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Sample records for amyloid fibril growth

  1. Amyloid fibrils

    PubMed Central

    Rambaran, Roma N

    2008-01-01

    Amyloid refers to the abnormal fibrous, extracellular, proteinaceous deposits found in organs and tissues. Amyloid is insoluble and is structurally dominated by β-sheet structure. Unlike other fibrous proteins it does not commonly have a structural, supportive or motility role but is associated with the pathology seen in a range of diseases known as the amyloidoses. These diseases include Alzheimer's, the spongiform encephalopathies and type II diabetes, all of which are progressive disorders with associated high morbidity and mortality. Not surprisingly, research into the physicochemical properties of amyloid and its formation is currently intensely pursued. In this chapter we will highlight the key scientific findings and discuss how the stability of amyloid fibrils impacts on bionanotechnology. PMID:19158505

  2. Surfaces Self-Assembly and Rapid Growth of Amyloid Fibrils

    NASA Astrophysics Data System (ADS)

    Lin, Yichih; Petersson, E. James; Fakhraai, Zahra

    2014-03-01

    The mechanism of surface-mediated fibrillization has been considered as a key issue in understanding the origins of the neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. In vitro, amyloid proteins fold through nucleation-elongation process. There is a critical concentration for early nucleating stage. However, some studies indicate that surfaces can modulate the fibril's formation under physiological conditions, even when the concentration is much lower than the critical concentration. Here, we use a label-free procedure to monitor the growth of fibrils across many length scales. We show that near a surface, the fibrillization process appears to bypass the nucleation step and fibrils grow through a self-assembly mechanism instead. We control and measure the pre-fibrillar morphology at different stages of this process on various surfaces. The interplay between the surface concentration and diffusion constant can help identify the detailed mechanisms of surface-mediated fibril growth, which remains largely unexplored. Our works provide a new insight in designing new probes and therapies. Supported by the National Institute On Aging of the National Institutes of Health under Award Number P30AG010124.

  3. Potassium-dependent oriented growth of amyloid β25 35 fibrils on mica

    NASA Astrophysics Data System (ADS)

    Karsai, Árpád; Grama, László; Murvai, Ünige; Soós, Katalin; Penke, Botond; Kellermayer, Miklós S. Z.

    2007-08-01

    Amyloid fibrils are important components of tissue deposits in neurodegenerative and protein misfolding diseases. Because modified amyloid peptide subunits can be generated by synthetic methods and the nanometer-scale fibrils are stable under diverse conditions, amyloid fibrils have been suggested for use in nanotechnology applications. However, well-controlled and oriented growth of amyloid fibrils has not yet been accomplished. Here we show that amyloid β 25-35 (Aβ 25-35), a toxic fragment of Alzheimer's beta peptide, forms trigonally oriented fibrils on mica. Oriented binding depends on an apparently cooperative interaction of a positively charged moiety on the Aβ 25-35 peptide with the K+-binding pocket of the mica lattice. Time-lapse in situ AFM revealed that the formation of oriented fibrils is the result of epitaxial polymerization rather than binding of already assembled fibrils from solution. By varying the K+ concentration the growth rate and the mesh size of the oriented amyloid fibril network may be tuned. The K+-controlled oriented assembly of Aβ 25-35 fibrils could be utilized in nanotechnology applications such as formation of oriented tracks for molecular devices and generation of nanoelectronic circuits.

  4. Amyloid Fibril Solubility.

    PubMed

    Rizzi, L G; Auer, S

    2015-11-19

    It is well established that amyloid fibril solubility is protein specific, but how solubility depends on the interactions between the fibril building blocks is not clear. Here we use a simple protein model and perform Monte Carlo simulations to directly measure the solubility of amyloid fibrils as a function of the interaction between the fibril building blocks. Our simulations confirms that the fibril solubility depends on the fibril thickness and that the relationship between the interactions and the solubility can be described by a simple analytical formula. The results presented in this study reveal general rules how side-chain-side-chain interactions, backbone hydrogen bonding, and temperature affect amyloid fibril solubility, which might prove to be a powerful tool to design protein fibrils with desired solubility and aggregation properties in general. PMID:26496385

  5. Membrane damage by human islet amyloid polypeptide through fibril growth at the membrane.

    PubMed

    Engel, Maarten F M; Khemtémourian, Lucie; Kleijer, Cécile C; Meeldijk, Hans J D; Jacobs, Jet; Verkleij, Arie J; de Kruijff, Ben; Killian, J Antoinette; Höppener, Jo W M

    2008-04-22

    Fibrillar protein deposits (amyloid) in the pancreatic islets of Langerhans are thought to be involved in death of the insulin-producing islet beta cells in type 2 diabetes mellitus. It has been suggested that the mechanism of this beta cell death involves membrane disruption by human islet amyloid polypeptide (hIAPP), the major constituent of islet amyloid. However, the molecular mechanism of hIAPP-induced membrane disruption is not known. Here, we propose a hypothesis that growth of hIAPP fibrils at the membrane causes membrane damage. We studied the kinetics of hIAPP-induced membrane damage in relation to hIAPP fibril growth and found that the kinetic profile of hIAPP-induced membrane damage is characterized by a lag phase and a sigmoidal transition, which matches the kinetic profile of hIAPP fibril growth. The observation that seeding accelerates membrane damage supports the hypothesis. In addition, variables that are well known to affect hIAPP fibril formation, i.e., the presence of a fibril formation inhibitor, hIAPP concentration, and lipid composition, were found to have the same effect on hIAPP-induced membrane damage. Furthermore, electron microscopy analysis showed that hIAPP fibrils line the surface of distorted phospholipid vesicles, in agreement with the notion that hIAPP fibril growth at the membrane and membrane damage are physically connected. Together, these observations point toward a mechanism in which growth of hIAPP fibrils, rather than a particular hIAPP species, is responsible for the observed membrane damage. This hypothesis provides an additional mechanism next to the previously proposed role of oligomers as the main cytotoxic species of amyloidogenic proteins. PMID:18408164

  6. Membrane damage by human islet amyloid polypeptide through fibril growth at the membrane

    PubMed Central

    Engel, Maarten F. M.; Khemtémourian, Lucie; Kleijer, Cécile C.; Meeldijk, Hans J. D.; Jacobs, Jet; Verkleij, Arie J.; de Kruijff, Ben; Killian, J. Antoinette; Höppener, Jo W. M.

    2008-01-01

    Fibrillar protein deposits (amyloid) in the pancreatic islets of Langerhans are thought to be involved in death of the insulin-producing islet β cells in type 2 diabetes mellitus. It has been suggested that the mechanism of this β cell death involves membrane disruption by human islet amyloid polypeptide (hIAPP), the major constituent of islet amyloid. However, the molecular mechanism of hIAPP-induced membrane disruption is not known. Here, we propose a hypothesis that growth of hIAPP fibrils at the membrane causes membrane damage. We studied the kinetics of hIAPP-induced membrane damage in relation to hIAPP fibril growth and found that the kinetic profile of hIAPP-induced membrane damage is characterized by a lag phase and a sigmoidal transition, which matches the kinetic profile of hIAPP fibril growth. The observation that seeding accelerates membrane damage supports the hypothesis. In addition, variables that are well known to affect hIAPP fibril formation, i.e., the presence of a fibril formation inhibitor, hIAPP concentration, and lipid composition, were found to have the same effect on hIAPP-induced membrane damage. Furthermore, electron microscopy analysis showed that hIAPP fibrils line the surface of distorted phospholipid vesicles, in agreement with the notion that hIAPP fibril growth at the membrane and membrane damage are physically connected. Together, these observations point toward a mechanism in which growth of hIAPP fibrils, rather than a particular hIAPP species, is responsible for the observed membrane damage. This hypothesis provides an additional mechanism next to the previously proposed role of oligomers as the main cytotoxic species of amyloidogenic proteins. PMID:18408164

  7. Nucleation of amyloid fibrils

    NASA Astrophysics Data System (ADS)

    Kashchiev, Dimo; Auer, Stefan

    2010-06-01

    We consider nucleation of amyloid fibrils in the case when the process occurs by the mechanism of direct polymerization of practically fully extended protein segments, i.e., β-strands, into β-sheets. Applying the classical nucleation theory, we derive a general expression for the work to form a nanosized amyloid fibril (protofilament) constituted of successively layered β-sheets. Analysis of this expression reveals that with increasing its size, the fibril transforms from one-dimensional to two-dimensional aggregate in order to preserve the equilibrium shape corresponding to minimal formation work. We determine the size of the fibril nucleus, the fibril nucleation work, and the fibril nucleation rate as explicit functions of the concentration and temperature of the protein solution. The results obtained are applicable to homogeneous nucleation, which occurs when the solution is sufficiently pure and/or strongly supersaturated.

  8. Polymorph-specific kinetics and thermodynamics of β-amyloid fibril growth

    PubMed Central

    Qiang, Wei; Kelley, Kevin; Tycko, Robert

    2013-01-01

    Amyloid fibrils formed by the 40-residue β-amyloid peptide (Aβ1–40) are highly polymorphic, with molecular structures that depend on the details of growth conditions. Underlying differences in physical properties are not well understood. Here, we investigate differences in growth kinetics and thermodynamic stabilities of two Aβ1–40 fibril polymorphs for which detailed structural models are available from solid state nuclear magnetic resonance (NMR) studies. Rates of seeded fibril elongation in the presence of excess soluble Aβ1–40 and shrinkage in the absence of soluble Aβ1–40 are determined with atomic force microscopy (AFM). From these rates, we derive polymorph-specific values for the soluble Aβ1–40 concentration at quasi-equilibrium, from which relative stabilities can be derived. The AFM results are supported by direct measurements by ultraviolet absorbance, using a novel dialysis system to establish quasi-equilibrium. At 24° C, the two polymorphs have significantly different elongation and shrinkage kinetics but similar thermodynamic stabilities. At 37° C, differences in kinetics are reduced, and thermodynamic stabilities are increased significantly. Fibril length distributions in AFM images provide support for an intermittent growth model, in which fibrils switch randomly between an "on" state (capable of elongation) and an "off" state (incapable of elongation). We also monitor interconversion between polymorphs at 24° C by solid state NMR, showing that the two-fold symmetric "agitated" () polymorph is more stable than the three-fold symmetric "quiescent" polymorph. Finally, we show that the two polymorphs have significantly different rates of fragmentation in the presence of shear forces, a difference that helps explain the observed predominance of the structure when fibrils are grown in agitated solutions. PMID:23627695

  9. Are amyloid fibrils molecular spandrels?

    PubMed

    Hane, Francis

    2013-11-15

    Amyloid-β, the protein implicated in Alzheimer's disease, along with a number of other proteins, has been shown to form amyloid fibrils. Fibril forming proteins share no common primary structure and have little known function. Furthermore, all proteins have the ability to form amyloid fibrils under certain conditions as the fibrillar structure lies at the global free energy minimum of proteins. This raises the question of the mechanism of the evolution of the amyloid fibril structure. Experimental evidence supports the hypothesis that the fibril structure is a by-product of the forces of protein folding and lies outside the bounds of evolutionary pressures. PMID:24140343

  10. Quality control system response to stochastic growth of amyloid fibrils.

    PubMed

    Pigolotti, Simone; Lizana, Ludvig; Otzen, Daniel; Sneppen, Kim

    2013-05-01

    We introduce a stochastic model describing aggregation of misfolded proteins and degradation by the protein quality control system in a single cell. Aggregate growth is contrasted by the cell quality control system, that attacks them at different stages of the growth process, with an efficiency that decreases with their size. Model parameters are estimated from experimental data. Two qualitatively different behaviors emerge: a homeostatic state, where the quality control system is stable and aggregates of large sizes are not formed, and an oscillatory state, where the quality control system periodically breaks down, allowing for formation of large aggregates. We discuss how these periodic breakdowns may constitute a mechanism for the development of neurodegenerative diseases. PMID:23524241

  11. Gold-Induced Fibril Growth: The Mechanism of Surface-Facilitated Amyloid Aggregation.

    PubMed

    Gladytz, Anika; Abel, Bernd; Risselada, Herre Jelger

    2016-09-01

    The question of how amyloid fibril formation is influenced by surfaces is crucial for a detailed understanding of the process in vivo. We applied a combination of kinetic experiments and molecular dynamics simulations to elucidate how (model) surfaces influence fibril formation of the amyloid-forming sequences of prion protein SUP35 and human islet amyloid polypeptide. The kinetic data suggest that structural reorganization of the initial peptide corona around colloidal gold nanoparticles is the rate-limiting step. The molecular dynamics simulations reveal that partial physisorption to the surface results in the formation of aligned monolayers, which stimulate the formation of parallel, critical oligomers. The general mechanism implies that the competition between the underlying peptide-peptide and peptide-surface interactions must strike a balance to accelerate fibril formation. PMID:27513605

  12. Structural fingerprints and their evolution during oligomeric vs. oligomer-free amyloid fibril growth

    PubMed Central

    Foley, Joseph; Hill, Shannon E.; Miti, Tatiana; Mulaj, Mentor; Ciesla, Marissa; Robeel, Rhonda; Persichilli, Christopher; Raynes, Rachel; Westerheide, Sandy; Muschol, Martin

    2013-01-01

    Deposits of fibrils formed by disease-specific proteins are the molecular hallmark of such diverse human disorders as Alzheimer's disease, type II diabetes, or rheumatoid arthritis. Amyloid fibril formation by structurally and functionally unrelated proteins exhibits many generic characteristics, most prominently the cross β-sheet structure of their mature fibrils. At the same time, amyloid formation tends to proceed along one of two separate assembly pathways yielding either stiff monomeric filaments or globular oligomers and curvilinear protofibrils. Given the focus on oligomers as major toxic species, the very existence of an oligomer-free assembly pathway is significant. Little is known, though, about the structure of the various intermediates emerging along different pathways and whether the pathways converge towards a common or distinct fibril structures. Using infrared spectroscopy we probed the structural evolution of intermediates and late-stage fibrils formed during in vitro lysozyme amyloid assembly along an oligomeric and oligomer-free pathway. Infrared spectroscopy confirmed that both pathways produced amyloid-specific β-sheet peaks, but at pathway-specific wavenumbers. We further found that the amyloid-specific dye thioflavin T responded to all intermediates along either pathway. The relative amplitudes of thioflavin T fluorescence responses displayed pathway-specific differences and could be utilized for monitoring the structural evolution of intermediates. Pathway-specific structural features obtained from infrared spectroscopy and Thioflavin T responses were identical for fibrils grown at highly acidic or at physiological pH values and showed no discernible effects of protein hydrolysis. Our results suggest that late-stage fibrils formed along either pathway are amyloidogenic in nature, but have distinguishable structural fingerprints. These pathway-specific fingerprints emerge during the earliest aggregation events and persist throughout the

  13. Structural fingerprints and their evolution during oligomeric vs. oligomer-free amyloid fibril growth

    NASA Astrophysics Data System (ADS)

    Foley, Joseph; Hill, Shannon E.; Miti, Tatiana; Mulaj, Mentor; Ciesla, Marissa; Robeel, Rhonda; Persichilli, Christopher; Raynes, Rachel; Westerheide, Sandy; Muschol, Martin

    2013-09-01

    Deposits of fibrils formed by disease-specific proteins are the molecular hallmark of such diverse human disorders as Alzheimer's disease, type II diabetes, or rheumatoid arthritis. Amyloid fibril formation by structurally and functionally unrelated proteins exhibits many generic characteristics, most prominently the cross β-sheet structure of their mature fibrils. At the same time, amyloid formation tends to proceed along one of two separate assembly pathways yielding either stiff monomeric filaments or globular oligomers and curvilinear protofibrils. Given the focus on oligomers as major toxic species, the very existence of an oligomer-free assembly pathway is significant. Little is known, though, about the structure of the various intermediates emerging along different pathways and whether the pathways converge towards a common or distinct fibril structures. Using infrared spectroscopy we probed the structural evolution of intermediates and late-stage fibrils formed during in vitro lysozyme amyloid assembly along an oligomeric and oligomer-free pathway. Infrared spectroscopy confirmed that both pathways produced amyloid-specific β-sheet peaks, but at pathway-specific wavenumbers. We further found that the amyloid-specific dye thioflavin T responded to all intermediates along either pathway. The relative amplitudes of thioflavin T fluorescence responses displayed pathway-specific differences and could be utilized for monitoring the structural evolution of intermediates. Pathway-specific structural features obtained from infrared spectroscopy and Thioflavin T responses were identical for fibrils grown at highly acidic or at physiological pH values and showed no discernible effects of protein hydrolysis. Our results suggest that late-stage fibrils formed along either pathway are amyloidogenic in nature, but have distinguishable structural fingerprints. These pathway-specific fingerprints emerge during the earliest aggregation events and persist throughout the

  14. Investigation of the growth mechanisms of diglyme plasma polymers on amyloid fibril networks

    NASA Astrophysics Data System (ADS)

    Li, Yali; Reynolds, Nicholas P.; Styan, Katie E.; Muir, Benjamin W.; Forsythe, John S.; Easton, Christopher D.

    2016-01-01

    Within the area of biomaterials research, the ability to tailor a materials surface chemistry while presenting a biomimetic topography is a useful tool for studying cell-surface and cell-cell interactions. For the study reported here we investigated the deposition of diglyme plasma polymer films (DGpp) onto amyloid fibril networks (AFNs), which have morphologies that mimic the extracellular matrix. We extend our previous work to observe that the nanoscale contours of the AFNs are well preserved even under thick layers of DGpp. The width of the surface features is positively correlated to the DGpp thickness. DGpp film growth conformed to the underlying fibril features, with a gradual smoothing out of the resultant surface topography. Further, to understand how the films grow on top of AFNs, X-ray photoelectron spectroscopy depth profiling was employed to determine the elemental composition within the coating, perpendicular to the plane of the substrate. It was found that AFNs partially fragment during the initial stage of plasma polymerisation, and these fragments then mix with the growing DGpp to form an intermixed interface region above the AFN. The findings in this study are likely applicable to situations where plasma polymerisation is used to apply an overcoat to adsorbed organic and/or biological molecules.

