Aqueous, Unfolded OmpA Forms Amyloid-Like Fibrils upon Self-Association.
ABSTRACT: Unfolded outer membrane beta-barrel proteins have been shown to self-associate in the absence of lipid bilayers. We previously investigated the formation of high molecular weight species by OmpA, with both the transmembrane domain alone and the full-length protein, and discovered that the oligomeric form contains non-native ?-sheet structure. We have further probed the conformation of self-associated OmpA by monitoring binding to Thioflavin T, a dye that is known to bind the cross-? a structure inherent in amyloid fibrils, and by observing the species by electron microscopy. The significant increase in fluorescence indicative of Thioflavin T binding and the appearance of fibrillar species by electron microscopy verify that the protein forms amyloid-like fibril structures upon oligomerization. These results are also consistent with our previous kinetic analysis of OmpA self-association that revealed a nucleated growth polymerization mechanism, which is frequently observed in amyloid formation. The discovery of OmpA's ability to form amyloid-like fibrils provides a new model protein with which to study fibrillization, and implicates periplasmic chaperone proteins as capable of inhibiting fibril formation.
Project description:The conformational strain diversity characterizing ?-synuclein (?-syn) amyloid fibrils is thought to determine the different clinical presentations of neurodegenerative diseases underpinned by a synucleinopathy. Experimentally, various ?-syn fibril polymorphs have been obtained from distinct fibrillization conditions by altering the medium constituents and were selected by amyloid monitoring using the probe thioflavin T (ThT). We report that, concurrent with classical ThT-positive products, fibrillization in saline also gives rise to polymorphs invisible to ThT (?<sup>-</sup>). The generation of ?<sup>-</sup> fibril polymorphs is stochastic and can skew the apparent fibrillization kinetics revealed by ThT. Their emergence has thus been ignored so far or mistaken for fibrillization inhibitions/failures. They present a yet undescribed atomic organization and show an exacerbated propensity toward self-replication in cortical neurons, and in living mice, their injection into the substantia nigra pars compacta triggers a synucleinopathy that spreads toward the dorsal striatum, the nucleus accumbens, and the insular cortex.
Project description:The hydrophobic A? peptide is highly aggregation prone; it first forms soluble oligomers, which then convert into the amyloid fibrils found in the cerebral plaques of Alzheimer's disease. It is generally understood that as the peptide concentration of A? increases, the fibrillization process is accelerated, but we examine the limits on this phenomenon. We found that once a threshold concentration of A? is exceeded, a stable oligomer is formed at the expense of fibril formation. The suppression of fibril formation was observed by amyloid-binding dye Thioflavin T and solution nuclear magnetic resonance (NMR). Small-angle X-ray scattering, size exclusion chromatography, and analytical ultracentrifugation demonstrated that A? peptides form a range of compact species, with a dimer being an early highly populated oligomer. Solution NMR allowed us to define the secondary structure of this A? dimer, which shows interlocking contacts between C-terminal peptide strands. Thus, we present a novel A? oligomer that resists conversion to fibrils and remains stable for more than one year.
Project description:<h4>Background</h4>It is known that in vivo human prion protein (PrP) have the tendency to form fibril deposits and are associated with infectious fatal prion diseases, while the rabbit PrP does not readily form fibrils and is unlikely to cause prion diseases. Although we have previously demonstrated that amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and macromolecular crowding has different effects on fibril formation of the rabbit/human PrPs, we do not know which domains of PrPs cause such differences. In this study, we have constructed two PrP chimeras, rabbit chimera and human chimera, and investigated how domain replacement affects fibril formation of the rabbit/human PrPs.<h4>Methodology/principal findings</h4>As revealed by thioflavin T binding assays and Sarkosyl-soluble SDS-PAGE, the presence of a strong crowding agent dramatically promotes fibril formation of both chimeras. As evidenced by circular dichroism, Fourier transform infrared spectroscopy, and proteinase K digestion assays, amyloid fibrils formed by human chimera have secondary structures and proteinase K-resistant features similar to those formed by the human PrP. However, amyloid fibrils formed by rabbit chimera have proteinase K-resistant features and secondary structures in crowded physiological environments different from those formed by the rabbit PrP, and secondary structures in dilute solutions similar to the rabbit PrP. The results from transmission electron microscopy show that macromolecular crowding caused human chimera but not rabbit chimera to form short fibrils and non-fibrillar particles.<h4>Conclusions/significance</h4>We demonstrate for the first time that the domains beyond PrP-H2H3 (?-strand 1, ?-helix 1, and ?-strand 2) have a remarkable effect on fibrillization of the rabbit PrP but almost no effect on the human PrP. Our findings can help to explain why amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and why macromolecular crowding has different effects on fibrillization of PrPs from different species.
