Congo Red Inhibits Proteoglycan and Serum Amyloid P Binding to Amyloid β Fibrils

Abstract: Various data suggest that Alzheimer's disease results from the accumulation of amyloid β (Aβ) peptide fibrils and the consequent formation of senile plaques in the cognitive regions of the brain. One approach to lowering senile plaque burden in Alzheimer's disease brain is to identify compounds that will increase the degradation of existing amyloid fibrils. Previous studies have shown that proteoglycans and serum amyloid P (SAP), molecules that localize to senile plaques, bind to Aβ fibrils and protect the amyloid peptide from proteolytic breakdown. Therefore, molecules that prevent the binding of SAP and/or proteoglycans to fibrillar Aβ might increase plaque degradation and prove useful in the treatment of Alzheimer's disease. The nature of SAP and proteoglycan binding to Aβ is defined further in the present study. SAP binds to both fibrillar and nonfibrillar forms of Aβ. However, only the former is rendered resistant to proteolysis after SAP association. It is interesting that both SAP and proteoglycan binding to Aβ fibrils can be inhibited by glycosaminoglycans and Congo red. Unexpectedly, Congo red protects fibrillar Aβ from breakdown, suggesting that this compound and other structurally related molecules are unlikely to be suitable for use in the treatment of Alzheimer's disease.     

Binding mode of Thioflavin T in insulin amyloid fibrils

Amyloid fibrils share various common structural features and their presence can be detected by Thioflavin T (ThT). In this paper, the binding mode of ThT to insulin amyloid fibrils was examined. Scatchard analysis and isothermal titration calorimetry (ITC) showed at least two binding site populations. The binding site population with the strongest binding was responsible for the characteristic ThT fluorescence. This binding had a capacity of about 0.1 moles of ThT bound per mole of insulin in fibril form. The binding capacity was unaffected by pH, but the affinity was lowest at low pH. Notably, presence of a third binding process prior to the other processes was suggested by ITC. Binding of ThT resulted in only minor changes in the fibril structure according to the X-ray diffraction patterns, where a slightly more dominant equatorial reflection at 16A relative to the intersheet distance of 11A was observed. No change in the interstrand distance of 4.8A was observed. On the basis of our results, we propose that ThT binds in cavities running parallel to the fibril axis, e.g., between the protofilaments forming the fibrils. Such cavities have been proposed previously in insulin fibrils and several other amyloid fibril models.     

Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils

Amyloid fibrils are associated with many maladies, including Alzheimer’s disease (AD). The isolation of amyloids from natural materials is very challenging because the extreme structural stability of amyloid fibrils makes it difficult to apply conventional protein science protocols to their purification. A protocol to isolate and detect amyloids is desired for the diagnosis of amyloid diseases and for the identification of new functional amyloids. Our aim was to develop a protocol to purify amyloid from organisms, based on the particular characteristics of the amyloid fold, such as its resistance to proteolysis and its capacity to be recognized by specific conformational antibodies. We used a two-step strategy with proteolytic digestion as the first step followed by immunoprecipitation using the amyloid conformational antibody LOC. We tested the efficacy of this method using as models amyloid fibrils produced in vitro, tissue extracts from C. elegans that overexpress Aβ peptide, and cerebrospinal fluid (CSF) from patients diagnosed with AD. We were able to immunoprecipitate Aβ_1–40 amyloid fibrils, produced in vitro and then added to complex biological extracts, but not α-synuclein and gelsolin fibrils. This method was useful for isolating amyloid fibrils from tissue homogenates from a C. elegans AD model, especially from aged worms. Although we were able to capture picogram quantities of Aβ_1–40 amyloid fibrils produced in vitro when added to complex biological solutions, we could not detect any Aβ amyloid aggregates in CSF from AD patients. Our results show that although immunoprecipitation using the LOC antibody is useful for isolating Aβ_1–40 amyloid fibrils, it fails to capture fibrils of other amyloidogenic proteins, such as α-synuclein and gelsolin. Additional research might be needed to improve the affinity of these amyloid conformational antibodies for an array of amyloid fibrils without compromising their selectivity before application of this protocol to the isolation of amyloids.     