  15. Nucleation of polymorphic amyloid fibrils.

    PubMed

    Auer, Stefan

    2015-03-10

    One and the same protein can self-assemble into amyloid fibrils with different morphologies. The phenomenon of fibril polymorphism is relevant biologically because different fibril polymorphs can have different toxicity, but there is no tool for predicting which polymorph forms and under what conditions. Here, we consider the nucleation of polymorphic amyloid fibrils occurring by direct polymerization of monomeric proteins into fibrils. We treat this process within the framework of our newly developed nonstandard nucleation theory, which allows prediction of the concentration dependence of the nucleation rate for different fibril polymorphs. The results highlight that the concentration dependence of the nucleation rate is closely linked with the protein solubility and a threshold monomer concentration below which fibril formation becomes biologically irrelevant. The relation between the nucleation rate, the fibril solubility, the threshold concentration, and the binding energies of the fibril building blocks within fibrils might prove a valuable tool for designing new experiments to control the formation of particular fibril polymorphs. PMID:25762329

  16. Metastable Amyloid Phases and their Conversion to Mature Fibrils

    NASA Astrophysics Data System (ADS)

    Muschol, Martin; Miti, Tatiana; Mulaj, Mentor; Schmit, Jeremy

    Self-assembly of proteins into amyloid fibrils plays a key role in both functional biological responses and pathogenic disorders which include Alzheimer's disease and type II diabetes. Amyloid fibril assembly frequently generates compact oligomeric and curvilinear polymeric intermediates which are implicated to be toxic to cells. Yet, the relation between these early-stage oligomeric aggregates and late-stage rigid fibrils, which are the hallmark structure of amyloid plaques, has remained unclear. Our measurements indicate that lysozyme amyloid oligomers and their curvilinear fibrils only form after crossing a salt and protein concentration dependent threshold. These oligomeric aggregates are structurally distinct from rigid fibrils and are metastable against nucleation and growth of rigid fibrils. Our experimental transition boundaries match well with colloidal model predictions accounting for salt-modulated charge repulsion. We also report our preliminary findings on the mechanism by which these metastable oligomeric phases are converted into stable amyloid fibrils.

  17. Shear flow promotes amyloid-{beta} fibrilization.

    PubMed

    Dunstan, Dave E; Hamilton-Brown, Paul; Asimakis, Peter; Ducker, William; Bertolini, Joseph

    2009-12-01

    The rate of formation of amyloid fibrils in an aqueous solution of amyloid-beta (Abeta) is greatly increased when the solution is sheared. When Abeta solution is stirred with a magnetic stirrer bar at 37 degrees C, a rapid increase in thioflavin T fluorescence is observed. Atomic Force Microscopy (AFM) images show the formation of aggregates, the growth of fibrils and the intertwining of the fibrils with time. Circular dichroism (CD) spectroscopy of samples taken after stirring shows a transition from random coil to alpha-helix to beta-sheet secondary structure over 20 h at 37 degrees C. The fluorescence, AFM and CD measurements are all consistent with the formation of amyloid fibrils. Quiescent, non-stirred solutions incubated at 37 degrees C showed no evidence of amyloid formation over a period of 3 days. Couette flow was found to accelerate the formation of amyloid fibrils demonstrating that the primary effect of stirring is not mixing but shearing. Only very small shear forces are applied to individual molecules in our experiments. Simple calculation suggests that the force is too small to support a hypothesis that shearing promotes partial unfolding of the protein as is observed. PMID:19850675

  18. Nanomechanical properties of single amyloid fibrils

    NASA Astrophysics Data System (ADS)

    Sweers, K. K. M.; Bennink, M. L.; Subramaniam, V.

    2012-06-01

    Amyloid fibrils are traditionally associated with neurodegenerative diseases like Alzheimer’s disease, Parkinson’s disease or Creutzfeldt-Jakob disease. However, the ability to form amyloid fibrils appears to be a more generic property of proteins. While disease-related, or pathological, amyloid fibrils are relevant for understanding the pathology and course of the disease, functional amyloids are involved, for example, in the exceptionally strong adhesive properties of natural adhesives. Amyloid fibrils are thus becoming increasingly interesting as versatile nanobiomaterials for applications in biotechnology. In the last decade a number of studies have reported on the intriguing mechanical characteristics of amyloid fibrils. In most of these studies atomic force microscopy (AFM) and atomic force spectroscopy play a central role. AFM techniques make it possible to probe, at nanometer length scales, and with exquisite control over the applied forces, biological samples in different environmental conditions. In this review we describe the different AFM techniques used for probing mechanical properties of single amyloid fibrils on the nanoscale. An overview is given of the existing mechanical studies on amyloid. We discuss the difficulties encountered with respect to the small fibril sizes and polymorphic behavior of amyloid fibrils. In particular, the different conformational packing of monomers within the fibrils leads to a heterogeneity in mechanical properties. We conclude with a brief outlook on how our knowledge of these mechanical properties of the amyloid fibrils can be exploited in the construction of nanomaterials from amyloid fibrils.

  19. Kinetic theory of amyloid fibril templating

    NASA Astrophysics Data System (ADS)

    Schmit, Jeremy D.

    2013-05-01

    The growth of amyloid fibrils requires a disordered or partially unfolded protein to bind to the fibril and adapt the same conformation and alignment established by the fibril template. Since the H-bonds stabilizing the fibril are interchangeable, it is inevitable that H-bonds form between incorrect pairs of amino acids which are either incorporated into the fibril as defects or must be broken before the correct alignment can be found. This process is modeled by mapping the formation and breakage of H-bonds to a one-dimensional random walk. The resulting microscopic model of fibril growth is governed by two timescales: the diffusion time of the monomeric proteins, and the time required for incorrectly bound proteins to unbind from the fibril. The theory predicts that the Arrhenius behavior observed in experiments is due to off-pathway states rather than an on-pathway transition state. The predicted growth rates are in qualitative agreement with experiments on insulin fibril growth rates as a function of protein concentration, denaturant concentration, and temperature. These results suggest a templating mechanism where steric clashes due to a single mis-aligned molecule prevent the binding of additional molecules.

  20. Urea modulation of β-amyloid fibril growth: Experimental studies and kinetic models

    PubMed Central

    Kim, Jin Ryoun; Muresan, Adrian; Lee, Ka Yee C.; Murphy, Regina M.

    2004-01-01

    Aggregation of β-amyloid (Aβ) into fibrillar deposits is widely believed to initiate a cascade of adverse biological responses associated with Alzheimer’s disease. Although it was once assumed that the mature fibril was the toxic form of Aβ, recent evidence supports the hypothesis that Aβ oligomers, intermediates in the fibrillogenic pathway, are the dominant toxic species. In this work we used urea to reduce the driving force for Aβ aggregation, in an effort to isolate stable intermediate species. The effect of urea on secondary structure, size distribution, aggregation kinetics, and aggregate morphology was examined. With increasing urea concentration, β-sheet content and the fraction of aggregated peptide decreased, the average size of aggregates was reduced, and the morphology of aggregates changed from linear to a globular/linear mixture and then to globular. The data were analyzed using a previously published model of Aβ aggregation kinetics. The model and data were consistent with the hypothesis that the globular aggregates were intermediates in the amyloidogenesis pathway rather than alternatively aggregated species. Increasing the urea concentration from 0.4 M to 2 M decreased the rate of filament initiation the most; between 2 M and 4 M urea the largest change was in partitioning between the nonamyloid and amyloid pathways, and between 4 M and 6 M urea, the most significant change was a reduction in the rate of filament elongation. PMID:15459334

  1. Amyloid Fibrils: Formation, Polymorphism, and Inhibition.

    PubMed

    Härd, Torleif

    2014-02-01

    Amyloid fibrils with cross-β spine basic architectures are prevalent and stable forms of peptides and proteins. Recent research has provided significant contributions to our understanding of the mechanisms of fibril formation and to the surprising diversity and persistence of structural polymorphism in amyloid fibrils. There have also been successful demonstrations of how molecules can be engineered to inhibit unwanted amyloid formation by different mechanisms. Future research in these areas will include investigations of mechanisms for primary nucleation and the structure of oligomeric intermediates, the general role of secondary nucleation events (autocatalysis), elucidation of the mechanisms and implications of preservation of structural morphology in amyloid propagation, and research into the largely unexplored phenomenon of cross-seeding, by which amyloid fibrils of one species induce the formation of amyloid by another species. PMID:26276617

  2. Polymorphism of Amyloid Fibrils In Vivo.

    PubMed

    Annamalai, Karthikeyan; Gührs, Karl-Heinz; Koehler, Rolf; Schmidt, Matthias; Michel, Henri; Loos, Cornelia; Gaffney, Patricia M; Sigurdson, Christina J; Hegenbart, Ute; Schönland, Stefan; Fändrich, Marcus

    2016-04-01

    Polymorphism is a wide-spread feature of amyloid-like fibrils formed in vitro, but it has so far remained unclear whether the fibrils formed within a patient are also affected by this phenomenon. In this study we show that the amyloid fibrils within a diseased individual can vary considerably in their three-dimensional architecture. We demonstrate this heterogeneity with amyloid fibrils deposited within different organs, formed from sequentially non-homologous polypeptide chains and affecting human or animals. Irrespective of amyloid type or source, we found in vivo fibrils to be polymorphic. These data imply that the chemical principles of fibril assembly that lead to such polymorphism are fundamentally conserved in vivo and in vitro. PMID:26954430

  3. Amyloid at the nanoscale: AFM and single-molecule investigations of early steps of aggregation and mature fibril growth, structure, and mechanics

    NASA Astrophysics Data System (ADS)

    Subramaniam, Vinod

    2013-03-01

    Misfolding and aggregation of proteins into nanometer-scale fibrillar assemblies is a hallmark of many neurodegenerative diseases. We have investigated the self-assembly of the human intrinsically disordered protein alpha-synuclein, involved in Parkinson's disease, into amyloid fibrils. A particularly relevant question is the role of early oligomeric aggregates in modulating the dynamics of protein nucleation and aggregation. We have used single molecule fluorescence spectroscopy to characterize conformational transitions of alpha-synuclein, and to gain insights into the structure and composition of oligomeric aggregates of alpha-synuclein. Quantitative atomic force microscopy and nanomechanical investigations provide information on amyloid fibril polymorphism and on nanoscale mechanical properties of mature fibrillar species, while conventional optical and super-resolution imaging have yielded insights into the growth of fibrils and into the assembly of suprafibrillar structures. We thank the Foundation for Fundamental Research on Matter (FOM), the Netherlands Organisation for Scientific Research (NWO), and the MESA+ Institute for Nanotechnology for support.

  4. Quenched Hydrogen Exchange NMR of Amyloid Fibrils.

    PubMed

    Alexandrescu, Andrei T

    2016-01-01

    Amyloid fibrils are associated with a number of human diseases. These aggregatively misfolded intermolecular β-sheet assemblies constitute some of the most challenging targets in structural biology because to their complexity, size, and insolubility. Here, protocols and controls are described for experiments designed to study hydrogen-bonding in amyloid fibrils indirectly, by transferring information about amide proton occupancy in the fibrils to the dimethyl sulfoxide-denatured state. Since the denatured state is amenable to solution NMR spectroscopy, the method can provide residue-level-resolution data on hydrogen exchange for the monomers that make up the fibrils. PMID:26453215

  5. Fibril Fragmentation Enhances Amyloid Cytotoxicity*♦

    PubMed Central

    Xue, Wei-Feng; Hellewell, Andrew L.; Gosal, Walraj S.; Homans, Steve W.; Hewitt, Eric W.; Radford, Sheena E.

    2009-01-01

    Fibrils associated with amyloid disease are molecular assemblies of key biological importance, yet how cells respond to the presence of amyloid remains unclear. Cellular responses may not only depend on the chemical composition or molecular properties of the amyloid fibrils, but their physical attributes such as length, width, or surface area may also play important roles. Here, we report a systematic investigation of the effect of fragmentation on the structural and biological properties of amyloid fibrils. In addition to the expected relationship between fragmentation and the ability to seed, we show a striking finding that fibril length correlates with the ability to disrupt membranes and to reduce cell viability. Thus, despite otherwise unchanged molecular architecture, shorter fibrillar samples show enhanced cytotoxic potential than their longer counterparts. The results highlight the importance of fibril length in amyloid disease, with fragmentation not only providing a mechanism by which fibril load can be rapidly increased but also creating fibrillar species of different dimensions that can endow new or enhanced biological properties such as amyloid cytotoxicity. PMID:19808677

  6. Molecular structures of amyloid and prion fibrils: consensus versus controversy.

    PubMed

    Tycko, Robert; Wickner, Reed B

    2013-07-16

    Many peptides and proteins self-assemble into amyloid fibrils. Examples include mammalian and fungal prion proteins, polypeptides associated with human amyloid diseases, and proteins that may have biologically functional amyloid states. To understand the propensity for polypeptides to form amyloid fibrils and to facilitate rational design of amyloid inhibitors and imaging agents, it is necessary to elucidate the molecular structures of these fibrils. Although fibril structures were largely mysterious 15 years ago, a considerable body of reliable structural information about amyloid fibril structures now exists, with essential contributions from solid state nuclear magnetic resonance (NMR) measurements. This Account reviews results from our laboratories and discusses several structural issues that have been controversial. In many cases, the amino acid sequences of amyloid fibrils do not uniquely determine their molecular structures. Self-propagating, molecular-level polymorphism complicates the structure determination problem and can lead to apparent disagreements between results from different laboratories, particularly when different laboratories study different polymorphs. For 40-residue β-amyloid (Aβ₁₋₄₀) fibrils associated with Alzheimer's disease, we have developed detailed structural models from solid state NMR and electron microscopy data for two polymorphs. These polymorphs have similar peptide conformations, identical in-register parallel β-sheet organizations, but different overall symmetry. Other polymorphs have also been partially characterized by solid state NMR and appear to have similar structures. In contrast, cryo-electron microscopy studies that use significantly different fibril growth conditions have identified structures that appear (at low resolution) to be different from those examined by solid state NMR. Based on solid state NMR and electron paramagnetic resonance (EPR) measurements, the in-register parallel β-sheet organization

  7. Compressive deformation of ultralong amyloid fibrils

    NASA Astrophysics Data System (ADS)

    Paparcone, Raffaella; Cranford, Steven; Buehler, Markus J.

    2010-12-01

    Involved in various neurodegenerative diseases, amyloid fibrils and plaques feature a hierarchical structure, ranging from the atomistic to the micrometer scale. At the atomistic level, a dense and organized hydrogen bond network is resembled in a beta-sheet rich secondary structure, which drives a remarkable stiffness in the range of 10-20GPa, larger than many other biological nanofibrils, a result confirmed by both experiment and theory. However, the understanding of how these exceptional mechanical properties transfer from the atomistic to the nanoscale remains unknown. Here we report a multiscale analysis that, from the atomistic-level structure of a single fibril, extends to the mesoscale level, reaching size scales of hundreds of nanometers. We use parameters directly derived from full atomistic simulations of A β (1-40) amyloid fibrils to parameterize a mesoscopic coarse-grained model, which is used to reproduce the elastic properties of amyloid fibrils. We then apply our mesoscopic model in an analysis of the buckling behavior of amyloid fibrils with different lengths and report a comparison with predictions from continuum beam theory. An important implication of our results is a severe reduction of the effective modulus due to buckling, an effect that could be important to interpret experimental results of ultra-long amyloid fibrils. Our model represents a powerful tool to mechanically characterize molecular structures on the order of hundreds of nanometers to micrometers on the basis of the underlying atomistic behavior. The work provides insight into structural and mechanical properties of amyloid fibrils and may enable further analysis of larger-scale assemblies such as amyloidogenic bundles or plaques as found in disease states.

  8. Magnetite nanoparticle interactions with insulin amyloid fibrils.

    PubMed

    Chen, Yun-Wen; Chang, Chiung-Wen; Hung, Huey-Shan; Kung, Mei-Lang; Yeh, Bi-Wen; Hsieh, Shuchen

    2016-10-14

    Accumulation of amyloid fibrils is one of the likely key factors leading to the development of Alzheimer's disease and other amyloidosis associated diseases. Magnetic nanoparticles (NPs) have been developed as promising medical materials for many medical applications. In this study, we have explored the effects of Fe3O4 NPs on the fibrillogenesis process of insulin fibrils. When Fe3O4 NPs were co-incubated with insulin, Fe3O4 NPs had no effect on the structural transformation into amyloid-like fibrils but had higher affinity toward insulin fibrils. We demonstrated that the zeta potential of insulin fibrils and Fe3O4 NPs were both positive, suggesting the binding forces between Fe3O4 NPs and insulin fibrils were van der Waals forces but not surface charge. Moreover, a different amount of Fe3O4 NPs added had no effect on secondary structural changes of insulin fibrils. These results propose the potential use of Fe3O4 NPs as therapeutic agents against diseases related to protein aggregation or contrast agents for magnetic resonance imaging. PMID:27585675

  9. Residue-specific, real-time characterization of lag-phase species and fibril growth during amyloid formation: a combined fluorescence and IR study of p-cyanophenylalanine analogs of islet amyloid polypeptide.

