyme, a 130-residue protein containing four disulfide bridges. The results demonstrate distinct similarities between the structures formed by the different classes of fibrils despite the contrasting nature of the polypeptide species involved. SH3 and lysozyme fibrils consist typically of four protofilaments, exhibiting a left-handed twist along the fibril axis. The substructure of TTR(10-19) fibrils is not resolved by atomic force microscopy and their uniform appearance is suggestive of a regular self-association of very thin filaments. We propose that the exact number and orientation of protofilaments within amyloid fibrils is dictated by packing of the regions of the polypeptide chains that are not directly involved in formation of the cross-beta core of the fibrils. The results obtained for these proteins, none of which is directly associated with any human disease, are closely similar to those of disease-related amyloid fibrils, supporting the concept that amyloid is a generic structure of polypeptide chains. The detailed architecture of an individual fibril, however, depends on the manner in which the protofilaments assemble into the fibrillar structure, which in turn is dependent on the sequence of the polypeptide and the conditions under which the fibril is formed.     

Amyloid insulin interaction with erythrocytes.

Erythrocyte membrane interactions with insulin fibrils (amyloid) have been investigated using centrifugation, fluorescence spectroscopy, light scattering, and flow cytometric techniques. The results indicate that insulin fibrils are having moderate affinity to erythrocyte membrane. However, analysis of the apparent dissociation constants of human erythrocyte membranes (leaky and resealed vesicles) with amyloid insulin reveal that the insulin binding is drastically reduced on attaining the fibrillar state compared with native insulin. To understand the role of insulin receptors on erythrocytes binding to amyloid, we have studied the interaction of biotinylated forms of denatured and amyloidic insulin with erythrocytes. FITC-streptavidin was used as a counter staining in flow cytometry measurements. We found that insulin fibrils bind 10 times more with erythrocyte membranes than with amylin and denatured insulin.     

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.     

An intersheet packing interaction in A beta fibrils mapped by disulfide cross-linking

Most models for the central cross-beta folding unit in amyloid fibrils of the Alzheimer's plaque protein Abeta align the peptides in register in H-bonded, parallel beta-sheet structure. Some models require the Abeta peptide to undergo a chain reversal when folding into the amyloid core, while other models feature very long extended chains, or zigzag chains, traversing the protofilament. In this paper we introduce the use of disulfide bond cross-linking to probe the fold within the core and the packing interactions between beta-sheets. In one approach, amyloid fibrils grown under reducing conditions from each of three double cysteine mutants (17/34, 17/35, and 17/36) of the Abeta(1-40) sequence were subjected to oxidizing conditions. Of these three mutants, only the Leu17Cys/Leu34Cys peptide could be cross-linked efficiently while resident in fibrils. In another approach, double Cys mutants were cross-linked as monomers before aggregation, and the resulting fibrils were assessed for stability, antibody binding, dye binding, and cross-seeding efficiency. Here too, fibrils from the 17/34 double Cys mutant most closely resemble wild-type Abeta(1-40) fibrils. These data support models of the Abeta fibril in which the Leu17 and Leu34 side chains of the same peptide pack against each other at the beta-sheet interface within the amyloid core. Related cross-linking strategies may reveal longer range spatial relationships. The ability of the cross-linked 17/35 double Cys mutant Abeta to also make amyloid fibrils illustrates a remarkable plasticity of the amyloid structure and suggests a structural mechanism for the generation of conformational variants of amyloid.     

Highly promiscuous nature of prion polymerization

The primary structure of the prion protein (PrP) is believed to be the key factor in regulating the species barrier of prion transmission. Because the strength of the species barrier was found to be affected by the prion strain, the extent to which the barrier can indeed be attributed to differences in the PrP primary structures of either donor and acceptor species remains unclear. In this study, we exploited the intrinsic property of PrP to polymerize spontaneously into disease-related amyloid conformations in the absence of a strain-specified template and analyzed polymerization of mouse and hamster full-length recombinant PrPs. Unexpectedly, we found no evidence of species specificity in cross-seeding polymerization assays. Even when both recombinant PrP variants were present in mixtures, preformed mouse or hamster fibrils displayed no selectivity in elongation reactions and consumed equally well both homologous and heterologous substrates. Analysis of individual fibrils revealed that fibrils can elongate in a bidirectional or unidirectional manner. Our work revealed that, in the absence of a cellular environment, post-translational modifications, or strain-specified conformational constraints, PrP fibrils are intrinsically promiscuous and capable of utilizing heterologous PrP variants as a substrate in a highly efficient manner. This study suggests that amyloid structures are capable of accommodating local perturbations arising because of a mismatch in amino acid sequences and highlights the promiscuous nature of the self-propagating activity of amyloid fibrils.     

Amyloid fibril formation from crude protein mixtures

Amyloid fibrils have potential as bionanomaterials. A bottleneck in their commercial use is the cost of the highly purified protein typically needed as a starting material. Thus, an understanding of the role of heterogeneity in the mixtures from which amyloid fibrils are formed may inform production of these structures from readily available impure starting materials. Insulin, a very well understood amyloid-forming protein, was modified by various reagents to explore whether amyloid fibrils could still form from a heterogeneous mixture of insulin derivatives. Aggregates were characterized by thioflavin T fluorescence and transmission electron microscopy. Using acetylation, reduction carboxymethylation, reduction pyridylethylation, trypsin digestion and chymotrypsin digestion, it was shown that amyloid fibrils can form from heterogeneous mixtures of modified insulin. The modifications changed both the rate of reaction and the yield of the final product, but led to fibrillar structures, some with interesting morphologies. Well defined, long, unbranched fibrils were observed in the crude reduced carboxymethylated insulin mixture and the crude reduced pyridylethylated insulin revealed the formation of "wavy" fibrils, compared with the straighter native insulin amyloid fibrils. Although trypsin digestion inhibited fibrils formation, chymotrypsin digestion of insulin produced a mixture of long and short fibrils under the same conditions. We conclude that amyloid fibrils may be successfully formed from heterogeneous mixtures and, further, that chemical modification may provide a simple means of manipulating protein fibril assembly for use in bionanotechnological applications, enabling some design of overall morphology in the bottom-up assembly of higher order protein structures from amyloid fibrils.