A Structural Model for Apolipoprotein C-II Amyloid Fibrils: Experimental Characterization and Molecular Dynamics Simulations

The self-assembly of specific proteins to form insoluble amyloid fibrils is a characteristic feature of a number of age-related and debilitating diseases. Lipid-free human apolipoprotein C-II (apoC-II) forms characteristic amyloid fibrils and is one of several apolipoproteins that accumulate in amyloid deposits located within atherosclerotic plaques. X-ray diffraction analysis of aligned apoC-II fibrils indicated a simple cross-β-structure composed of two parallel β-sheets. Examination of apoC-II fibrils using transmission electron microscopy, scanning transmission electron microscopy, and atomic force microscopy indicated that the fibrils are flat ribbons composed of one apoC-II molecule per 4.7-Å rise of the cross-β-structure. Cross-linking results using single-cysteine substitution mutants are consistent with a parallel in-register structural model for apoC-II fibrils. Fluorescence resonance energy transfer analysis of apoC-II fibrils labeled with specific fluorophores provided distance constraints for selected donor-acceptor pairs located within the fibrils. These findings were used to develop a simple ‘letter-G-like’ β-strand-loop-β-strand model for apoC-II fibrils. Fully solvated all-atom molecular dynamics (MD) simulations showed that the model contained a stable cross-β-core with a flexible connecting loop devoid of persistent secondary structure. The time course of the MD simulations revealed that charge clusters in the fibril rearrange to minimize the effects of same-charge interactions inherent in parallel in-register models. Our structural model for apoC-II fibrils suggests that apoC-II monomers fold and self-assemble to form a stable cross-β-scaffold containing relatively unstructured connecting loops.     

Analysis of Core Region from Egg White Lysozyme Forming Amyloid Fibrils

Some of the lysozyme mutants in humans cause systemic amyloidosis. Hen egg white lysozyme (HEWL) has been well studied as a model protein of amyloid fibrils formation. We previously identified an amyloid core region consisting of nine amino acids (designated as the K peptide), which is present at 54-62 in HEWL. The K peptide, with tryptophan at its C- terminus, has the ability of self-aggregation. In the present work we focused on its structural properties in relation to the formation of fibrils. The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C. A substantial number of fibrils were generated under this pH condition and incubation period. Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils. Tryptophan 62 in lysozyme was suggested to be especially crucial to forming amyloid fibrils. We also show that amyloid fibrils formation of the K peptide requires not only tryptophan 62 but also a certain length containing hydrophobic amino acids. A core region is involved in the significant formation of amyloid fibrils of lysozyme.     

An Efficient Kinetic Model for Assemblies of Amyloid Fibrils and Its Application to Polyglutamine Aggregation

Protein polymerization consists in the aggregation of single monomers into polymers that may fragment. Fibrils assembly is a key process in amyloid diseases. Up to now, protein aggregation was commonly mathematically simulated by a polymer size-structured ordinary differential equations (ODE) system, which is infinite by definition and therefore leads to high computational costs. Moreover, this Ordinary Differential Equation-based modeling approach implies biological assumptions that may be difficult to justify in the general case. For example, whereas several ordinary differential equation models use the assumption that polymerization would occur at a constant rate independently of polymer size, it cannot be applied to certain protein aggregation mechanisms. Here, we propose a novel and efficient analytical method, capable of modelling and simulating amyloid aggregation processes. This alternative approach consists of an integro-Partial Differential Equation (PDE) model of coalescence-fragmentation type that was mathematically derived from the infinite differential system by asymptotic analysis. To illustrate the efficiency of our approach, we applied it to aggregation experiments on polyglutamine polymers that are involved in Huntington’s disease. Our model demonstrates the existence of a monomeric structural intermediate acting as a nucleus and deriving from a non polymerizing monomer (). Furthermore, we compared our model to previously published works carried out in different contexts and proved its accuracy to describe other amyloid aggregation processes.     