    PubMed

    Marek, Peter; Mukherjee, Sudipta; Zanni, Martin T; Raleigh, Daniel P

    2010-07-23

    Amyloid formation normally exhibits a lag phase followed by a growth phase, which leads to amyloid fibrils. Characterization of the species populated during the lag phase is experimentally challenging, but is critical since the most toxic entities may be pre-fibrillar species. p-Cyanophenylalanine (F(C[triple bond]N)) fluorescence is used to probe the nature of lag-phase species populated during the formation of amyloid by human islet amyloid polypeptide. The polypeptide contains two phenylalanines at positions 15 and 23 and a single tyrosine located at the C-terminus. Each aromatic residue was separately replaced by F(C[triple bond]N). The substitutions do not perturb amyloid formation relative to wild-type islet amyloid polypeptide as detected using thioflavin T fluorescence and electron microscopy. F(C[triple bond]N) fluorescence is high when the cyano group is hydrogen bonded and low when it is not. It can also be quenched via Förster resonance energy transfer to tyrosine. Fluorescence intensity was monitored in real time and revealed that all three positions remained exposed to solvent during the lag phase but less exposed than unstructured model peptides. The time course of amyloid formation as monitored by thioflavin T fluorescence and F(C[triple bond]N) fluorescence is virtually identical. Fluorescence quenching experiments confirmed that each residue remains exposed during the lag phase. These results place significant constraints on the nature of intermediates that are populated during the lag phase and indicate that significant sequestering of the aromatic side chains does not occur until beta-structure sufficient to bind thioflavin T has developed. Seeding studies and analysis of maximum rates confirm that sequestering of the cyano groups occurs concomitantly with the development of thioflavin T binding capability. Overall, the process of amyloid formation and growth appears to be remarkably homogenous in terms of side-chain ordering. F(C[triple bond

  10. Amyloid fibrils as a nanoscaffold for enzyme immobilization.

    PubMed

    Pilkington, Sarah M; Roberts, Sarah J; Meade, Susie J; Gerrard, Juliet A

    2010-01-01

    Amyloid fibrils are a misfolded state, formed by many proteins when subjected to denaturing conditions. Their constituent amino acids make them ideally suited as a readily functionalized nanoscaffold for enzyme immobilization and their strength, stability, and nanometer size are attractive features for exploitation in the creation of new bionanomaterials. We report successful functionalization of amyloid fibrils by conjugation to glucose oxidase (GOD) using glutaraldehyde. GOD retained activity upon attachment and successful cross-linking was determined using electrophoresis, centrifugation, sucrose gradient centrifugation, and TEM. The resulting functionalized enzyme scaffold was then incorporated into a model poly(vinyl alcohol) (PVOH) film, to create a new bionanomaterial. The antibacterial effect of the functionalized film was then tested on E. coli, the growth of which was inhibited, demonstrating the incorporation of GOD antibacterial activity into the PVOH film. The incorporation of the GOD-functionalized amyloid fibrils into PVOH provides an excellent 'proof of concept' model for the creation of a new bionanomaterial using a functionalized amyloid fibril scaffold. PMID:19918761

  11. Atomistic theory of amyloid fibril nucleation

    NASA Astrophysics Data System (ADS)

    Cabriolu, Raffaela; Kashchiev, Dimo; Auer, Stefan

    2010-12-01

    We consider the nucleation of amyloid fibrils at the molecular level when the process takes place by a direct polymerization of peptides or protein segments into β-sheets. Employing the atomistic nucleation theory (ANT), we derive a general expression for the work to form a nanosized amyloid fibril (protofilament) composed of successively layered β-sheets. The application of this expression to a recently studied peptide system allows us to determine the size of the fibril nucleus, the fibril nucleation work, and the fibril nucleation rate as functions of the supersaturation of the protein solution. Our analysis illustrates the unique feature of ANT that the size of the fibril nucleus is a constant integer in a given supersaturation range. We obtain the ANT nucleation rate and compare it with the rates determined previously in the scope of the classical nucleation theory (CNT) and the corrected classical nucleation theory (CCNT). We find that while the CNT nucleation rate is orders of magnitude greater than the ANT one, the CCNT and ANT nucleation rates are in very good quantitative agreement. The results obtained are applicable to homogeneous nucleation, which occurs when the protein solution is sufficiently pure and/or strongly supersaturated.

  12. Simulations of nucleation and elongation of amyloid fibrils

    PubMed Central

    Zhang, Jianing; Muthukumar, M.

    2009-01-01

    We present a coarse-grained model for the growth kinetics of amyloid fibrils from solutions of peptides and address the fundamental mechanism of nucleation and elongation by using a lattice Monte Carlo procedure. We reproduce the three main characteristics of nucleation of amyloid fibrils: (1) existence of lag time, (2) occurrence of a critical concentration, and (3) seeding. We find the nucleation of amyloid fibrils to require a quasi-two-dimensional configuration, where a second layer of β sheet must be formed adjunct to a first layer, which in turn leads to a highly cooperative nucleation barrier. The elongation stage is found to involve the Ostwald ripening (evaporation-condensation) mechanism, whereby bigger fibrils grow at the expense of smaller ones. This new mechanism reconciles the debate as to whether protofibrils are precursors or monomer reservoirs. We have systematically investigated the roles of time, peptide concentration, temperature, and seed size. In general, we find that there are two kinds of lag time arising from two different mechanisms. For higher temperatures or low enough concentrations close to the disassembly boundary, the fibrillization follows the nucleation mechanism. However, for low temperatures, where the nucleation time is sufficiently short, there still exists an apparent lag time due to slow Ostwald ripening mechanism. Consequently, the lag time is nonmonotonic with temperature, with the shortest lag time occurring at intermediate temperatures, which in turn depend on the peptide concentration. While the nucleation dominated regime can be controlled by seeding, the Ostwald ripening regime is insensitive to seeding. Simulation results from our coarse-grained model on the fibril size, lag time, elongation rate, and solubility are consistent with available experimental observations on many specific amyloid systems. PMID:19173542

  13. Scanning ion conductance microscopy studies of amyloid fibrils at nanoscale

    NASA Astrophysics Data System (ADS)

    Zhang, Shuai; Cho, Sang-Joon; Busuttil, Katerina; Wang, Chen; Besenbacher, Flemming; Dong, Mingdong

    2012-05-01

    Atomic force microscopy (AFM) has developed to become a very versatile nano-scale technique to reveal the three-dimensional (3D) morphology of amyloid aggregates under physiological conditions. However, the imaging principle of AFM is based on measuring the `force' between a sharp tip and a given nanostructure, which may cause mechanical deformation of relatively soft objects. To avoid the deformation, scanning ion conductance microscopy (SICM) is an alternative scanning probe microscopy technique, operating with alternating current mode. Here we can indeed reveal the 3D morphology of amyloid fibrils and it is capable of exploring proteins with nanoscale resolution. Compared with conventional AFM, we show that SICM can provide precise height measurements of amyloid protein aggregates, a feature that enables us to obtain unique insight into the detailed nucleation and growth mechanisms behind amyloid self-assembly.

  14. Fibril structure of human islet amyloid polypeptide.

    PubMed

    Bedrood, Sahar; Li, Yiyu; Isas, J Mario; Hegde, Balachandra G; Baxa, Ulrich; Haworth, Ian S; Langen, Ralf

    2012-02-17

    Misfolding and amyloid fibril formation by human islet amyloid polypeptide (hIAPP) are thought to be important in the pathogenesis of type 2 diabetes, but the structures of the misfolded forms remain poorly understood. Here we developed an approach that combines site-directed spin labeling with continuous wave and pulsed EPR to investigate local secondary structure and to determine the relative orientation of the secondary structure elements with respect to each other. These data indicated that individual hIAPP molecules take up a hairpin fold within the fibril. This fold contains two β-strands that are much farther apart than expected from previous models. Atomistic structural models were obtained using computational refinement with EPR data as constraints. The resulting family of structures exhibited a left-handed helical twist, in agreement with the twisted morphology observed by electron microscopy. The fibril protofilaments contain stacked hIAPP monomers that form opposing β-sheets that twist around each other. The two β-strands of the monomer adopt out-of-plane positions and are staggered by about three peptide layers (∼15 Å). These results provide a mechanism for hIAPP fibril formation and could explain the remarkable stability of the fibrils. Thus, the structural model serves as a starting point for understanding and preventing hIAPP misfolding. PMID:22187437

  15. Nanopore analysis of amyloid fibrils formed by lysozyme aggregation.

    PubMed

    Martyushenko, Nikolay; Bell, Nicholas A W; Lamboll, Robin D; Keyser, Ulrich F

    2015-07-21

    The measurement of single particle size distributions of amyloid fibrils is crucial for determining mechanisms of growth and toxicity. Nanopore sensing is an attractive solution for this problem since it gives information on aggregates' shapes with relatively high throughput for a single particle technology. In this paper we study the translocation of lysozyme fibrils through quartz glass nanopores. We demonstrate that, under appropriate salt and pH conditions, lysozyme fibrils translocate through bare quartz nanopores without causing significant clogging. This enables us to measure statistics on tens of thousands of translocations of lysozyme fibrils with the same nanopore and track their development over a time course of aggregation spanning 24 h. Analysis of our events shows that the statistics are consistent with a simple bulk conductivity model for the passage of rods with a fixed cross sectional area through a conical glass nanopore. PMID:25994201

  16. [Cytotoxicity of amyloid fibrils of X-protein].

    PubMed

    Marsagishvili, L G; Shpagina, M D; Shatalin, Iu V; Shubina, V S; Naumov, A A; Potselueva, M M; Podlubnaia, Z A

    2006-01-01

    It is known that amyloid oligomers, protofibrils, and fibrils induce cell death, and antibiotic tetracycline inhibits the fibrillization of beta amyloid peptides and other amyloidogenic proteins and disassembles their pre-formed fibrils. Earlier we have demonstrated that sarcomeric cytoskeletal proteins of the titin family (X-, C-, and H-proteins) are capable to form in vitro amyloid fibrils, and tetracycline effectively destroys these fibrils. Here we show that the viability of polymorphonuclear leukocytes in the presence of X-protein amyloids depends on the concentration of amyloid fibrils of X-protein and the time of incubation. In addition to the disaggregation of X-protein fibrils, tetracycline eliminated the cytotoxic effect of the protein. The antibiotic itself did not show a toxic effect, and the cell viability in its presence even increased. Our results evidence the potential of this approach for evaluating the effectiveness of drugs preventing or treating amyloidoses. PMID:17131815

  17. Physical and structural basis for polymorphism in amyloid fibrils

    PubMed Central

    Tycko, Robert

    2014-01-01

    As our understanding of the molecular structures of amyloid fibrils has matured over the past 15 years, it has become clear that, while amyloid fibrils do have well-defined molecular structures, their molecular structures are not uniquely determined by the amino acid sequences of their constituent peptides and proteins. Self-propagating molecular-level polymorphism is a common phenomenon. This article reviews current information about amyloid fibril structures, variations in molecular structures that underlie amyloid polymorphism, and physical considerations that explain the development and persistence of amyloid polymorphism. Much of this information has been obtained through solid state nuclear magnetic resonance measurements. The biological significance of amyloid polymorphism is also discussed briefly. Although this article focuses primarily on studies of fibrils formed by amyloid-β peptides, the same principles apply to many amyloid-forming peptides and proteins. PMID:25179159

  18. Universal Behavior in the Mesoscale Properties of Amyloid Fibrils

    NASA Astrophysics Data System (ADS)

    Assenza, Salvatore; Adamcik, Jozef; Mezzenga, Raffaele; De Los Rios, Paolo

    2014-12-01

    Amyloid fibrils are ubiquitous proteinaceous aggregates occurring in vivo and in vitro, with an invariant structural fingerprint at the molecular length scale. However, interpretation of their mesoscopic architectures is complicated by diverse observable polymorphic states. We here present a constitutive model for amyloid fibrils based on the minimization of the total energy per fibril. The model is benchmarked on real amyloid fibrils studied by atomic force microscopy. We use multistranded β -lactoglobulin amyloid fibrils as a model system exhibiting a rich polymorphism. The constitutive model quantitatively recapitulates the main mesoscopic topological features of amyloid fibrils, that is, the evolution of fibril periodicity as a function of the ionic strength of the solution and of the fibril width. A universal mesoscopic structural signature of the fibrils emerges from this picture, predicting a general, parameter-free law for the periodicity of the fibrils, that depends solely on the number of protofilaments per fibril. These predictions are validated experimentally and conclusively highlight the role of competing electrostatic and elastic contributions as the main players in the establishment of amyloid fibrils structure.

  19. Amyloid fibril formation by macrophage migration inhibitory factor

    SciTech Connect

    Lashuel, Hilal A. . E-mail: hilal.lashuel@epfl.ch; Aljabari, Bayan; Sigurdsson, Einar M.; Metz, Christine N.; Leng Lin; Callaway, David J.E.; Bucala, Richard

    2005-12-16

    We demonstrate herein that human macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine expressed in the brain and not previously considered to be amyloidogenic, forms amyloid fibrils similar to those derived from the disease associated amyloidogenic proteins {beta}-amyloid and {alpha}-synuclein. Acid denaturing conditions were found to readily induce MIF to undergo amyloid fibril formation. MIF aggregates to form amyloid-like structures with a morphology that is highly dependent on pH. The mechanism of MIF amyloid formation was probed by electron microscopy, turbidity, Thioflavin T binding, circular dichroism spectroscopy, and analytical ultracentrifugation. The fibrillar structures formed by MIF bind Congo red and exhibit the characteristic green birefringence under polarized light. These results are consistent with the notion that amyloid fibril formation is not an exclusive property of a select group of amyloidogenic proteins, and contribute to a better understanding of the factors which govern protein conformational changes and amyloid fibril formation in vivo.

  20. Amyloid fibrils as functionalizable components of nanocomposite materials.

    PubMed

    Rao, Shiva P; Meade, Susie J; Healy, Jackie P; Sutton, Kevin H; Larsen, Nigel G; Staiger, Mark P; Gerrard, Juliet A

    2012-01-01

    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. PMID:22002950

  1. Correlation between nanomechanics and polymorphic conformations in amyloid fibrils.

    PubMed

    Usov, Ivan; Mezzenga, Raffaele

    2014-11-25

    Amyloid fibrils occur in diverse morphologies, but how polymorphism affects the resulting mechanical properties is still not fully appreciated. Using formalisms from the theory of elasticity, we propose an original way of averaging the second area moment of inertia for non-axisymmetric fibrils, which constitutes the great majority of amyloid fibrils. By following this approach, we derive theoretical expressions for the bending properties of the most common polymorphic forms of amyloid fibrils (twisted ribbons, helical ribbons, and nanotubes), and we benchmark the predictions to experimental cases. These results not only allow an accurate estimation of the amyloid fibrils' elastic moduli but also bring insight into the structure-property relationships in the nanomechanics of amyloid systems, such as in the closure of helical ribbons into nanotubes. PMID:25275956

  2. Destroying activity of magnetoferritin on lysozyme amyloid fibrils

    NASA Astrophysics Data System (ADS)

    Kopcansky, Peter; Siposova, Katarina; Melnikova, Lucia; Bednarikova, Zuzana; Timko, Milan; Mitroova, Zuzana; Antosova, Andrea; Garamus, Vasil M.; Petrenko, Viktor I.; Avdeev, Mikhail V.; Gazova, Zuzana

    2015-03-01

    Presence of protein amyloid aggregates (oligomers, protofilaments, fibrils) is associated with many diseases as diabetes mellitus or Alzheimer's disease. The interaction between lysozyme amyloid fibrils and magnetoferritin loaded with different amount of iron atoms (168 or 532 atoms) has been investigated by small-angle X-rays scattering and thioflavin T fluorescence measurements. Results suggest that magnetoferritin caused an iron atom-concentration dependent reduction of lysozyme fibril size.

  3. Toxic species in amyloid disorders: Oligomers or mature fibrils

    PubMed Central

    Verma, Meenakshi; Vats, Abhishek; Taneja, Vibha

    2015-01-01

    Protein aggregation is the hallmark of several neurodegenerative disorders. These protein aggregation (fibrillization) disorders are also known as amyloid disorders. The mechanism of protein aggregation involves conformation switch of the native protein, oligomer formation leading to protofibrils and finally mature fibrils. Mature fibrils have long been considered as the cause of disease pathogenesis; however, recent evidences suggest oligomeric intermediates formed during fibrillization to be toxic. In this review, we have tried to address the ongoing debate for these toxic amyloid species. We did an extensive literature search and collated information from Pubmed (http://www.ncbi.nlm.nih.gov) and Google search using various permutations and combinations of the following keywords: Neurodegeneration, amyloid disorders, protein aggregation, fibrils, oligomers, toxicity, Alzheimer's Disease, Parkinson's Disease. We describe different instances showing the toxicity of mature fibrils as well as oligomers in Alzheimer's Disease and Parkinson's Disease. Distinct structural framework and morphology of amyloid oligomers suggests difference in toxic effect between oligomers and fibrils. We highlight the difference in structure and proposed toxicity pathways for fibrils and oligomers. We also highlight the evidences indicating that intermediary oligomeric species can act as potential diagnostic biomarker. Since the formation of these toxic species follow a common structural switch among various amyloid disorders, the protein aggregation events can be targeted for developing broad-range therapeutics. The therapeutic trials based on the understanding of different protein conformers (monomers, oligomers, protofibrils and fibrils) in amyloid cascade are also described. PMID:26019408

  4. Computational Assembly of Polymorphic Amyloid Fibrils Reveals Stable Aggregates

    PubMed Central

    Smaoui, Mohamed Raef; Poitevin, Frédéric; Delarue, Marc; Koehl, Patrice; Orland, Henri; Waldispühl, Jérôme

    2013-01-01

    Amyloid proteins aggregate into polymorphic fibrils that damage tissues of the brain, nerves, and heart. Experimental and computational studies have examined the structural basis and the nucleation of short fibrils, but the ability to predict and precisely quantify the stability of larger aggregates has remained elusive. We established a complete classification of fibril shapes and developed a tool called CreateFibril to build such complex, polymorphic, modular structures automatically. We applied stability landscapes, a technique we developed to reveal reliable fibril structural parameters, to assess fibril stability. CreateFibril constructed HET-s, Aβ, and amylin fibrils up to 17 nm in length, and utilized a novel dipolar solvent model that captured the effect of dipole-dipole interactions between water and very large molecular systems to assess their aqueous stability. Our results validate experimental data for HET-s and Aβ, and suggest novel (to our knowledge) findings for amylin. In particular, we predicted the correct structural parameters (rotation angles, packing distances, hydrogen bond lengths, and helical pitches) for the one and three predominant HET-s protofilaments. We reveal and structurally characterize all known Aβ polymorphic fibrils, including structures recently classified as wrapped fibrils. Finally, we elucidate the predominant amylin fibrils and assert that native amylin is more stable than its amyloid form. CreateFibril and a database of all stable polymorphic fibril models we tested, along with their structural energy landscapes, are available at http://amyloid.cs.mcgill.ca. PMID:23442919

  5. Cell Adhesion on Amyloid Fibrils Lacking Integrin Recognition Motif.

    PubMed

    Jacob, Reeba S; George, Edna; Singh, Pradeep K; Salot, Shimul; Anoop, Arunagiri; Jha, Narendra Nath; Sen, Shamik; Maji, Samir K