Project description:Amyloid fibril formation is central to the etiology of a wide range of serious human diseases, such as Alzheimer's disease and prion diseases. Despite an ever growing collection of amyloid fibril structures found in the Protein Data Bank (PDB) and numerous clinical trials, therapeutic strategies remain elusive. One contributing factor to the lack of progress on this challenging problem is incomplete understanding of the mechanisms by which these locally ordered protein aggregates self-assemble in solution. Many current models of amyloid deposition diseases posit that the most toxic species are oligomers that form either along the pathway to forming fibrils or in competition with their formation, making it even more critical to understand the kinetics of fibrillization. A recently introduced topological model for aggregation based on network Hamiltonians is capable of recapitulating the entire process of amyloid fibril formation, beginning with thousands of free monomers and ending with kinetically accessible and thermodynamically stable amyloid fibril structures. The model can be parameterized to match the five topological classes encompassing all amyloid fibril structures so far discovered in the PDB. This paper introduces a set of network statistical and topological metrics for quantitative analysis and characterization of the fibrillization mechanisms predicted by the network Hamiltonian model. The results not only provide insight into different mechanisms leading to similar fibril structures, but also offer targets for future experimental exploration into the mechanisms by which fibrils form.
Project description:Formation of amyloid fibrils by A?42 protein is a pathological hallmark of Alzheimer's disease. A?42 fibrillization is a nucleation-dependent polymerization process, in which nucleation is the rate-limiting step. Structural knowledge of the fibril nucleus is important to understand the molecular mechanism of A? aggregation and is also critical for successful modulation of the fibrillization process. Here, we used a scanning mutagenesis approach to study the role of each residue position in A?42 fibrillization kinetics. The side chain we used to replace the native residue is a nitroxide spin label called R1, which was introduced using site-directed spin labeling. In this systematic study, all residue positions of A?42 sequence were studied, and we identified six key residues for the A?42 fibril formation: H14, E22, D23, G33, G37, and G38. Our results suggest that charges at positions 22 and 23 and backbone flexibilities at positions 33, 37, and 38 play key roles in A?42 fibrillization kinetics. Our results also suggest that the formation of a ?-strand at residues 15-21 is an important feature in A?42 fibril nucleus. In overall evaluation of all of the mutational effects on fibrillization kinetics, we found that the thioflavin T fluorescence at the aggregation plateau is a poor indicator of aggregation rates.
Project description:<h4>Background</h4>The misfolding of amyloidogenic proteins including human Tau protein, human prion protein, and human ?-synuclein is involved in neurodegenerative diseases such as Alzheimer disease, prion disease, and Parkinson disease. Although a lot of research on such amyloidogenic proteins has been done, we do not know the determinants that drive these proteins to form fibrils and thereby induce neurodegenerative diseases. In this study, we want to know the role of fibril-forming motifs from such amyloidogenic proteins in the fibrillization of human Tau protein.<h4>Methodology/principal findings</h4>As evidenced by thioflavin T binding and turbidity assays, transmission electron microscopy, and circular dichroism, fibril-forming motifs are essential and sufficient for the fibrillization of microtubule-associated protein Tau: only when both of its fibril-forming motifs, PHF6 and PHF6*, are deleted can recombinant human Tau fragment Tau(244-372) lose its ability to form fibrils, and the insertion of unrelated fibril-forming motifs from other amyloidogenic proteins, such as human prion protein, yeast prion protein, human ?-synuclein, and human amyloid ?, into the disabled Tau protein can retrieve its ability to form fibrils. Furthermore, this retrieval is independent of the insertion location on Tau(244-372).<h4>Conclusions/significance</h4>We demonstrate for the first time that insertion of fibril-forming motifs can replace PHF6/PHF6* motifs, driving human Tau protein to form fibrils with different morphologies and different kinetic parameters. Our results suggest that fibril-forming motifs play a key role in the fibrillization of human Tau protein and could be the determinants of amyloidogenic proteins tending to misfold, thereby causing the initiation and development of neurodegenerative diseases. Our study also touches on the importance of amyloid "strains": changes to the amyloidgenic driver region results in altered structural morphologies at the macromolecular level.