Study on the binding of Thioflavin T to beta-sheet-rich and non-beta-sheet cavities

Amyloid fibril formation plays a role in more than 20 diseases including Alzheimer's disease. In vitro detection of these fibrils is often performed using Thioflavin T (ThT), though the ThT binding mode is largely unknown. In the present study, spectral properties of ThT in binding environments representing beta-sheet-rich and non-beta-sheet cavities were examined. Acetylcholinesterase and gamma-cyclodextrin induced a characteristic ThT fluorescence similar to that with amyloid fibrils, whereas beta-cyclodextrin and the beta-sheet-rich transthyretin did not. The cavities of acetylcholinesterase and gamma-cyclodextrin were of similar diameter and only these cavities could accommodate two ThT ions according to molecular modelling. Binding stoichiometry studies also showed a possible binding of two ThT ions. Thus, the characteristic ThT fluorescence is induced in cavities with a diameter of 8-9A and a length able to accommodate the entire length of the ThT ion. The importance of a cavity diameter capable of binding two ThT ions, among others, indicates that an excimer formation is a plausible mechanism for the characteristic fluorescence. We propose a similar ThT binding mode in amyloid fibrils, where cavities of an appropriate size running parallel to the fibril axis have previously been proposed in several amyloid fibril models.     

Nanoscale Mechanical Characterisation of Amyloid Fibrils Discovered in a Natural Adhesive

Using the atomic force microscope, we have investigated the nanoscale mechanical response of the attachment adhesive of the terrestrial alga Prasiola linearis (Prasiolales, Chlorophyta). We were able to locate and extend highly ordered mechanical structures directly from the natural adhesive matrix of the living plant. The in vivo mechanical response of the structured biopolymer often displayed the repetitive sawtooth force-extension characteristics of a material exhibiting high mechanical strength at the molecular level. Mechanical and histological evidence leads us to propose a mechanism for mechanical strength in our sample based on amyloid fibrils. These proteinaceous, pleated β-sheet complexes are usually associated with neurodegenerative diseases. However, we now conclude that the amyloid protein quaternary structures detected in our material should be considered as a possible generic mechanism for mechanical strength in natural adhesives.     

Burial of the Polymorphic Residue 129 in Amyloid Fibrils of Prion Stop Mutants

Misfolding of the natively α-helical prion protein into a β-sheet rich isoform is related to various human diseases such as Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker syndrome. In humans, the disease phenotype is modified by a methionine/valine polymorphism at codon 129 of the prion protein gene. Using a combination of hydrogen/deuterium exchange coupled to NMR spectroscopy, hydroxyl radical probing detected by mass spectrometry, and site-directed mutagenesis, we demonstrate that stop mutants of the human prion protein have a conserved amyloid core. The 129 residue is deeply buried in the amyloid core structure, and its mutation strongly impacts aggregation. Taken together the data support a critical role of the polymorphic residue 129 of the human prion protein in aggregation and disease.     

Polyanionic Candidate Microbicides Accelerate the Formation of Semen-Derived Amyloid Fibrils to Enhance HIV-1 Infection

Polyanionic candidate microbicides, including cellulose sulfate, carrageenan, PRO 2000, were proven ineffective in preventing HIV-1 transmission and even cellulose sulfate showed increased risk of HIV acquisition in the Phase III efficacy trials. Semen plays critical roles in HIV-1 sexual transmission. Specifically, amyloid fibrils formed by fragments of prostatic acidic phosphatase (PAP) in semen termed semen-derived enhancer of virus infection (SEVI) could drastically enhance HIV-1 infection. Here we investigated the interaction between polyanions and PAP248-286, a prototype peptide of SEVI, to understand the possible cause of polyanionic candidate microbicides to fail in clinical trials. We found anionic polymers could efficiently promote SEVI fibril formation, most likely mediated by the natural electrostatic interaction between polyanions and PAP248-286, as revealed by acid native PAGE and Western blot. The overall anti-HIV-1 activity of polyanions in the presence or absence of PAP248-286 or semen was evaluated. In the viral infection assay, the supernatants of polyanions/PAP248-286 or polyanions/semen mixtures containing the free, unbound polyanionic molecules showed a general reduction in antiviral efficacy, while the pellets containing amyloid fibrils formed by the polyanion-bound PAP248-286 showed aggravated enhancement of viral infection. Collectively, from the point of drug-host protein interaction, our study revealed that polyanions facilitate SEVI fibril formation to promote HIV-1 infection, thus highlighting a molecular mechanism underlying the failure of polyanions in clinical trials and the importance of drug-semen interaction in evaluating the anti-HIV-1 efficacy of candidate microbicides.     