    2016-03-01

    Amyloids are highly ordered, cross-β-sheet-rich protein/peptide aggregates associated with both human diseases and native functions. Given the well established ability of amyloids in interacting with cell membranes, we hypothesize that amyloids can serve as universal cell-adhesive substrates. Here, we show that, similar to the extracellular matrix protein collagen, amyloids of various proteins/peptides support attachment and spreading of cells via robust stimulation of integrin expression and formation of integrin-based focal adhesions. Additionally, amyloid fibrils are also capable of immobilizing non-adherent red blood cells through charge-based interactions. Together, our results indicate that both active and passive mechanisms contribute to adhesion on amyloid fibrils. The present data may delineate the functional aspect of cell adhesion on amyloids by various organisms and its involvement in human diseases. Our results also raise the exciting possibility that cell adhesivity might be a generic property of amyloids. PMID:26742841

  6. Self-assembled amyloid fibrils with controllable conformational heterogeneity

    PubMed Central

    Lee, Gyudo; Lee, Wonseok; Lee, Hyungbeen; Lee, Chang Young; Eom, Kilho; Kwon, Taeyun

    2015-01-01

    Amyloid fibrils are a hallmark of neurodegenerative diseases and exhibit a conformational diversity that governs their pathological functions. Despite recent findings concerning the pathological role of their conformational diversity, the way in which the heterogeneous conformations of amyloid fibrils can be formed has remained elusive. Here, we show that microwave-assisted chemistry affects the self-assembly process of amyloid fibril formation, which results in their conformational heterogeneity. In particular, microwave-assisted chemistry allows for delicate control of the thermodynamics of the self-assembly process, which enabled us to tune the molecular structure of β-lactoglobulin amyloid fibrils. The heterogeneous conformations of amyloid fibrils, which can be tuned with microwave-assisted chemistry, are attributed to the microwave-driven thermal energy affecting the electrostatic interaction during the self-assembly process. Our study demonstrates how microwave-assisted chemistry can be used to gain insight into the origin of conformational heterogeneity of amyloid fibrils as well as the design principles showing how the molecular structures of amyloid fibrils can be controlled. PMID:26592772

  7. Self-assembled amyloid fibrils with controllable conformational heterogeneity.

    PubMed

    Lee, Gyudo; Lee, Wonseok; Lee, Hyungbeen; Lee, Chang Young; Eom, Kilho; Kwon, Taeyun

    2015-01-01

    Amyloid fibrils are a hallmark of neurodegenerative diseases and exhibit a conformational diversity that governs their pathological functions. Despite recent findings concerning the pathological role of their conformational diversity, the way in which the heterogeneous conformations of amyloid fibrils can be formed has remained elusive. Here, we show that microwave-assisted chemistry affects the self-assembly process of amyloid fibril formation, which results in their conformational heterogeneity. In particular, microwave-assisted chemistry allows for delicate control of the thermodynamics of the self-assembly process, which enabled us to tune the molecular structure of β-lactoglobulin amyloid fibrils. The heterogeneous conformations of amyloid fibrils, which can be tuned with microwave-assisted chemistry, are attributed to the microwave-driven thermal energy affecting the electrostatic interaction during the self-assembly process. Our study demonstrates how microwave-assisted chemistry can be used to gain insight into the origin of conformational heterogeneity of amyloid fibrils as well as the design principles showing how the molecular structures of amyloid fibrils can be controlled. PMID:26592772

  8. Self-assembled amyloid fibrils with controllable conformational heterogeneity

    NASA Astrophysics Data System (ADS)

    Lee, Gyudo; Lee, Wonseok; Lee, Hyungbeen; Lee, Chang Young; Eom, Kilho; Kwon, Taeyun

    2015-11-01

    Amyloid fibrils are a hallmark of neurodegenerative diseases and exhibit a conformational diversity that governs their pathological functions. Despite recent findings concerning the pathological role of their conformational diversity, the way in which the heterogeneous conformations of amyloid fibrils can be formed has remained elusive. Here, we show that microwave-assisted chemistry affects the self-assembly process of amyloid fibril formation, which results in their conformational heterogeneity. In particular, microwave-assisted chemistry allows for delicate control of the thermodynamics of the self-assembly process, which enabled us to tune the molecular structure of β-lactoglobulin amyloid fibrils. The heterogeneous conformations of amyloid fibrils, which can be tuned with microwave-assisted chemistry, are attributed to the microwave-driven thermal energy affecting the electrostatic interaction during the self-assembly process. Our study demonstrates how microwave-assisted chemistry can be used to gain insight into the origin of conformational heterogeneity of amyloid fibrils as well as the design principles showing how the molecular structures of amyloid fibrils can be controlled.

  9. Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

    SciTech Connect

    McWilliams-Koeppen, Helen P.; Foster, James S.; Hackenbrack, Nicole; Ramirez-Alvarado, Marina; Donohoe, Dallas; Williams, Angela; Macy, Sallie; Wooliver, Craig; Wortham, Dale; Morrell-Falvey, Jennifer; Foster, Carmen M.; Kennel, Stephen J.; Wall, Jonathan S.

    2015-09-22

    Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. Ultimately, these data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.

  10. Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

    DOE PAGESBeta

    McWilliams-Koeppen, Helen P.; Foster, James S.; Hackenbrack, Nicole; Ramirez-Alvarado, Marina; Donohoe, Dallas; Williams, Angela; Macy, Sallie; Wooliver, Craig; Wortham, Dale; Morrell-Falvey, Jennifer; et al

    2015-09-22

    Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presencemore » of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. Ultimately, these data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.« less

  11. Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

    PubMed Central

    McWilliams-Koeppen, Helen P.; Foster, James S.; Hackenbrack, Nicole; Ramirez-Alvarado, Marina; Donohoe, Dallas; Williams, Angela; Macy, Sallie; Wooliver, Craig; Wortham, Dale; Morrell-Falvey, Jennifer; Foster, Carmen M.; Kennel, Stephen J.; Wall, Jonathan S.

    2015-01-01

    Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. These data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils. PMID:26393799

  12. PMEL Amyloid Fibril Formation: The Bright Steps of Pigmentation.

    PubMed

    Bissig, Christin; Rochin, Leila; van Niel, Guillaume

    2016-01-01

    In pigment cells, melanin synthesis takes place in specialized organelles, called melanosomes. The biogenesis and maturation of melanosomes is initiated by an unpigmented step that takes place prior to the initiation of melanin synthesis and leads to the formation of luminal fibrils deriving from the pigment cell-specific pre-melanosomal protein (PMEL). In the lumen of melanosomes, PMEL fibrils optimize sequestration and condensation of the pigment melanin. Interestingly, PMEL fibrils have been described to adopt a typical amyloid-like structure. In contrast to pathological amyloids often associated with neurodegenerative diseases, PMEL fibrils represent an emergent category of physiological amyloids due to their beneficial cellular functions. The formation of PMEL fibrils within melanosomes is tightly regulated by diverse mechanisms, such as PMEL traffic, cleavage and sorting. These mechanisms revealed increasing analogies between the formation of physiological PMEL fibrils and pathological amyloid fibrils. In this review we summarize the known mechanisms of PMEL fibrillation and discuss how the recent understanding of physiological PMEL amyloid formation may help to shed light on processes involved in pathological amyloid formation. PMID:27589732

  13. Elongation dynamics of amyloid fibrils: A rugged energy landscape picture

    NASA Astrophysics Data System (ADS)

    Lee, Chiu Fan; Loken, James; Jean, Létitia; Vaux, David J.

    2009-10-01

    Protein amyloid fibrils are a form of linear protein aggregates that are implicated in many neurodegenerative diseases. Here, we study the dynamics of amyloid fibril elongation by performing Langevin dynamic simulations on a coarse-grained model of peptides. Our simulation results suggest that the elongation process is dominated by a series of local minimum due to frustration in monomer-fibril interactions. This rugged energy landscape picture indicates that the amount of recycling of monomers at the fibrils’ ends before being fibrilized is substantially reduced in comparison to the conventional two-step elongation model. This picture, along with other predictions discussed, can be tested with current experimental techniques.

  14. Solid-State NMR Studies of Amyloid Fibril Structure

    NASA Astrophysics Data System (ADS)

    Tycko, Robert

    2011-05-01

    Current interest in amyloid fibrils stems from their involvement in neurodegenerative and other diseases and from their role as an alternative structural state for many peptides and proteins. Solid-state nuclear magnetic resonance (NMR) methods have the unique capability of providing detailed structural constraints for amyloid fibrils, sufficient for the development of full molecular models. In this article, recent progress in the application of solid-state NMR to fibrils associated with Alzheimer's disease, prion fibrils, and related systems is reviewed, along with relevant developments in solid-state NMR techniques and technology.

  15. Solid State NMR Studies of Amyloid Fibril Structure

    PubMed Central

    Tycko, Robert

    2011-01-01

    Current interest in amyloid fibrils stems from their involvement in neurodegenerative and other diseases and from their role as an alternative structural state for many peptides and proteins. Solid state NMR methods have the unique capability of providing detailed structural constraints for amyloid fibrils, sufficient for the development of full molecular models. In this article, recent progress in the application of solid state NMR to fibrils associated with Alzheimer’s disease, prion fibrils, and related systems is reviewed, along with relevant developments in solid state NMR techniques and technology. PMID:21219138

  16. Raft lipids as common components of human extracellular amyloid fibrils

    PubMed Central

    Gellermann, Gerald P.; Appel, Thomas R.; Tannert, Astrid; Radestock, Anja; Hortschansky, Peter; Schroeckh, Volker; Leisner, Christian; Lütkepohl, Tim; Shtrasburg, Shmuel; Röcken, Christoph; Pras, Mordechai; Linke, Reinhold P.; Diekmann, Stephan; Fändrich, Marcus

    2005-01-01

    Amyloid fibrils are fibrillar polypeptide aggregates from several degenerative human conditions, including Alzheimer's and Creutzfeldt-Jakob diseases. Analysis of amyloid fibrils derived from various human diseases (AA, ATTR, Aβ2M, ALλ, and ALκ amyloidosis) shows that these are associated with a common lipid component that has a conserved chemical composition and that is specifically rich in cholesterol and sphingolipids, the major components of cellular lipid rafts. This pattern is not notably affected by the purification procedure, and no tight lipid interactions can be detected when preformed fibrils are mixed with lipids. By contrast, the early and prefibrillar aggregates formed in an AA amyloid-producing cell system interact with the raft marker ganglioside-1, and amyloid formation is impaired by addition of cholesterol-reducing agents. These data suggest the existence of common cellular mechanisms in the generation of different types of clinical amyloid deposits. PMID:15851687

  17. On the lag phase in amyloid fibril formation

    PubMed Central

    Arosio, Paolo; Knowles, Tuomas P. J.

    2015-01-01

    The formation of nanoscale amyloid fibrils from normally soluble peptides and proteins is a common form of self-assembly phenomenon that has fundamental connections with biological functions and human diseases. The kinetics of this process has been widely studied and exhibits on a macroscopic level three characteristic stages: a lag phase, a growth phase and a final plateau regime. The question of which molecular events take place during each one of these phases has been a central element in the quest for a mechanism of amyloid formation. In this review, we discuss the nature and molecular origin of the lag-phase in amyloid formation by making use of tools and concepts from physical chemistry, in particular from chemical reaction kinetics. We discuss how, in macroscopic samples, it has become apparent that the lag-phase is not a waiting time for nuclei to form. Rather, multiple parallel processes exist and typically millions of primary nuclei form during the lag phase from monomers in solution. Thus, the lag-time represents a time that is required for the nuclei that are formed early on in the reaction to grow and proliferate in order to reach an aggregate concentration that is readily detected in bulk assays. In many cases, this proliferation takes place through secondary nucleation, where fibrils may present a catalytic surface for the formation of new aggregates. Fibrils may also break (fragmentation) and thereby provide new ends for elongation. Thus, at least two – primary nucleation and elongation – and in many systems at least four – primary nucleation, elongation, secondary nucleation and fragmentation – microscopic processes occur during the lag phase. Moreover, these same processes occur during all three phases of the macroscopic aggregation process, albeit at different rates as governed by rate constants and by the concentration of reacting species at each point in time. PMID:25719972

  18. Molecular Mechanism of Thioflavin-T Binding to Amyloid Fibrils

    PubMed Central

    Biancalana, Matthew; Koide, Shohei

    2010-01-01

    Intense efforts to detect, diagnose, and analyze the kinetic and structural properties of amyloid fibrils have generated a powerful toolkit of amyloid-specific molecular probes. Since its first description in 1959, the fluorescent dye Thioflavin-T (ThT) has become among the most widely used “gold standards” for selectively staining and identifying amyloid fibrils both in vivo and in vitro. The large enhancement of its fluorescence emission upon binding to fibrils makes ThT a particularly powerful and convenient tool. Despite its widespread use in clinical and basic science applications, the molecular mechanism for the ability of ThT to recognize diverse types of amyloid fibrils and for the dye’s characteristic fluorescence has only begun to be elucidated. Here, we review recent progress in the understanding of ThT-fibril interactions at an atomic resolution. These studies have yielded important insights into amyloid structures and the processes of fibril formation, and they also offer guidance for designing the next generation of amyloid assembly diagnostics, inhibitors, and therapeutics. PMID:20399286

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

    PubMed Central

    Kato, Yusuke; Yagi, Hisashi; Kaji, Yuichi; Oshika, Tetsuro; Goto, Yuji

    2013-01-01

    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. PMID:23861389

  20. Molecular basis for amyloid fibril formation and stability

    PubMed Central

    Makin, O. Sumner; Atkins, Edward; Sikorski, Pawel; Johansson, Jan; Serpell, Louise C.

    2005-01-01

    The molecular structure of the amyloid fibril has remained elusive because of the difficulty of growing well diffracting crystals. By using a sequence-designed polypeptide, we have produced crystals of an amyloid fiber. These crystals diffract to high resolution (1 Å) by electron and x-ray diffraction, enabling us to determine a detailed structure for amyloid. The structure reveals that the polypeptides form fibrous crystals composed of antiparallel β-sheets in a cross-β arrangement, characteristic of all amyloid fibers, and allows us to determine the side-chain packing within an amyloid fiber. The antiparallel β-sheets are zipped together by means of π-bonding between adjacent phenylalanine rings and salt-bridges between charge pairs (glutamic acid–lysine), thus controlling and stabilizing the structure. These interactions are likely to be important in the formation and stability of other amyloid fibrils. PMID:15630094

  1. Gallic acid, one of the components in many plant tissues, is a potential inhibitor for insulin amyloid fibril formation.

    PubMed

    Jayamani, Jayaraman; Shanmugam, Ganesh

    2014-10-01

    Proteins under stressful conditions can lead to the formation of an ordered self-assembled structure, referred to as amyloid fibrils, to which many neurodegenerative diseases such as Type II diabetes, Alzheimer's, Parkinson's, Huntington's, etc., are attributed. Inhibition of amyloid fibril formation using natural products is one of the main therapeutic strategies to prevent the progression of these diseases. Polyphenols are the mostly consumed as antioxidants in a human nutrition. Herein, we have studied the effect of a simple polyphenol, gallic acid (GA), one of the main components in plant tissues, especially in tea leaves, on the insulin amyloid fibril formation. Different biophysical characterizations such as turbidity, atomic force microscopy (AFM), Thioflavin T (ThT) assays, circular dichroism, and Fourier transform-infrared spectroscopy have been used to analyze the inhibition of amyloid fibril formation. The occurrence of fibrils in an AFM image and ThT fluorescence enhancement confirms the formation of insulin amyloid fibrils when incubated under acidic pH 2 at 65 °C. In the presence of GA, absence of fibrils in AFM image and no change in the intensity of ThT fluorescence confirms the inhibition of insulin amyloid fibrils by GA. Spectroscopic results reveal that GA inhibits the conformational transition of α-helix → β-sheet, which is generally induced during the insulin fibril formation. It was found that the inhibitory effect of GA is concentration dependent and non-linear. Based on the observed results, we propose that GA interacts with native insulin, preventing nuclei formation, which is essential for fibril growth, thereby inhibiting the amyloid fibril formation. The present results thus demonstrate that GA can effectively inhibit insulin amyloid fibril formation in vitro. PMID:25105923

  2. Superresolution Imaging of Amyloid Fibrils with Binding-Activated Probes

    PubMed Central

    2013-01-01

    Protein misfolding into amyloid-like aggregates underlies many neurodegenerative diseases. Thus, insights into the structure and function of these amyloids will provide valuable information on the pathological mechanisms involved and aid in the design of improved drugs for treating amyloid-based disorders. However, determining the structure of endogenous amyloids at high resolution has been difficult. Here we employ binding-activated localization microscopy (BALM) to acquire superresolution images of α-synuclein amyloid fibrils with unprecedented optical resolution. We propose that BALM imaging can be extended to study the structure of other amyloids, for differential diagnosis of amyloid-related diseases and for discovery of drugs that perturb amyloid structure for therapy. PMID:23594172

  3. Isotropic-nematic phase transition in amyloid fibrilization

    NASA Astrophysics Data System (ADS)

    Lee, Chiu Fan

    2009-09-01

    We carry out a theoretical study on the isotropic-nematic phase transition and phase separation in amyloid fibril solutions. Borrowing the thermodynamic model employed in the study of cylindrical micelles, we investigate the variations in the fibril length distribution and phase behavior with respect to changes in the protein concentration, fibril’s rigidity, and binding energy. We then relate our theoretical findings to the nematic ordering experimentally observed in Hen Lysozyme fibril solution.

  4. Dynamic behavior of small heat shock protein inhibition on amyloid fibrillization of a small peptide (SSTSAA) from RNase A

    SciTech Connect

    Xi, Dong; Dong, Xiao; Deng, Wei; Lai, Luhua

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Mechanism of small heat shock protein inhibition on fibril formation was studied. Black-Right-Pointing-Pointer Peptide SSTSAA with modified ends was used for amyloid fibril formation. Black-Right-Pointing-Pointer FRET signal was followed during the fibril formation. Black-Right-Pointing-Pointer Mj HSP16.5 inhibits fibril formation when introduced in the lag phase. Black-Right-Pointing-Pointer Mj HSP16.5 slows down fibril formation when introduced after the lag phase. -- Abstract: Small heat shock proteins, a class of molecular chaperones, are reported to inhibit amyloid fibril formation in vitro, while the mechanism of inhibition remains unknown. In the present study, we investigated the mechanism by which Mj HSP16.5 inhibits amyloid fibril formation of a small peptide (SSTSAA) from RNase A. A model peptide (dansyl-SSTSAA-W) was designed by introducing a pair of fluorescence resonance energy transfer (FRET) probes into the peptide, allowing for the monitoring of fibril formation by this experimental model. Mj HSP16.5 completely inhibited fibril formation of the model peptide at a molar ratio of 1:120. The dynamic process of fibril formation, revealed by FRET, circular dichroism, and electron microscopy, showed a lag phase of about 2 h followed by a fast growth period. The effect of Mj HSP16.5 on amyloid fibril formation was investigated by adding it into the incubation solution during different growth phases. Adding Mj HSP16.5 to the incubating peptide before or during the lag phase completely inhibited fibril formation. However, introducing Mj HSP16.5 after the lag phase only slowed down the fibril formation process by adhering to the already formed fibrils. These findings provide insight into the inhibitory roles of small heat shock proteins on amyloid fibril formation at the molecular level.