Project description:Several degenerative amyloid diseases, with no fully effective treatment, affect millions of people worldwide. These pathologies-amyloidoses-are known to be associated with the formation of ordered protein aggregates and highly stable and insoluble amyloid fibrils, which are deposited in multiple tissues and organs. The disruption of preformed amyloid aggregates and fibrils is one possible therapeutic strategy against amyloidosis; however, only a few compounds have been identified as possible fibril disruptors in vivo to date. To properly identify chemical compounds as potential fibril disruptors, a reliable, fast, and economic screening protocol must be developed. For this purpose, three amyloid fibril formation protocols using transthyretin (TTR), a plasma protein involved in several amyloidoses, were studied using thioflavin-T fluorescence assays, circular dichroism (CD), turbidity, dynamic light scattering (DLS), and transmission electron microscopy (TEM), in order to characterize and select the most appropriate fibril formation protocol. Saturation transfer difference nuclear magnetic resonance spectroscopy (STD NMR) was successfully used to study the interaction of doxycycline, a known amyloid fibril disruptor, with preformed wild-type TTR (TTRwt) aggregates and fibrils. DLS and TEM were also used to characterize the effect of doxycycline on TTRwt amyloid species disaggregation. A comparison of the TTR amyloid morphology formed in different experimental conditions is also presented.
Project description:Amyloid fibril formation is associated with numerous neurodegenerative diseases. To elucidate the mechanism of fibril formation, the thioflavin T (ThT) fluorescence assay is widely used. ThT is a fluorescent dye that selectively binds to amyloid fibrils and exhibits fluorescence enhancement, which enables quantitative analysis of the fibril formation process. However, the detailed binding mechanism has remained unclear. Here we acquire real-time profiles of fibril formation of superoxide dismutase 1 (SOD1) using high-sensitivity Rheo-NMR spectroscopy and detect weak and strong interactions between ThT and SOD1 fibrils in a time-dependent manner. Real-time information on the interaction between ThT and fibrils will contribute to the understanding of the binding mechanism of ThT to fibrils. In addition, our method provides an alternative way to analyze fibril formation.
Project description:Amyloid fibril formation is a distinctive hallmark of a number of degenerative diseases. In this process, protein monomers self-assemble to form insoluble structures that are generally referred to as amyloid fibrils. We have induced in vitro amyloid fibril formation of a PDZ domain by combining mechanical agitation and high ionic strength under conditions otherwise close to physiological (pH 7.0, 37 degrees C, no added denaturants). The resulting aggregates enhance the fluorescence of the thioflavin T dye via a sigmoidal kinetic profile. Both infrared spectroscopy and circular dichroism spectroscopy detect the formation of a largely intermolecular beta-sheet structure. Atomic force microscopy shows straight, rod-like fibrils that are similar in appearance and height to mature amyloid-like fibrils. Under these conditions, before aggregation, the protein domain adopts an essentially native-like structure and an even higher conformational stability (DeltaG(U-F)(H2O)). These results show a new method for converting initially folded proteins into amyloid-like aggregates. The methodological approach used here does not require denaturing conditions; rather, it couples agitation with a high ionic strength. Such an approach offers new opportunities to investigate protein aggregation under conditions in which a globular protein is initially folded, and to elucidate the physical forces that promote amyloid fibril formation.
Project description:Amyloid fibrils are a class of insoluble protein nanofibers that are formed via the self-assembly of a wide range of peptides and proteins. They are increasingly exploited for a broad range of applications in bionanotechnology, such as biosensing and drug delivery, as nanowires, hydrogels, and thin films. Amyloid fibrils have been prepared from many proteins, but there has been no definitive characterization of amyloid fibrils from hemoglobin to date. Here, nanofiber formation was carried out under denaturing conditions using solutions of apo-hemoglobin extracted from bovine waste blood. A characteristic amyloid fibril morphology was confirmed by transmission electron microscopy (TEM) and atomic force microscopy (AFM), with mean fibril dimensions of approximately 5 nm diameter and up to several microns in length. The thioflavin T assay confirmed the presence of ?-sheet structures in apo-hemoglobin fibrils, and X-ray fiber diffraction showed the characteristic amyloid cross-? quaternary structure. Apo-hemoglobin nanofibers demonstrated high stability over a range of temperatures (-20 to 80 °C) and pHs (2-10), and were stable in the presence of organic solvents and trypsin, confirming their potential as nanomaterials with versatile applications. This study conclusively demonstrates the formation of amyloid fibrils from hemoglobin for the first time, and also introduces a cost-effective method for amyloid fibril manufacture using meat industry by-products.