Benzalkonium Chloride Accelerates the Formation of the Amyloid Fibrils of Corneal Dystrophy-associated Peptides

Corneal dystrophies are genetic disorders resulting in progressive corneal clouding due to the deposition of amyloid fibrils derived from keratoepithelin, also called transforming growth factor β-induced protein (TGFBI). The formation of amyloid fibrils is often accelerated by surfactants such as sodium dodecyl sulfate (SDS). Most eye drops contain benzalkonium chloride (BAC), a cationic surfactant, as a preservative substance. In the present study, we aimed to reveal the role of BAC in the amyloid fibrillation of keratoepithelin-derived peptides in vitro. We used three types of 22-residue synthetic peptides covering Leu110-Glu131 of the keratoepithelin sequence: an R-type peptide with wild-type R124, a C-type peptide with C124 associated with lattice corneal dystrophy type I, and a H-type peptide with H124 associated with granular corneal dystrophy type II. The time courses of spontaneous amyloid fibrillation and seed-dependent fibril elongation were monitored in the presence of various concentrations of BAC or SDS using thioflavin T fluorescence. BAC and SDS accelerated the fibrillation of all synthetic peptides in the absence and presence of seeds. Optimal acceleration occurred near the CMC, which suggests that the unstable and dynamic interactions of keratoepithelin peptides with amphipathic surfactants led to the formation of fibrils. These results suggest that eye drops containing BAC may deteriorate corneal dystrophies and that those without BAC are preferred especially for patients with corneal dystrophies.     

Propensity to Form Amyloid Fibrils Is Encoded as Excitations in the Free Energy Landscape of Monomeric Proteins

Protein aggregation, linked to many of diseases, is initiated when monomers access rogue conformations that are poised to form amyloid fibrils. We show, using simulations of src SH3 domain, that mechanical force enhances the population of the aggregation-prone (N^?) states, which are rarely populated under force free native conditions but are encoded in the spectrum of native fluctuations. The folding phase diagrams of SH3 as a function of denaturant concentration ([C]), mechanical force (f), and temperature exhibit an apparent two-state behavior, without revealing the presence of the elusive N^? states. Interestingly, the phase boundaries separating the folded and unfolded states at all [C] and f fall on a master curve, which can be quantitatively described using an analogy to superconductors in a magnetic field. The free energy profiles as a function of the molecular extension (R), which are accessible in pulling experiments, (R), reveal the presence of a native-like N^? with a disordered solvent-exposed amino-terminal β-strand. The structure of the N^? state is identical with that found in Fyn SH3 by NMR dispersion experiments. We show that the timescale for fibril formation can be estimated from the population of the N^? state, determined by the free energy gap separating the native structure and the N^? state, a finding that can be used to assess fibril forming tendencies of proteins. The structures of the N^? state are used to show that oligomer formation and likely route to fibrils occur by a domain-swap mechanism in SH3 domain.     