  5. Amyloid fibrils composed of hexameric peptides attenuate neuroinflammation.

    PubMed

    Kurnellas, Michael P; Adams, Chris M; Sobel, Raymond A; Steinman, Lawrence; Rothbard, Jonathan B

    2013-04-01

    The amyloid-forming proteins tau, αB crystallin, and amyloid P protein are all found in lesions of multiple sclerosis (MS). Our previous work established that amyloidogenic peptides from the small heat shock protein αB crystallin (HspB5) and from amyloid β fibrils, characteristic of Alzheimer's disease, were therapeutic in experimental autoimmune encephalomyelitis (EAE), reflecting aspects of the pathology of MS. To understand the molecular basis for the therapeutic effect, we showed a set of amyloidogenic peptides composed of six amino acids, including those from tau, amyloid β A4, major prion protein (PrP), HspB5, amylin, serum amyloid P, and insulin B chain, to be anti-inflammatory and capable of reducing serological levels of interleukin-6 and attenuating paralysis in EAE. The chaperone function of the fibrils correlates with the therapeutic outcome. Fibrils composed of tau 623-628 precipitated 49 plasma proteins, including apolipoprotein B-100, clusterin, transthyretin, and complement C3, supporting the hypothesis that the fibrils are active biological agents. Amyloid fibrils thus may provide benefit in MS and other neuroinflammatory disorders. PMID:23552370

  6. Amyloid Fibrils Composed of Hexameric Peptides Attenuate Neuroinflammation

    PubMed Central

    Kurnellas, Michael P.; Adams, Chris M.; Sobel, Raymond A.; Steinman, Lawrence; Rothbard, Jonathan B.

    2013-01-01

    Amyloid forming proteins Tau, alpha B crystallin, and amyloid P protein are all found in lesions of multiple sclerosis (MS). Our previous work established that amyloidogenic peptides from the small heat shock protein, alpha B crystallin(HspB5), and from amyloid β fibrils, characteristic of Alzheimer’s disease, were therapeutic in experimental autoimmune encephalomyelitis (EAE), reflecting aspects of the pathology of MS. To understand the molecular basis for the therapeutic effect, a set of amyloidogenic peptides composed of six amino acids, including those from tau, amyloid β A4, major prion protein (PrP), HspB5, amylin, serum amyloid P (SAP), and insulin B chain were shown to be anti-inflammatory, capable of reducing serological levels of IL-6, and attenuating paralysis in EAE. The chaperone function of the fibrils correlates with the therapeutic outcome. Fibrils composed of Tau 623–628 precipitated 49 plasma proteins, including apolipoprotein B-100, clusterin, transthyretin, and complement C3, supporting the hypothesis that the fibrils are active biological agents. Amyloid fibrils thus may provide benefit in MS and other neuroinflammatory disorders. PMID:23552370

  7. Antimicrobial Protegrin-1 Forms Amyloid-Like Fibrils with Rapid Kinetics Suggesting a Functional Link

    PubMed Central

    Jang, Hyunbum; Arce, Fernando Teran; Mustata, Mirela; Ramachandran, Srinivasan; Capone, Ricardo; Nussinov, Ruth; Lal, Ratnesh

    2011-01-01

    Protegrin-1 (PG-1) is an 18 residues long, cysteine-rich β-sheet antimicrobial peptide (AMP). PG-1 induces strong cytotoxic activities on cell membrane and acts as a potent antibiotic agent. Earlier we reported that its cytotoxicity is mediated by its channel-forming ability. In this study, we have examined the amyloidogenic fibril formation properties of PG-1 in comparison with a well-defined amyloid, the amyloid-β (Aβ1–42) peptide. We have used atomic force microscopy (AFM) and thioflavin-T staining to investigate the kinetics of PG-1 fibrils growth and molecular dynamics simulations to elucidate the underlying mechanism. AFM images of PG-1 on a highly hydrophilic surface (mica) show fibrils with morphological similarities to Aβ1–42 fibrils. Real-time AFM imaging of fibril growth suggests that PG-1 fibril growth follows a relatively fast kinetics compared to the Aβ1–42 fibrils. The AFM results are in close agreement with results from thioflavin-T staining data. Furthermore, the results indicate that PG-1 forms fibrils in solution. Significantly, in contrast, we do not detect fibrillar structures of PG-1 on an anionic lipid bilayer 2-dioleoyl-sn-glycero-3-phospho-L-serine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; only small PG-1 oligomers can be observed. Molecular dynamics simulations are able to identify the presence of these small oligomers on the membrane bilayer. Thus, our current results show that cytotoxic AMP PG-1 is amyloidogenic and capable of forming fibrils. Overall, comparing β-rich AMPs and amyloids such as Aβ, in addition to cytotoxicity and amyloidogenicity, they share a common structural motif, and are channel forming. These combined properties support a functional relationship between amyloidogenic peptides and β-sheet-rich cytolytic AMPs, suggesting that amyloids channels may have an antimicrobial function. PMID:21463591

  8. Peptide concentration alters intermediate species in amyloid β fibrillation kinetics

    SciTech Connect

    Garvey, M.; Morgado, I.

    2013-04-12

    Highlights: ► Aβ(1–40) aggregation in vitro has been monitored at different concentrations. ► Aβ(1–40) fibrillation does not always follow conventional kinetic mechanisms. ► We demonstrate non-linear features in the kinetics of Aβ(1–40) fibril formation. ► At high Aβ(1–40) concentrations secondary processes dictate fibrillation speed. ► Intermediate species may play significant roles on final amyloid fibril development. -- Abstract: The kinetic mechanism of amyloid aggregation remains to be fully understood. Investigations into the species present in the different kinetic phases can assist our comprehension of amyloid diseases and further our understanding of the mechanism behind amyloid β (Aβ) (1–40) peptide aggregation. Thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM) have been used in combination to monitor Aβ(1–40) aggregation in vitro at both normal and higher than standard concentrations. The observed fibrillation behaviour deviates, in several respects, from standard concepts of the nucleation–polymerisation models and shows such features as concentration-dependent non-linear effects in the assembly mechanism. Aβ(1–40) fibrillation kinetics do not always follow conventional kinetic mechanisms and, specifically at high concentrations, intermediate structures become populated and secondary processes may further modify the fibrillation mechanism.

  9. Amyloid fibril disruption by ultrasonic cavitation: nonequilibrium molecular dynamics simulations.

    PubMed

    Okumura, Hisashi; Itoh, Satoru G

    2014-07-30

    We describe the disruption of amyloid fibrils of Alzheimer's amyloid-β peptides by ultrasonic cavitation. For this purpose, we performed nonequilibrium all-atom molecular dynamics simulations with sinusoidal pressure and visualized the process with movies. When the pressure is negative, a bubble is formed, usually at hydrophobic residues in the transmembrane region. Most β-strands maintain their secondary structures in the bubble. When the pressure becomes positive, the bubble collapses, and water molecules crash against the hydrophilic residues in the nontransmembrane region to disrupt the amyloid. Shorter amyloids require longer sonication times for disruption because they do not have enough hydrophobic residues to serve as a nucleus to form a bubble. These results agree with experiments in which monodispersed amyloid fibrils were obtained by ultrasonication. PMID:24987794

  10. Ultrafast propagation of β-amyloid fibrils in oligomeric cloud

    PubMed Central

    Ogi, Hirotsugu; Fukukshima, Masahiko; Hamada, Hiroki; Noi, Kentaro; Hirao, Masahiko; Yagi, Hisashi; Goto, Yuji

    2014-01-01

    Interaction between monomer peptides and seeds is essential for clarifying the fibrillation mechanism of amyloid β (Aβ) peptides. We monitored the deposition reaction of Aβ1–40 peptides on immobilized seeds grown from Aβ1–42, which caused formation of oligomers in the early stage. The deposition reaction and fibrillation procedure were monitored throughout by novel total-internal-reflection-fluorescence microscopy with a quartz-crystal microbalance (TIRFM-QCM) system. This system allows simultaneous evaluation of the amount of deposited peptides on the surface seeds by QCM and fibril nucleation and elongation by TIRFM. Most fibrils reached other nuclei, forming the fibril network across the nucleus hubs in a short time. We found a fibril-elongation rate two-orders-of-magnitude higher in an oligomeric cloud than reported values, indicating ultrafast transition of oligomers into fibrils. PMID:25376301

  11. Plasmodium falciparum Merozoite Surface Protein 2 is Unstructured and Forms Amyloid-Like Fibrils

    PubMed Central

    Adda, Christopher G.; Murphy, Vince J.; Sunde, Margaret; Waddington, Lynne J.; Jesse, Schloegel; Talbo, Gert H.; Vingas, Kleo; Kienzle, Vivian; Masciantonio, Rosella; Howlett, Geoffrey J.; Hodder, Anthony N.; Foley, Michael; Anders, Robin F.

    2009-01-01

    Several merozoite surface proteins are being assessed as potential components of a vaccine against Plasmodium falciparum, the cause of the most serious form of human malaria. One of these proteins, merozoite surface protein 2 (MSP2), is unusually hydrophilic and contains tandem sequence repeats, characteristics of intrinsically unstructured proteins. A range of physicochemical studies have confirmed that recombinant forms of MSP2 are largely unstructured. Both dimorphic types of MSP2 (3D7 and FC27) are equivalently extended in solution and form amyloid-like fibrils although with different kinetics and structural characteristics. These fibrils have a regular underlying β-sheet structure and both fibril types stain with Congo Red, but only the FC27 fibrils stain with Thioflavin T. 3D7 MSP2 fibrils seeded the growth of fibrils from 3D7 or FC27 MSP2 monomer indicating the involvement of a conserved region of MSP2 in fibril formation. Consistent with this, digestion of fibrils with proteinase K generated resistant peptides, which included the N-terminal conserved region of MSP2. A monoclonal antibody that reacted preferentially with monomeric recombinant MSP2 did not react with the antigen in situ on the merozoite surface. Glutaraldehyde cross-linking of infected erythrocytes generated MSP2 oligomers similar to those formed by polymeric recombinant MSP2. We conclude that MSP2 oligomers containing intermolecular β-strand interactions similar to those in amyloid fibrils may be a component of the fibrillar surface coat on P. falciparum merozoites. PMID:19450733

  12. Plasmodium falciparum merozoite surface protein 2 is unstructured and forms amyloid-like fibrils.

    PubMed

    Adda, Christopher G; Murphy, Vince J; Sunde, Margaret; Waddington, Lynne J; Schloegel, Jesse; Talbo, Gert H; Vingas, Kleo; Kienzle, Vivian; Masciantonio, Rosella; Howlett, Geoffrey J; Hodder, Anthony N; Foley, Michael; Anders, Robin F

    2009-08-01

    Several merozoite surface proteins are being assessed as potential components of a vaccine against Plasmodium falciparum, the cause of the most serious form of human malaria. One of these proteins, merozoite surface protein 2 (MSP2), is unusually hydrophilic and contains tandem sequence repeats, characteristics of intrinsically unstructured proteins. A range of physicochemical studies has confirmed that recombinant forms of MSP2 are largely unstructured. Both dimorphic types of MSP2 (3D7 and FC27) are equivalently extended in solution and form amyloid-like fibrils although with different kinetics and structural characteristics. These fibrils have a regular underlying beta-sheet structure and both fibril types stain with Congo Red, but only the FC27 fibrils stain with Thioflavin T. 3D7 MSP2 fibrils seeded the growth of fibrils from 3D7 or FC27 MSP2 monomer indicating the involvement of a conserved region of MSP2 in fibril formation. Consistent with this, digestion of fibrils with proteinase K generated resistant peptides, which included the N-terminal conserved region of MSP2. A monoclonal antibody that reacted preferentially with monomeric recombinant MSP2 did not react with the antigen in situ on the merozoite surface. Glutaraldehyde cross-linking of infected erythrocytes generated MSP2 oligomers similar to those formed by polymeric recombinant MSP2. We conclude that MSP2 oligomers containing intermolecular beta-strand interactions similar to those in amyloid fibrils may be a component of the fibrillar surface coat on P. falciparum merozoites. PMID:19450733

  13. On the adsorption of magnetite nanoparticles on lysozyme amyloid fibrils.

    PubMed

    Majorosova, Jozefina; Petrenko, Viktor I; Siposova, Katarina; Timko, Milan; Tomasovicova, Natalia; Garamus, Vasil M; Koralewski, Marceli; Avdeev, Mikhail V; Leszczynski, Błażej; Jurga, Stefan; Gazova, Zuzana; Hayryan, Shura; Hu, Chin-Kun; Kopcansky, Peter

    2016-10-01

    An adsorption of magnetic nanoparticles (MNP) from electrostatically stabilized aqueous ferrofluids on amyloid fibrils of hen egg white lysozyme (HEWL) in 2mg/mL acidic dispersions have been detected for the MNP concentration range of 0.01-0.1vol.%. The association of the MNP with amyloid fibrils has been characterized by transmission electron microscopy (TEM), small-angle X-ray scattering (SAXS) and magneto-optical measurements. It has been observed that the extent of adsorption is determined by the MNP concentration. When increasing the MNP concentration the formed aggregates of magnetic particles repeat the general rod-like structure of the fibrils. The effect is not observed when MNP are mixed with the solution of lysozyme monomers. The adsorption has been investigated with the aim to clarify previously found disaggregation activity of MNP in amyloid fibrils dispersions and to get deeper insight into interaction processes between amyloids and MNP. The observed effect is also discussed with respect to potential applications for ordering lysozyme amyloid fibrils in a liquid crystal phase under external magnetic fields. PMID:27451367

  14. A generic crystallization-like model that describes the kinetics of amyloid fibril formation.

    PubMed

    Crespo, Rosa; Rocha, Fernando A; Damas, Ana M; Martins, Pedro M

    2012-08-31

    Associated with neurodegenerative disorders such as Alzheimer, Parkinson, or prion diseases, the conversion of soluble proteins into amyloid fibrils remains poorly understood. Extensive "in vitro" measurements of protein aggregation kinetics have been reported, but no consensus mechanism has emerged until now. This contribution aims at overcoming this gap by proposing a theoretically consistent crystallization-like model (CLM) that is able to describe the classic types of amyloid fibrillization kinetics identified in our literature survey. Amyloid conversion represented as a function of time is shown to follow different curve shapes, ranging from sigmoidal to hyperbolic, according to the relative importance of the nucleation and growth steps. Using the CLM, apparently unrelated data are deconvoluted into generic mechanistic information integrating the combined influence of seeding, nucleation, growth, and fibril breakage events. It is notable that this complex assembly of interdependent events is ultimately reduced to a mathematically simple model, whose two parameters can be determined by little more than visual inspection. The good fitting results obtained for all cases confirm the CLM as a good approximation to the generalized underlying principle governing amyloid fibrillization. A perspective is presented on possible applications of the CLM during the development of new targets for amyloid disease therapeutics. PMID:22767606

  15. A Generic Crystallization-like Model That Describes the Kinetics of Amyloid Fibril Formation*♦

    PubMed Central

    Crespo, Rosa; Rocha, Fernando A.; Damas, Ana M.; Martins, Pedro M.

    2012-01-01

    Associated with neurodegenerative disorders such as Alzheimer, Parkinson, or prion diseases, the conversion of soluble proteins into amyloid fibrils remains poorly understood. Extensive “in vitro” measurements of protein aggregation kinetics have been reported, but no consensus mechanism has emerged until now. This contribution aims at overcoming this gap by proposing a theoretically consistent crystallization-like model (CLM) that is able to describe the classic types of amyloid fibrillization kinetics identified in our literature survey. Amyloid conversion represented as a function of time is shown to follow different curve shapes, ranging from sigmoidal to hyperbolic, according to the relative importance of the nucleation and growth steps. Using the CLM, apparently unrelated data are deconvoluted into generic mechanistic information integrating the combined influence of seeding, nucleation, growth, and fibril breakage events. It is notable that this complex assembly of interdependent events is ultimately reduced to a mathematically simple model, whose two parameters can be determined by little more than visual inspection. The good fitting results obtained for all cases confirm the CLM as a good approximation to the generalized underlying principle governing amyloid fibrillization. A perspective is presented on possible applications of the CLM during the development of new targets for amyloid disease therapeutics. PMID:22767606

  16. Structural requirements of glycosaminoglycans for facilitating amyloid fibril formation of human serum amyloid A.

    PubMed

    Takase, Hiroka; Tanaka, Masafumi; Yamamoto, Aki; Watanabe, Shiori; Takahashi, Sanae; Nadanaka, Satomi; Kitagawa, Hiroshi; Yamada, Toshiyuki; Mukai, Takahiro

    2016-06-01

    Serum amyloid A (SAA) is a precursor protein of amyloid fibrils. Given that heparan sulfate (HS), a glycosaminoglycan (GAG), is detected in amyloid deposits, it has been suggested that GAG is a key component of amyloid fibril formation. We previously reported that heparin (an analog of HS) facilitates the fibril formation of SAA, but the structural requirements remain unknown. In the present study, we investigated the structural requirements of GAGs for facilitating the amyloid fibril formation of SAA. Spectroscopic analyses using structurally diverse GAG analogs suggested that the fibril formation of SAA was facilitated irrespective of the backbone structure of GAGs; however, the facilitating effect was strongly correlated with the degree of sulfation. Microscopic analyses revealed that the morphologies of SAA aggregates were modulated by the GAGs. The HS molecule, which is less sulfated than heparin but contains highly sulfated domains, exhibited a relatively high potential to facilitate fibril formation compared to other GAGs. The length dependence of fragmented heparins on the facilitating effect suggested that a high density of sulfate groups is also required. These results indicate that not only the degree of sulfation but also the lengths of sulfated domains in GAG play important roles in fibril formation of SAA. PMID:27097047

  17. Amyloid Fibrillation of Insulin under Water-Limited Conditions

    PubMed Central

    Choi, Tae Su; Lee, Jong Wha; Jin, Kyeong Sik; Kim, Hugh I.

    2014-01-01

    Amyloid fibrillation in water-organic mixtures has been widely studied to understand the effect of protein-solvent interactions on the fibrillation process. In this study, we monitored insulin fibrillation in formamide and its methyl derivatives (formamide, N-methyl formamide, N,N-dimethyl formamide) in the presence and absence of water. These model solvent systems mimic the cellular environment by providing denaturing conditions and a hydrophobic environment with limited water content. Thioflavin T (ThT) assay revealed that binary mixtures of water with formamide and its methyl derivatives enhanced fibrillation rates and β-sheet abundance, whereas organic solvents suppressed insulin fibrillation. We utilized solution small-angle x-ray scattering (SAXS) and differential scanning calorimetry (DSC) to investigate the correlation between protein-solvent interactions and insulin fibrillation. SAXS experiments combined with simulated annealing of the protein indicated that the degree of denaturation of the hydrophobic core region at residues B11–B17 determines the fibrillation rate. In addition, DSC experiments suggested a crucial role of hydrophobic interactions in the fibrillation process. These results imply that an environment with limited water, which imitates a lipid membrane system, accelerates protein denaturation and the formation of intermolecular hydrophobic interactions during amyloid fibrillation. PMID:25418175

  18. Brazilin inhibits amyloid β-protein fibrillogenesis, remodels amyloid fibrils and reduces amyloid cytotoxicity.

    PubMed

    Du, Wen-Jie; Guo, Jing-Jing; Gao, Ming-Tao; Hu, Sheng-Quan; Dong, Xiao-Yan; Han, Yi-Fan; Liu, Fu-Feng; Jiang, Shaoyi; Sun, Yan

    2015-01-01

    Soluble amyloid β-protein (Aβ) oligomers, the main neurotoxic species, are predominantly formed from monomers through a fibril-catalyzed secondary nucleation. Herein, we virtually screened an in-house library of natural compounds and discovered brazilin as a dual functional compound in both Aβ42 fibrillogenesis inhibition and mature fibril remodeling, leading to significant reduction in Aβ42 cytotoxicity. The potent inhibitory effect of brazilin was proven by an IC50 of 1.5 ± 0.3 μM, which was smaller than that of (-)-epigallocatechin gallate in Phase III clinical trials and about one order of magnitude smaller than those of curcumin and resveratrol. Most importantly, it was found that brazilin redirected Aβ42 monomers and its mature fibrils into unstructured Aβ aggregates with some β-sheet structures, which could prevent both the primary nucleation and the fibril-catalyzed secondary nucleation. Molecular simulations demonstrated that brazilin inhibited Aβ42 fibrillogenesis by directly binding to Aβ42 species via hydrophobic interactions and hydrogen bonding and remodeled mature fibrils by disrupting the intermolecular salt bridge Asp23-Lys28 via hydrogen bonding. Both experimental and computational studies revealed a different working mechanism of brazilin from that of known inhibitors. These findings indicate that brazilin is of great potential as a neuroprotective and therapeutic agent for Alzheimer's disease. PMID:25613018

  19. Brazilin inhibits amyloid β-protein fibrillogenesis, remodels amyloid fibrils and reduces amyloid cytotoxicity

    NASA Astrophysics Data System (ADS)

    Du, Wen-Jie; Guo, Jing-Jing; Gao, Ming-Tao; Hu, Sheng-Quan; Dong, Xiao-Yan; Han, Yi-Fan; Liu, Fu-Feng; Jiang, Shaoyi; Sun, Yan

    2015-01-01

    Soluble amyloid β-protein (Aβ) oligomers, the main neurotoxic species, are predominantly formed from monomers through a fibril-catalyzed secondary nucleation. Herein, we virtually screened an in-house library of natural compounds and discovered brazilin as a dual functional compound in both Aβ42 fibrillogenesis inhibition and mature fibril remodeling, leading to significant reduction in Aβ42 cytotoxicity. The potent inhibitory effect of brazilin was proven by an IC50 of 1.5 +/- 0.3 μM, which was smaller than that of (-)-epigallocatechin gallate in Phase III clinical trials and about one order of magnitude smaller than those of curcumin and resveratrol. Most importantly, it was found that brazilin redirected Aβ42 monomers and its mature fibrils into unstructured Aβ aggregates with some β-sheet structures, which could prevent both the primary nucleation and the fibril-catalyzed secondary nucleation. Molecular simulations demonstrated that brazilin inhibited Aβ42 fibrillogenesis by directly binding to Aβ42 species via hydrophobic interactions and hydrogen bonding and remodeled mature fibrils by disrupting the intermolecular salt bridge Asp23-Lys28 via hydrogen bonding. Both experimental and computational studies revealed a different working mechanism of brazilin from that of known inhibitors. These findings indicate that brazilin is of great potential as a neuroprotective and therapeutic agent for Alzheimer's disease.

  20. Generalization of Filament Braiding Model for Amyloid Fibril Assembly

    NASA Astrophysics Data System (ADS)

    Pope, Maighdlin; Ionescu-Zanetti, Cristian; Khurana, Ritu; Carter, Sue

    2001-03-01

    Research into the formation of amyloid fibrils is motivated by their association with several prominent diseases, among these Alzheimer's Disease, Parkinson's Disease and amyloidosis. Previous work in monitering the aggregation of immunoglobulin light chains to form amyloid fibrils suggests a braided structure where filaments and protofibrils wind together to form Type I and Type II fibrils. Non-contact atomic force microscopy is used to image and explore the kinetics of several other amyloid fibril forming proteins in an effort to generalize the filament braiding model. Included in this study are insulin and the B1 domain of G. Both of these have been shown to form fibrils in vitro. Alpha-synuclein is also included in this study. It is involved in the formation of Lewy bodies in Parkinson's Disease. The fourth protein used in this comparitive study is human amylin that is the cause of a systemic amyloidosis. Results from these four proteins and their associated fibrils are compared to the Ig light chain fibril structure in an effort to show the universality of the filament braiding model.

  1. Nucleus factory on cavitation bubble for amyloid β fibril

    PubMed Central

    Nakajima, Kichitaro; Ogi, Hirotsugu; Adachi, Kanta; Noi, Kentaro; Hirao, Masahiko; Yagi, Hisashi; Goto, Yuji

    2016-01-01

    Structural evolution from monomer to fibril of amyloid β peptide is related to pathogenic mechanism of Alzheimer disease, and its acceleration is a long-running problem in drug development. This study reveals that ultrasonic cavitation bubbles behave as catalysts for nucleation of the peptide: The nucleation reaction is highly dependent on frequency and pressure of acoustic wave, and we discover an optimum acoustical condition, at which the reaction-rate constant for nucleation is increased by three-orders-of magnitudes. A theoretical model is proposed for explaining highly frequency and pressure dependent nucleation reaction, where monomers are captured on the bubble surface during its growth and highly condensed by subsequent bubble collapse, so that they are transiently exposed to high temperatures. Thus, the dual effects of local condensation and local heating contribute to dramatically enhance the nucleation reaction. Our model consistently reproduces the frequency and pressure dependences, supporting its essential applicability. PMID:26912021

  2. Nucleus factory on cavitation bubble for amyloid β fibril.

    PubMed

    Nakajima, Kichitaro; Ogi, Hirotsugu; Adachi, Kanta; Noi, Kentaro; Hirao, Masahiko; Yagi, Hisashi; Goto, Yuji

    2016-01-01

    Structural evolution from monomer to fibril of amyloid β peptide is related to pathogenic mechanism of Alzheimer disease, and its acceleration is a long-running problem in drug development. This study reveals that ultrasonic cavitation bubbles behave as catalysts for nucleation of the peptide: The nucleation reaction is highly dependent on frequency and pressure of acoustic wave, and we discover an optimum acoustical condition, at which the reaction-rate constant for nucleation is increased by three-orders-of magnitudes. A theoretical model is proposed for explaining highly frequency and pressure dependent nucleation reaction, where monomers are captured on the bubble surface during its growth and highly condensed by subsequent bubble collapse, so that they are transiently exposed to high temperatures. Thus, the dual effects of local condensation and local heating contribute to dramatically enhance the nucleation reaction. Our model consistently reproduces the frequency and pressure dependences, supporting its essential applicability. PMID:26912021

  3. Nucleus factory on cavitation bubble for amyloid β fibril

    NASA Astrophysics Data System (ADS)

    Nakajima, Kichitaro; Ogi, Hirotsugu; Adachi, Kanta; Noi, Kentaro; Hirao, Masahiko; Yagi, Hisashi; Goto, Yuji

    2016-02-01

    Structural evolution from monomer to fibril of amyloid β peptide is related to pathogenic mechanism of Alzheimer disease, and its acceleration is a long-running problem in drug development. This study reveals that ultrasonic cavitation bubbles behave as catalysts for nucleation of the peptide: The nucleation reaction is highly dependent on frequency and pressure of acoustic wave, and we discover an optimum acoustical condition, at which the reaction-rate constant for nucleation is increased by three-orders-of magnitudes. A theoretical model is proposed for explaining highly frequency and pressure dependent nucleation reaction, where monomers are captured on the bubble surface during its growth and highly condensed by subsequent bubble collapse, so that they are transiently exposed to high temperatures. Thus, the dual effects of local condensation and local heating contribute to dramatically enhance the nucleation reaction. Our model consistently reproduces the frequency and pressure dependences, supporting its essential applicability.

  4. Aqueous, Unfolded OmpA Forms Amyloid-Like Fibrils upon Self-Association

    PubMed Central

    Danoff, Emily J.; Fleming, Karen G.

    2015-01-01

    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 β-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-β 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. PMID:26196893

  5. Seeding-dependent maturation of beta2-microglobulin amyloid fibrils at neutral pH.

    PubMed

    Kihara, Miho; Chatani, Eri; Sakai, Miyo; Hasegawa, Kazuhiro; Naiki, Hironobu; Goto, Yuji

    2005-03-25

    Beta2-microglobulin (beta2-m) is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Recent studies have focused on the mechanism by which amyloid fibrils are formed under physiological conditions, which had been difficult to reproduce quantitatively. Yamamoto et al. (Yamamoto, S., Hasegawa, K., Yamaguchi, I., Tsutsumi, S., Kardos, J., Goto, Y., Gejyo, F. & Naiki, H. (2004) Biochemistry 43, 11075-11082) showed that a combination of seed fibrils prepared under acidic conditions and a low concentration of sodium dodecyl sulfate below its critical micelle concentration enabled extensive fibril formation at pH 7.0. Here, we found that repeated self-seeding at pH 7.0 with fibrils formed at the same pH causes a marked acceleration of growth, indicating the maturation of fibrils. The observed maturation can be simulated by assuming the existence of two types of fibrils with different growth rates. Importantly, some mutations of beta2-m or the addition of a low concentration of urea, both destabilizing the native conformation, were not enough to extend the fibrils at pH 7.0, and a low concentration of sodium dodecyl sulfate (i.e. 0.5 mM) was essential. Thus, even though the first stage fibrils in patients are unstable and require stabilizing factors to remain at neutral pH, they can adapt to a neutral pH with repeated self-seeding, implying a mechanism of development of amyloid deposition after a long latent period in patients. PMID:15659393

  6. Amyloid-like Fibril Formation by Trypsin in Aqueous Ethanol. Inhibition of Fibrillation by PEG.

    PubMed

    Kotormán, Márta; Simon, Mária L; Borics, Attila; Szabó, Márton Richárd; Szabó, Kitti; Szögi, Titanilla; Fülöp, Lívia

    2015-01-01

    The formation of amyloid-like fibrils was studied by using the well-known serine protease trypsin as a model protein in the presence of ethanol as organic solvent. Trypsin forms amyloid-like fibrils in aqueous ethanol at pH = 7.0. The dye Congo red (CR) was used to detect the presence of amyloid-like fibrils in the samples. The binding of CR to fibrils led to an increase in absorption intensity and a red shift in the absorption band of CR. Thioflavin T (ThT) and 8-anilino-1- naphthalenesulfonic acid (ANS) binding assays were employed to characterize amyloid-like fibril formation. The ThT binding assay revealed that the protein exhibited maximum aggregation in 60% (v/v) ethanol after incubation for 24 h at 24 (o)C. The ANS binding results indicated that the hydrophobic residues were more exposed to the solvent in the aggregated form of the protein. The effects of polyethylene glycol (PEG) on the formation of amyloid-like fibrils was studied in vitro. The aggregation of trypsin was followed via the kinetics of aggregation, the far-UV circular dichroism (CD) and transmission electron microscopy (TEM) in the presence and absence of PEG. The CD measurements indicated that the protein aggregates have a cross-beta structure in 60% ethanol. TEM revealed that trypsin forms fibrils with a thread-like structure. The inhibitory effect of PEG on the aggregation of trypsin increased with rising PEG concentration. PEG therefore inhibits the formation of amyloid-like fibrils of trypsin in aqueous ethanol. PMID:26428300

  7. In vitro fibrillization of Alzheimer's amyloid-β peptide (1-42)

    NASA Astrophysics Data System (ADS)

    Tiiman, Ann; Krishtal, Jekaterina; Palumaa, Peep; Tõugu, Vello

    2015-09-01

    The amyloid deposition in the form of extracellular fibrillar aggregates of amyloid-β (Aβ) peptide is a critical pathological event in Alzheimer's disease. Here, we report a systematic investigation of the effects of environmental factors on the kinetics of Aβ fibrillization in vitro. The effects of Aβ42 peptide concentration, temperature, pH, added solvents and the ratio of Aβ40 and Aβ42 on the peptide fibrillization under agitated conditions was studied. The analysis show that the rate of fibril growth by monomer addition is not limited by diffusion but by rearrangement in the monomer structure, which is enhanced by low concentrations of fluorinated alcohols and characterized by the activation energy of 12 kcal/mol. Fibrillization rate decreases at pH values below 7.0 where simultaneous protonation of His 13 and 14 inhibits fibril formation. The lag period for Aβ42 was only twofold shorter and the fibril growth rate twofold faster than those of Aβ40. Lag period was shortened and the fibrillization rate was increased only at 90% content of Aβ42.

  8. Population of non-native states of lysozyme variants drives amyloid fibril formation

    PubMed Central

    Mossuto, Maria F.; Cremades, Nunilo; Kumita, Janet R.; Dumoulin, Mireille; Welland, Mark E.; Knowles, Tuomas P.J.; Salvatella, Xavier; Dobson, Christopher M.

    2016-01-01

    The propensity of protein molecules to self-assemble into highly ordered, fibrillar aggregates lies at the heart of the understanding of many disorders such as Alzheimer’s disease or lysozyme systemic amyloidosis. In this article we use highly accurate kinetic measurements of amyloid fibril growth in combination with spectroscopic tools to quantify the effect of modifications in the solution conditions and the amino acid sequence of human lysozyme on its propensity to form amyloid fibrils under acidic conditions. We elucidate and quantify the correlation between the rate of amyloid growth and the population of non-native states, and show that changes in amyloidogenicity are almost entirely due to alterations in the stability of the native state, while other regions of the global free energy surface remain largely unmodified. These results provide insight into the complex dynamics of a macromolecule on a multidimensional energy landscape, and point the way for a better understanding of misfolding diseases. PMID:21528861

  9. Atomic Resolution Structure of Monomorphic Aβ42 Amyloid Fibrils.

    PubMed

    Colvin, Michael T; Silvers, Robert; Ni, Qing Zhe; Can, Thach V; Sergeyev, Ivan; Rosay, Melanie; Donovan, Kevin J; Michael, Brian; Wall, Joseph; Linse, Sara; Griffin, Robert G

    2016-08-01

    Amyloid-β (Aβ) is a 39-42 residue protein produced by the cleavage of the amyloid precursor protein (APP), which subsequently aggregates to form cross-β amyloid fibrils that are a hallmark of Alzheimer's disease (AD). The most prominent forms of Aβ are Aβ1-40 and Aβ1-42, which differ by two amino acids (I and A) at the C-terminus. However, Aβ42 is more neurotoxic and essential to the etiology of AD. Here, we present an atomic resolution structure of a monomorphic form of AβM01-42 amyloid fibrils derived from over 500 (13)C-(13)C, (13)C-(15)N distance and backbone angle structural constraints obtained from high field magic angle spinning NMR spectra. The structure (PDB ID: 5KK3 ) shows that the fibril core consists of a dimer of Aβ42 molecules, each containing four β-strands in a S-shaped amyloid fold, and arranged in a manner that generates two hydrophobic cores that are capped at the end of the chain by a salt bridge. The outer surface of the monomers presents hydrophilic side chains to the solvent. The interface between the monomers of the dimer shows clear contacts between M35 of one molecule and L17 and Q15 of the second. Intermolecular (13)C-(15)N constraints demonstrate that the amyloid fibrils are parallel in register. The RMSD of the backbone structure (Q15-A42) is 0.71 ± 0.12 Å and of all heavy atoms is 1.07 ± 0.08 Å. The structure provides a point of departure for the design of drugs that bind to the fibril surface and therefore interfere with secondary nucleation and for other therapeutic approaches to mitigate Aβ42 aggregation. PMID:27355699

  10. Imaging and quantification of amyloid fibrillation in the cell nucleus.

    PubMed

    Arnhold, Florian; Scharf, Andrea; von Mikecz, Anna

    2015-01-01

    Xenobiotics, as well as intrinsic processes such as cellular aging, contribute to an environment that constantly challenges nuclear organization and function. While it becomes increasingly clear that proteasome-dependent proteolysis is a major player, the topology and molecular mechanisms of nuclear protein homeostasis remain largely unknown. We have shown previously that (1) proteasome-dependent protein degradation is organized in focal microenvironments throughout the nucleoplasm and (2) heavy metals as well as nanoparticles induce nuclear protein fibrillation with amyloid characteristics. Here, we describe methods to characterize the landscape of intranuclear amyloid on the global and local level in different systems such as cultures of mammalian cells and the soil nematode Caenorhabditis elegans. Application of discrete mathematics to imaging data is introduced as a tool to develop pattern recognition of intracellular protein fibrillation. Since stepwise fibrillation of otherwise soluble proteins to insoluble amyloid-like protein aggregates is a hallmark of neurodegenerative protein-misfolding disorders including Alzheimer's disease, CAG repeat diseases, and the prion encephalopathies, investigation of intracellular amyloid may likewise aid to a better understanding of the pathomechanisms involved. We consider aggregate profiling as an important experimental approach to determine if nuclear amyloid has toxic or protective roles in various disease processes. PMID:25311131

  11. Amyloid Cardiomyopathy in Hereditary Transthyretin V30M Amyloidosis - Impact of Sex and Amyloid Fibril Composition

    PubMed Central

    Arvidsson, Sandra; Pilebro, Björn; Westermark, Per; Lindqvist, Per; Suhr, Ole B.