A New Methodology of Spatial Cross-Correlation Analysis

Spatial correlation modeling comprises both spatial autocorrelation and spatial cross-correlation processes. The spatial autocorrelation theory has been well-developed. It is necessary to advance the method of spatial cross-correlation analysis to supplement the autocorrelation analysis. This paper presents a set of models and analytical procedures for spatial cross-correlation analysis. By analogy with Moran’s index newly expressed in a spatial quadratic form, a theoretical framework is derived for geographical cross-correlation modeling. First, two sets of spatial cross-correlation coefficients are defined, including a global spatial cross-correlation coefficient and local spatial cross-correlation coefficients. Second, a pair of scatterplots of spatial cross-correlation is proposed, and the plots can be used to visually reveal the causality behind spatial systems. Based on the global cross-correlation coefficient, Pearson’s correlation coefficient can be decomposed into two parts: direct correlation (partial correlation) and indirect correlation (spatial cross-correlation). As an example, the methodology is applied to the relationships between China’s urbanization and economic development to illustrate how to model spatial cross-correlation phenomena. This study is an introduction to developing the theory of spatial cross-correlation, and future geographical spatial analysis might benefit from these models and indexes.     

Role of Glycosaminoglycan Sulfation in the Formation of Immunoglobulin Light Chain Amyloid Oligomers and Fibrils

Primary amyloidosis (AL) results from overproduction of unstable monoclonal immunoglobulin light chains (LCs) and the deposition of insoluble fibrils in tissues, leading to fatal organ disease. Glycosaminoglycans (GAGs) are associated with AL fibrils and have been successfully targeted in the treatment of other forms of amyloidosis. We investigated the role of GAGs in LC fibrillogenesis. Ex vivo tissue amyloid fibrils were extracted and examined for structure and associated GAGs. The GAGs were detected along the length of the fibril strand, and the periodicity of heparan sulfate (HS) along the LC fibrils generated in vitro was similar to that of the ex vivo fibrils. To examine the role of sulfated GAGs on AL oligomer and fibril formation in vitro, a κ1 LC purified from urine of a patient with AL amyloidosis was incubated in the presence or absence of GAGs. The fibrils generated in vitro at physiologic concentration, temperature, and pH shared morphologic characteristics with the ex vivo κ1 amyloid fibrils. The presence of HS and over-O-sulfated-heparin enhanced the formation of oligomers and fibrils with HS promoting the most rapid transition. In contrast, GAGs did not enhance fibril formation of a non-amyloidogenic κ1 LC purified from urine of a patient with multiple myeloma. The data indicate that the characteristics of the full-length κ1 amyloidogenic LC, containing post-translational modifications, possess key elements that influence interactions of the LC with HS. These findings highlight the importance of the variable and constant LC regions in GAG interaction and suggest potential therapeutic targets for treatment.     

Thioflavin T fluorescence anisotropy: an alternative technique for the study of amyloid aggregation

The process of amyloid polymerisation raises keen interest in particular because of the biomedical impact of this process. A variety of analytical methods have been developed to monitor amyloid formation. Thioflavin T (ThT) is the most commonly used dye for detection of amyloid aggregation. Nevertheless, ThT fluorescence enhancement is strongly dependent of fibril morphology. In this study using the HET-s prion fibril model, we show that amyloid formation can be monitored by measuring ThT fluorescence anisotropy. Kinetic parameters obtained by this method are identical to those determined by CD spectrometry. We propose that ThT anisotropy represent an interesting, simple and alternative technique to analyze the amyloid formation process.     

Binding of Amyloid Beta-Protein to Intracellular Brain Proteins in Rat and Human

Amyloid beta-protein (A), in its soluble form, is known to bind several circulatory proteins such as apolipoprotein (apo) E, apo J and transthyretin. However, the binding of A to intracellular proteins has not been studied. We have developed an overlay assay to study A binding to intracellular brain proteins. The supernatants from both rat and human brains were found to contain several proteins that bind to A 1–40 and A 1–42. No major difference was observed in the A binding-proteins from brain supernatants of patients with Alzheimer's disease and normal age-matched controls. Binding studies using shorter amyloid beta-peptides and competitive overlay assays showed that the binding site of A to brain proteins resides between 12–28 amino acid sequence of A. The presence of several intracellular A-binding (AB) proteins suggests that these proteins may either protect A from its fibrillization or alternatively promote A polymerization. Identification of these proteins and their binding affinities for A are needed to assess their potential role in the pathogenesis of Alzheimer's disease.