    2015-01-01

    Purpose Transthyretin V30M (ATTR V30M) amyloidosis is a phenotypically diverse disease with symptoms ranging from predominant neuropathy to exclusive cardiac manifestations. The aims of this study were to determine the dispersion of the two types of fibrils found in Swedish ATTR V30M patients -Type A consisting of a mixture of truncated and full length ATTR fibrils and type B fibrils consisting of full length fibrils, and to estimate the severity of cardiac dysfunction in relation to fibril composition and sex. Material and Methods Echocardiographic data were analysed in 107 Swedish ATTR V30M patients with their fibril composition determined as either type A or type B. Measurements of left ventricular (LV) dimensions and evaluation of systolic and diastolic function including speckle tracking derived strain were performed. Patients were grouped according to fibril type and sex. Multivariate linear regression was utilised to determine factors of significant impact on LV thickness. Results There was no significant difference in proportions of the two types of fibrils between men and women. In patients with type A fibrils, women had significantly lower median septal (p = 0.007) and posterior wall thicknesses (p = 0.010), lower median LV mass indexed to height (p = 0.008), and higher septal strain (p = 0.037), as compared to males. These differences were not apparent in patients with type B fibrils. Multiple linear regression analysis revealed that fibril type, sex and age all had significant impact on LV septal thickness. Conclusion This study demonstrates a clear difference between sexes in the severity of amyloid heart disease in ATTR V30M amyloidosis patients. Even though type A fibrils were associated with more advanced amyloid heart disease compared to type B, women with type A fibrils generally developed less cardiac infiltration than men. The differences may explain the better outcome for liver transplanted late-onset female patients compared to males. PMID

  12. Hierarchical ordering of amyloid fibrils on the mica surface

    NASA Astrophysics Data System (ADS)

    Zhou, Xingfei; Zhang, Yingying; Zhang, Feng; Pillai, Saju; Liu, Jianhua; Li, Rong; Dai, Bin; Li, Bin; Zhang, Yi

    2013-05-01

    The aggregation of amyloid peptides into ordered fibrils is closely associated with many neurodegenerative diseases. The surfaces of cell membranes and biomolecules are believed to play important roles in modulation of peptide aggregation under physiological conditions. Experimental studies of fibrillogenesis at the molecular level in vivo, however, are inherently challenging, and the molecular mechanisms of how surface affects the structure and ordering of amyloid fibrils still remain elusive. Herein we have investigated the aggregation behavior of insulin peptides within water films adsorbed on the mica surface. AFM measurements revealed that the structure and orientation of fibrils were significantly affected by the mica lattice and the peptide concentration. At low peptide concentration (~0.05 mg mL-1), there appeared a single layer of short and well oriented fibrils with a mean height of 1.6 nm. With an increase of concentration to a range of 0.2-2.0 mg mL-1, a different type of fibrils with a mean height of 3.8 nm was present. Interestingly, when the concentration was above 2.0 mg mL-1, the thicker fibrils exhibited two-dimensional liquid-crystal-like ordering probably caused by the combination of entropic and electrostatic forces. These results could help us gain better insight into the effects of the substrate on amyloid fibrillation.The aggregation of amyloid peptides into ordered fibrils is closely associated with many neurodegenerative diseases. The surfaces of cell membranes and biomolecules are believed to play important roles in modulation of peptide aggregation under physiological conditions. Experimental studies of fibrillogenesis at the molecular level in vivo, however, are inherently challenging, and the molecular mechanisms of how surface affects the structure and ordering of amyloid fibrils still remain elusive. Herein we have investigated the aggregation behavior of insulin peptides within water films adsorbed on the mica surface. AFM

  13. Aggregate Geometry in Amyloid Fibril Nucleation

    NASA Astrophysics Data System (ADS)

    Irbäck, Anders; Jónsson, Sigurður Æ.; Linnemann, Niels; Linse, Björn; Wallin, Stefan

    2013-02-01

    We present and study a minimal structure-based model for the self-assembly of peptides into ordered β-sheet-rich fibrils. The peptides are represented by unit-length sticks on a cubic lattice and interact by hydrogen bonding and hydrophobicity forces. Using Monte Carlo simulations with >105 peptides, we show that fibril formation occurs with sigmoidal kinetics in the model. To determine the mechanism of fibril nucleation, we compute the joint distribution in length and width of the aggregates at equilibrium, using an efficient cluster move and flat-histogram techniques. This analysis, based on simulations with 256 peptides in which aggregates form and dissolve reversibly, shows that the main free-energy barriers that a nascent fibril has to overcome are associated with changes in width.

  14. Decoding vibrational states of Concanavalin A amyloid fibrils.

    PubMed

    Piccirilli, Federica; Schirò, Giorgio; Vetri, Valeria; Lupi, Stefano; Perucchi, Andrea; Militello, Valeria

    2015-04-01

    Amyloid and amyloid-like fibrils are a general class of protein aggregates and represent a central topic in life sciences for their involvement in several neurodegenerative disorders and their unique mechanical and supramolecular morphological properties. Both their biological role and their physical properties, including their high mechanical stability and thermodynamic inertia, are related to the structural arrangement of proteins in the aggregates at molecular level. Significant variations may exist in the supramolecular organization of the commonly termed cross-β structure that constitutes the amyloid core. In this context, a fine knowledge of the structural details in fibrils may give significant information on the assembly process and on possible ways of tuning or inhibiting it. Here we propose a simple method based on the combined use of Fourier transform infrared spectroscopy and Fourier transform Raman spectroscopy to accurately reveal structural details in the fibrillar aggregates, side-chain exposure and intermolecular interactions. Interestingly, coupled analysis of mid-infrared spectra reveals antiparallel β-sheet orientation in ConA fibrils. We also report the comparison between THz absorption spectra of Concanavalin A in its native and fibrillar state at different hydration levels, allowing obtaining corroboration of peaks assignation in this range and information on the effect of amyloid supramolecular arrangement on the network dynamics of hydration water. PMID:25776525

  15. Amyloid fibril protein nomenclature: 2012 recommendations from the Nomenclature Committee of the International Society of Amyloidosis.

    PubMed

    Sipe, Jean D; Benson, Merrill D; Buxbaum, Joel N; Ikeda, Shu-ichi; Merlini, Giampaolo; Saraiva, Maria J M; Westermark, Per

    2012-12-01

    The Nomenclature Committee of the International Society of Amyloidosis (ISA) met during the XIIIth International Symposium, May 6-10, 2012, Groningen, The Netherlands, to formulate recommendations on amyloid fibril protein nomenclature and to consider newly identified candidate amyloid fibril proteins for inclusion in the ISA Amyloid Fibril Protein Nomenclature List. The need to promote utilization of consistent and up to date terminology for both fibril chemistry and clinical classification of the resultant disease syndrome was emphasized. Amyloid fibril nomenclature is based on the chemical identity of the amyloid fibril forming protein; clinical classification of the amyloidosis should be as well. Although the importance of fibril chemistry to the disease process has been recognized for more than 40 years, to this day the literature contains clinical and histochemical designations that were used when the chemical diversity of amyloid diseases was poorly understood. Thus, the continued use of disease classifications such as familial amyloid neuropathy and familial amyloid cardiomyopathy generates confusion. An amyloid fibril protein is defined as follows: the protein must occur in body tissue deposits and exhibit both affinity for Congo red and green birefringence when Congo red stained deposits are viewed by polarization microscopy. Furthermore, the chemical identity of the protein must have been unambiguously characterized by protein sequence analysis when so is practically possible. Thus, in nearly all cases, it is insufficient to demonstrate mutation in the gene of a candidate amyloid protein; the protein itself must be identified as an amyloid fibril protein. Current ISA Amyloid Fibril Protein Nomenclature Lists of 30 human and 10 animal fibril proteins are provided together with a list of inclusion bodies that, although intracellular, exhibit some or all of the properties of the mainly extracellular amyloid fibrils. PMID:23113696

  16. Conformational stability of PrP amyloid fibrils controls their smallest possible fragment size.

    PubMed

    Sun, Ying; Makarava, Natallia; Lee, Cheng-I; Laksanalamai, Pongpan; Robb, Frank T; Baskakov, Ilia V

    2008-02-29

    Fibril fragmentation is considered to be an essential step in prion replication. Recent studies have revealed a strong correlation between the incubation period to prion disease and conformational stability of synthetic prions. To gain insight into the molecular mechanism that accounts for this correlation, we proposed that the conformational stability of prion fibrils controls their intrinsic fragility or the size of the smallest possible fibrillar fragments. Using amyloid fibrils produced from full-length mammalian prion protein under three growth conditions, we found a correlation between conformational stability and the smallest possible fragment sizes. Specifically, the fibrils that were conformationally less stable were found to produce shorter pieces upon fragmentation. Site-specific denaturation experiments revealed that the fibril conformational stability was controlled by the region that acquires a cross-beta-sheet structure. Using atomic force microscopy imaging, we found that fibril fragmentation occurred in both directions--perpendicular to and along the fibrillar axis. Two mechanisms of fibril fragmentation were identified: (i) fragmentation caused by small heat shock proteins, including alpha B-crystallin, and (ii) fragmentation due to mechanical stress arising from adhesion of the fibril to a surface. This study provides new mechanistic insight into the prion replication mechanism and offers a plausible explanation for the correlation between conformational stability of synthetic prions and incubation time to prion disease. PMID:18206163

  17. Walnut extract inhibits the fibrillization of amyloid beta-protein, and also defibrillizes its preformed fibrils.

    PubMed

    Chauhan, Neha; Wang, K C; Wegiel, Jerzy; Malik, Mazhar N

    2004-08-01

    Fibrillar amyloid beta-protein (Abeta) is the principal component of amyloid plaques in the brains of patients with Alzheimer's disease. We have studied the effect of walnut extract on Abeta fibrillization by Thioflavin T fluorescence spectroscopy and electron microscopy. The walnut extract not only inhibited Abeta fibril formation in a concentration and time- dependent manner but it was also able to defibrillize Abeta preformed fibrils. Over 90% inhibition of Abeta fibrillization was observed with 5 microl of methanolic extract of walnut (MEOW) both after 2 and 3 days of incubation. The maximum defibrillization (91.6%) was observed when preformed Abeta fibrils were incubated with 10 microl of MEOW for 2 days. These results suggest that walnuts may reduce the risk or delay the onset of Alzheimer's disease by maintaining Abeta in the soluble form. Further studies showed that anti-amyloidogenic compound in walnut is an organic compound of molecular weight less than 10 kDa, which is neither a lipid nor a protein. Chloroform extract of walnut had no effect on Abeta fibrillization while MEOW and its 10 kDa filtrate inhibited Abeta fibrillization equally. It is proposed that polyphenolic compounds (such as flavonoids) present in walnuts may be responsible for its anti-amyloidogenic activity. PMID:15975066

  18. Aggregation of fibrils and plaques in amyloid molecular systems

    NASA Astrophysics Data System (ADS)

    Nicodemi, Mario; de Candia, Antonio; Coniglio, Antonio

    2009-10-01

    Amyloidlike proteins form highly organized aggregates, such as fibrils and plaques, preceded by the assembly of a wide range of unstructured oligomers and protofibrils. Despite their importance in a number of human neurodegenerative diseases, a comprehensive understanding of their kinetics and thermodynamics is still missing. We investigate, by computer simulations, a realistic model of amyloid molecules interacting via the experimentally determined Derjaguin-Landau-Verwey-Overbeek potential and derive its phase diagram. We show that fibrils and plaques, along with their precursors, correspond to different equilibrium and metastable thermodynamics phases and discuss the dynamical mechanisms leading to the nucleation and self-assembly of large scale structures.

  19. Early Events in the Amyloid Formation of the A546T Mutant of Transforming Growth Factor Beta Induced Protein (TGFBIp) in Corneal Dystrophies Compared to the Non-Fibrillating R555W and R555Q Mutants

    PubMed Central

    Koldsø, Heidi; Andersen, Ole Juul; Nikolajsen, Camilla Lund; Scavenius, Carsten; Sørensen, Charlotte S.; Underhaug, Jarl; Runager, Kasper; Nielsen, Niels Chr.; Enghild, Jan J.; Schiøtt, Birgit

    2016-01-01

    The human transforming growth factor beta induced protein (TGFBIp) is involved in several types of corneal dystrophies where protein aggregation and amyloid fibril formation severely impairs vision. Most disease-causing mutations are located in the last of four homologous fasciclin-1 (FAS1) domains of the protein, and it has been shown that when isolated, the fourth FAS1 domain (FAS1–4) mimics the behavior of full-length TGFBIp. In this study, we use molecular dynamics simulations and principal component analysis to study the wild type FAS1–4 domain along with three disease-causing mutations (R555W, R555Q, and A546T) to decipher any internal difference in dynamical properties of the domains that may explain their varied stabilities and aggregation properties. In addition, we use a protein-protein docking method in combination with chemical cross-linking experiments and mass spectrometry of the cross-linked species to obtain information about interaction faces between identical FAS1–4 domains. The results show that the pathogenic mutations A546T and R555W affect the packing in the hydrophobic core of FAS1–4 in different directions. We further show that the FAS1–4 monomers associate using their β-rich regions consistent with peptides observed to be part of the amyloid fibril core in lattice corneal dystrophy patients. PMID:26305369

  20. Designed amyloid beta peptide fibril - a tool for high-throughput screening of fibril inhibitors.

    PubMed

    Dolphin, Gunnar T; Ouberai, Myriam; Dumy, Pascal; Garcia, Julian

    2007-11-01

    Amyloid beta peptide (Abeta) fibril formation is widely believed to be the causative event of Alzheimer's disease pathogenesis. Therapeutic approaches are therefore in development that target various sites in the production and aggregation of Abeta. Herein we present a high-throughput screening tool to generate novel hit compounds that block Abeta fibril formation. This tool is an application for our fibril model (Abeta(16-37)Y(20)K(22)K(24))(4), which is a covalent assembly of four Abeta fragments. With this tool, screening studies are complete within one hour, as opposed to days with native Abeta(1-40). A Z' factor of 0.84+/-0.03 was determined for fibril formation and inhibition, followed by the reporter molecule thioflavin T. Herein we also describe the analysis of a broad range of reported inhibitors and non-inhibitors of Abeta fibril formation to test the validity of the system. PMID:17876751

  1. Nonequilibrium and generalized-ensemble molecular dynamics simulations for amyloid fibril

    NASA Astrophysics Data System (ADS)

    Okumura, Hisashi

    2015-12-01

    Amyloids are insoluble and misfolded fibrous protein aggregates and associated with more than 20 serious human diseases. We perform all-atom molecular dynamics simulations of amyloid fibril assembly and disassembly.

  2. Nonequilibrium and generalized-ensemble molecular dynamics simulations for amyloid fibril

    SciTech Connect

    Okumura, Hisashi

    2015-12-31

    Amyloids are insoluble and misfolded fibrous protein aggregates and associated with more than 20 serious human diseases. We perform all-atom molecular dynamics simulations of amyloid fibril assembly and disassembly.

  3. Engineering amyloid fibrils from β-solenoid proteins for biomaterials applications.

    PubMed

    Peralta, Maria D R; Karsai, Arpad; Ngo, Alice; Sierra, Catherine; Fong, Kai T; Hayre, Natha Robert; Mirzaee, Nima; Ravikumar, Krishnakumar Mayuram; Kluber, Alexander J; Chen, Xi; Liu, Gang-yu; Toney, Michael D; Singh, Rajiv R; Cox, Daniel Lee

    2015-01-27

    Nature provides numerous examples of self-assembly that can potentially be implemented for materials applications. Considerable attention has been given to one-dimensional cross-β or amyloid structures that can serve as templates for wire growth or strengthen materials such as glue or cement. Here, we demonstrate controlled amyloid self-assembly based on modifications of β-solenoid proteins. They occur naturally in several contexts (e.g., antifreeze proteins, drug resistance proteins) but do not aggregate in vivo due to capping structures or distortions at their ends. Removal of these capping structures and regularization of the ends of the spruce budworm and rye grass antifreeze proteins yield micron length amyloid fibrils with predictable heights, which can be a platform for biomaterial-based self-assembly. The design process, including all-atom molecular dynamics simulations, purification, and self-assembly procedures are described. Fibril formation with the predicted characteristics is supported by evidence from thioflavin-T fluorescence, circular dichroism, dynamic light scattering, and atomic force microscopy. Additionally, we find evidence for lateral assembly of the modified spruce budworm antifreeze fibrils with sufficient incubation time. The kinetics of polymerization are consistent with those for other amyloid formation reactions and are relatively fast due to the preformed nature of the polymerization nucleus. PMID:25562726

  4. Does Thioflavin-T Detect Oligomers Formed During Amyloid Fibril Assembly

    NASA Astrophysics Data System (ADS)

    Persichilli, Christopher; Hill, Shannon E.; Mast, Jason; Muschol, Martin

    2011-03-01

    Recent results have shown that oligomeric intermediates of amyloid fibril assembly represent the main toxic species in disorders such as Alzheimer's disease and type II diabetes. Thioflavin-T (ThT) is among the most commonly used indicator dyes for mature amyloid fibrils in vitro. We used ThT to monitor amyloid fibril formation of lysozyme (HEWL), and correlated ThT fluorescence to concurrent dynamic light scattering and atomic force microscopy measurements. Specifically, we tested the ability of ThT to discern among oligomer-free vs. oligomeric fibril assembly pathways. We found that ThT fluorescence did not detect oligomer growth; however, fluorescence increases did coincide with the formation of monomeric filaments in the oligomer-free assembly pathway. This implies that ThT fluorescence is not generally suitable for the detection of oligomeric intermediates. The results further suggest different internal structures for oligomeric vs. monomeric filaments. This research was supported, in part, by funding through the Byrd Alzheimer's Institute (ARG-2007-22) and the BITT-Florida Center of Excellence for M.M., an NSF-REU grant (DMR-1004873) for C. P. and an NSF-IGERT fellowship for S.H.

  5. Stimulated emission depletion microscopy to study amyloid fibril formation

    NASA Astrophysics Data System (ADS)

    Mahou, Pierre; Curry, Nathan; Pinotsi, Dorothea; Kaminski Schierle, Gabriele; Kaminski, Clemens

    2015-03-01

    Aggregation of misfolded proteins is a characteristic hallmark of many neurodegenerative disorders, such as Parkinson's, Alzheimer's and Huntington's diseases. The ability to observe these aggregation processes and the corresponding structures formed in vitro or in situ is therefore a key requirement to understand the molecular mechanisms of these diseases. We report here on the implementation and application of Stimulated Emission Depletion (STED) microscopy to visualize the formation of amyloid fibrils in vitro.

  6. Self-Assembly of Amyloid Fibrils That Display Active Enzymes

    PubMed Central

    Zhou, Xiao-Ming; Entwistle, Aiman; Zhang, Hong; Jackson, Antony P; Mason, Thomas O; Shimanovich, Ulyana; Knowles, Tuomas P J; Smith, Andrew T; Sawyer, Elizabeth B; Perrett, Sarah

    2014-01-01

    Enzyme immobilization is an important strategy to enhance the stability and recoverability of enzymes and to facilitate the separation of enzymes from reaction products. However, enzyme purification followed by separate chemical steps to allow immobilization on a solid support reduces the efficiency and yield of the active enzyme. Here we describe polypeptide constructs that self-assemble spontaneously into nanofibrils with fused active enzyme subunits displayed on the amyloid fibril surface. We measured the steady-state kinetic parameters for the appended enzymes in situ within fibrils and compare these with the identical protein constructs in solution. Finally, we demonstrated that the fibrils can be recycled and reused in functional assays both in conventional batch processes and in a continuous-flow microreactor. PMID:25937845

  7. Glycosaminoglycans in extracts of cardiac amyloid fibrils from familial amyloid cardiomyopathy of Danish origin related to variant transthyretin Met 111.

    PubMed

    Magnus, J H; Stenstad, T; Kolset, S O; Husby, G

    1991-07-01

    We have previously demonstrated an association between secondary AA type amyloid fibrils and glycosaminoglycans (GAGs) in human liver. The present study was aimed at investigating whether a similar association could be demonstrated in isolated cardiac amyloid fibrils from a unique Danish family with amyloid cardiomyopathy related to variant transthyretin (TTR) with a single amino acid substitution of a methionin for leucine at position 111 (TTR Met 111). Using gel filtration and ion exchange chromatography, significant amounts of GAGs were detected in close association with purified myocardial amyloid fibrils, whereas only trace amounts of polysaccharides were present in the corresponding normal preparation. The GAGs were identified as 50% chondroitin sulfate, 33% heparin/heparan sulfate, and 17% hyaluronan. With the methods used the amyloid associated GAGs appeared as high molecular weight free polysaccharide chains, and not as part of intact proteoglycans (PGs) in the fibril extracts. We conclude that the association between purified amyloid fibrils and GAGs may be a general feature of amyloid deposits. Also, we suggest that the proportion of different GAGs in the amyloid deposits may depend both on the organ or tissues affected and the type of proteins making up the fibrils. PMID:2068532

  8. Tensile deformation and failure of amyloid and amyloid-like protein fibrils

    NASA Astrophysics Data System (ADS)

    Solar, Max; Buehler, Markus J.

    2014-03-01

    Here we report a series of full atomistic molecular dynamics simulations of six amyloid or amyloid-like protein fibrils in order to systematically understand the effect of different secondary structure motifs on the mechanical tensile and failure response of cross-\\beta protein fibrils. We find a similar failure behavior across the six structures; an initial failure event occurs at small strains involving cooperative rupture of a group of hydrogen bonds, followed by a slow one-by-one hydrogen bond rupture process as the remaining \\beta -sheets peel off with very low applied stress. We also find that the ultimate tensile strength of the protein fibrils investigated scales directly with the number of hydrogen bonds per unit area which break in the initial rupture event. Our results provide insights into structure-property relationships in protein fibrils important for disease and engineering applications and lay the groundwork for the development of materials selection criteria for the design of de novo amyloid-based functional biomaterials.

  9. Electron tomography reveals the fibril structure and lipid interactions in amyloid deposits.

    PubMed

    Kollmer, Marius; Meinhardt, Katrin; Haupt, Christian; Liberta, Falk; Wulff, Melanie; Linder, Julia; Handl, Lisa; Heinrich, Liesa; Loos, Cornelia; Schmidt, Matthias; Syrovets, Tatiana; Simmet, Thomas; Westermark, Per; Westermark, Gunilla T; Horn, Uwe; Schmidt, Volker; Walther, Paul; Fändrich, Marcus

    2016-05-17

    Electron tomography is an increasingly powerful method to study the detailed architecture of macromolecular complexes or cellular structures. Applied to amyloid deposits formed in a cell culture model of systemic amyloid A amyloidosis, we could determine the structural morphology of the fibrils directly in the deposit. The deposited fibrils are arranged in different networks, and depending on the relative fibril orientation, we can distinguish between fibril meshworks, fibril bundles, and amyloid stars. These networks are frequently infiltrated by vesicular lipid inclusions that may originate from the death of the amyloid-forming cells. Our data support the role of nonfibril components for constructing fibril deposits and provide structural views of different types of lipid-fibril interactions. PMID:27140609

  10. Effect of acid predissolution on fibril size and fibril flexibility of synthetic beta-amyloid peptide.

    PubMed Central

    Shen, C L; Fitzgerald, M C; Murphy, R M

    1994-01-01

    beta-amyloid peptide (A beta) is the major protein component of senile plaques and cerebrovascular amyloid deposits in Alzheimer's patients. Several researchers have demonstrated that A beta is neurotoxic in in vitro and in vivo systems. Peptide aggregation state and/or conformation might play a significant role in determining the toxicity of the peptide. The size and flexibility of fibrils formed from the synthetic peptide beta (1-39), corresponding to the first 39 residues of A beta, were determined. Samples were prepared either directly from lyophilized peptide or diluted from a 10 mg/ml stock solution in 0.1% trifluoroacetic acid (TFA). All samples had a final peptide concentration of 0.5 mg/ml, a final pH of 7.4, and a final NaCl concentration of 0.14 M. The molecular weight and linear density of the fibrils increased with increasing pre-incubation time in TFA, based on static light scattering measurements. Analysis of the angular dependence of the intensity of scattered light indicated that the fibrils were semi-flexible chains and that the fibril flexibility decreased with increasing pre-incubation time in TFA. There was a concomitant change in phase behavior from precipitation to gelation with the decrease in fibril flexibility. Images FIGURE 3 PMID:7811938

  11. Toward a Molecular Theory of Early and Late Events in Monomer to Amyloid Fibril Formation

    NASA Astrophysics Data System (ADS)

    Straub, John E.; Thirumalai, D.

    2011-05-01

    Quantitative understanding of the kinetics of fibril formation and the molecular mechanism of transition from monomers to fibrils is needed to obtain insights into the growth of amyloid fibrils and more generally self-assembly multisubunit protein complexes. Significant advances using computations of protein aggregation in a number of systems have established generic and sequence-specific aspects of the early steps in oligomer formation. Theoretical considerations, which view oligomer and fibril growth as diffusion in a complex energy landscape, and computational studies, involving minimal lattice and coarse-grained models, have revealed general principles governing the transition from monomeric protein to ordered fibrillar aggregates. Detailed atomistic calculations have explored the early stages of the protein aggregation pathway for a number of amyloidogenic proteins, most notably amyloid β- (Aβ-) protein and fragments from proteins linked to various diseases. These computational studies have provided insights into the role of sequence, role of water, and specific interatomic interactions underlying the thermodynamics and dynamics of elementary kinetic steps in the aggregation pathway. Novel methods are beginning to illustrate the structural basis for the production of Aβ-peptides through interactions with secretases in the presence of membranes. We show that a variety of theoretical approaches, ranging from scaling arguments to minimal models to atomistic simulations, are needed as a complement to experimental studies probing the principles governing protein aggregation.

  12. Force generation by the growth of amyloid aggregates

    PubMed Central

    Herling, Therese W.; Garcia, Gonzalo A.; Michaels, Thomas C. T.; Grentz, Wolfgang; Dean, James; Shimanovich, Ulyana; Gang, Hongze; Müller, Thomas; Kav, Batuhan; Terentjev, Eugene M.; Dobson, Christopher M.; Knowles, Tuomas P. J.

    2015-01-01

    The generation of mechanical forces are central to a wide range of vital biological processes, including the function of the cytoskeleton. Although the forces emerging from the polymerization of native proteins have been studied in detail, the potential for force generation by aberrant protein polymerization has not yet been explored. Here, we show that the growth of amyloid fibrils, archetypical aberrant protein polymers, is capable of unleashing mechanical forces on the piconewton scale for individual filaments. We apply microfluidic techniques to measure the forces released by amyloid growth for two systems: insulin and lysozyme. The level of force measured for amyloid growth in both systems is comparable to that observed for actin and tubulin, systems that have evolved to generate force during their native functions and, unlike amyloid growth, rely on the input of external energy in the form of nucleotide hydrolysis for maximum force generation. Furthermore, we find that the power density released from growing amyloid fibrils is comparable to that of high-performance synthetic polymer actuators. These findings highlight the potential of amyloid structures as active materials and shed light on the criteria for regulation and reversibility that guide molecular evolution of functional polymers. PMID:26195762

  13. Fibpredictor: a computational method for rapid prediction of amyloid fibril structures.

    PubMed

    Tabatabaei Ghomi, Hamed; Topp, Elizabeth M; Lill, Markus A

    2016-09-01

    Amyloid fibrils are important in diseases such as Alzheimer's disease and Parkinson's disease, and are also a common instability in peptide and protein drug products. Despite their importance, experimental structures of amyloid fibrils in atomistic detail are rare. To address this limitation, we have developed a novel, rapid computational method to predict amyloid fibril structures (Fibpredictor). The method combines β-sheet model building, β-sheet replication, and symmetry operations with side-chain prediction and statistical scoring functions. When applied to nine amyloid fibrils with experimentally determined structures, the method predicted the correct structures of amyloid fibrils and enriched those among the top-ranked structures. These models can be used as the initial heuristic structures for more complicated computational studies. Fibpredictor is available at http://nanohub.org/resources/fibpredictor . PMID:27502172

  14. Localization microscopy for the study of amyloid fibril formation

    NASA Astrophysics Data System (ADS)

    Pinotsi, Dorothea; Kaminski Schierle, Gabriele S.; Rees, Eric; Kaminski, Clemens F.

    2013-09-01

    Super-resolution microscopy has emerged as a powerful and non-invasive tool for the study of molecular processes both in vitro, but also as they occur in live cells. Here we present the application of direct stochastic optical reconstruction microscopy (dSTORM), a super-resolution technique based on single molecule localization, to determine the morphology of protein aggregates and of small extra- and intracellular structures. The technique reveals details down to 20 nm providing information on scales much smaller than the wavelength of the probing light. We use dSTORM in the study of amyloid fibril self-assembly processes associated with neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. We show that the aggregation process can be followed kinetically and observe the emergence of amyloid structures in time as they occur in vitro. As an all optical technique, there is translation potential from studies in vitro to in vivo applications.

  15. Binuclear ruthenium(II) complexes for amyloid fibrils recognition

    NASA Astrophysics Data System (ADS)

    Hanczyc, Piotr

    2014-12-01

    Metal-organic compounds represent a unique class of biomarkers with promising photophysical properties useful for imaging. Here interactions of insulin fibrils with two binuclear complexes [μ-(11,11‧-bidppz)(phen)4Ru2]4+ (1) and [μ-C4(cpdppz)(phen)4Ru2]4+ (2) are studied by linear dichroism (LD) and fluorescence. These ruthenium(II) compounds could provide a new generation of amyloid binding chromophores with long lived lifetimes, good luminescence quantum yields for the bound molecules and photo-stability useful in multiphoton luminescence imaging.

  16. Quantification of amyloid fibrils using size exclusion chromatography coupled with online fluorescence and ultraviolet detection.

    PubMed

    Randrianjatovo-Gbalou, Irina; Marcato-Romain, Claire-Emmanuelle; Girbal-Neuhauser, Elisabeth

    2015-11-01

    An amyloid fibrils investigation within biofilm samples requires distinguishing the amyloid β-sheet structure of these proteins and quantifying them. In this study, the property of amyloids to incorporate the fluorescent dye Thioflavin T has been exploited to propose a method of quantification. The experimental protocol includes the preparation of amyloids from commercial κ-casein (κCN) and their fractionation through size exclusion chromatography (SEC) to provide calibration curves from fluorescence and absorbance signals. Finally, a bacterial biofilm extract was injected into SEC, and the amyloid fibrils could be expressed as equivalent κCN, representing approximately 21% of the total proteins. PMID:26239215

  17. Structure-based design of non-natural amino-acid inhibitors of amyloid fibril formation

    SciTech Connect

    Sievers, Stuart A.; Karanicolas, John; Chang, Howard W.; Zhao, Anni; Jiang, Lin; Zirafi, Onofrio; Stevens, Jason T.; Münch, Jan; Baker, David; Eisenberg, David

    2011-09-20

    Many globular and natively disordered proteins can convert into amyloid fibrils. These fibrils are associated with numerous pathologies as well as with normal cellular functions, and frequently form during protein denaturation. Inhibitors of pathological amyloid fibril formation could be useful in the development of therapeutics, provided that the inhibitors were specific enough to avoid interfering with normal processes. Here we show that computer-aided, structure-based design can yield highly specific peptide inhibitors of amyloid formation. Using known atomic structures of segments of amyloid fibrils as templates, we have designed and characterized an all-D-amino-acid inhibitor of the fibril formation of the tau protein associated with Alzheimer's disease, and a non-natural L-amino-acid inhibitor of an amyloid fibril that enhances sexual transmission of human immunodeficiency virus. Our results indicate that peptides from structure-based designs can disrupt the fibril formation of full-length proteins, including those, such as tau protein, that lack fully ordered native structures. Because the inhibiting peptides have been designed on structures of dual-{beta}-sheet 'steric zippers', the successful inhibition of amyloid fibril formation strengthens the hypothesis that amyloid spines contain steric zippers.

  18. Looking for a generic inhibitor of amyloid-like fibril formation among flavone derivatives

    PubMed Central

    Šneideris, Tomas; Baranauskienė, Lina; Cannon, Jonathan G.; Rutkienė, Rasa; Meškys, Rolandas

    2015-01-01

    A range of diseases is associated with amyloid fibril formation. Despite different proteins being responsible for each disease, all of them share similar features including beta-sheet-rich secondary structure and fibril-like protein aggregates. A number of proteins can form amyloid-like fibrils in vitro, resembling structural features of disease-related amyloids. Given these generic structural properties of amyloid and amyloid-like fibrils, generic inhibitors of fibril formation would be of interest for treatment of amyloid diseases. Recently, we identified five outstanding inhibitors of insulin amyloid-like fibril formation among the pool of 265 commercially available flavone derivatives. Here we report testing of these five compounds and of epi-gallocatechine-3-gallate (EGCG) on aggregation of alpha-synuclein and beta-amyloid. We used a Thioflavin T (ThT) fluorescence assay, relying on halftimes of aggregation as the measure of inhibition. This method avoids large numbers of false positive results. Our data indicate that four of the five flavones and EGCG inhibit alpha-synuclein aggregation in a concentration-dependent manner. However none of these derivatives were able to increase halftimes of aggregation of beta-amyloid. PMID:26421240

  19. Amyloid fibrils containing fragmented ATTR may be the standard fibril composition in ATTR amyloidosis.

    PubMed

    Ihse, Elisabet; Rapezzi, Claudio; Merlini, Giampaolo; Benson, Merrill D; Ando, Yukio; Suhr, Ole B; Ikeda, Shu-Ichi; Lavatelli, Francesca; Obici, Laura; Quarta, Candida C; Leone, Ornella; Jono, Hirofumi; Ueda, Mitsuharu; Lorenzini, Massimiliano; Liepnieks, Juris; Ohshima, Toshinori; Tasaki, Masayoshi; Yamashita, Taro; Westermark, Per

    2013-09-01

    Abstract The clinical phenotype of familial ATTR amyloidosis depends to some extent on the particular mutation, but differences exist also within mutations. We have previously described that two types of amyloid fibril compositions exist among Swedish ATTRV30M amyloidosis patients, one consisting of a mixture of intact and fragmented ATTR (type A) and one consisting of mainly intact ATTR (type B). The fibril types are correlated to phenotypic differences. Patients with ATTR fragments have a late onset and develop cardiomyopathy, while patients without fragments have an early onset and less myocardial involvement. The present study aimed to determine whether this correlation between fibril type and phenotype is valid for familial ATTR amyloidosis in general. Cardiac or adipose tissues from 63 patients carrying 29 different TTR non-V30M mutations as well as 13 Japanese ATTRV30M patients were examined. Fibril type was determined by western blotting and compared to the patients' age of onset and degree of cardiomyopathy. All ATTR non-V30M patients had a fibril composition with ATTR fragments, except two ATTRY114C patients. No clear conclusions could be drawn about a phenotype to fibril type correlation among ATTR non-V30M patients. In contrast, Japanese ATTRV30M patients showed a similar correlation as previously described for Swedish ATTRV30M patients. This study shows that a fibril composition with fragmented ATTR is very common in ATTR amyloidosis, and suggests that fibrils composed of only full-length ATTR is an exception found only in a subset of patients. PMID:23713495

  20. Freeze-Thaw Cycling Induced Isotropic-Nematic Coexistence of Amyloid Fibrils Suspensions.

    PubMed

    Zhao, Jianguo; Bolisetty, Sreenath; Adamcik, Jozef; Han, Jun; Fernández-Ronco, María P; Mezzenga, Raffaele

    2016-03-15

    Amyloid fibrils are charged semiflexible assemblies with very large aspect ratio (length to diameter, L/D). Because of this large aspect ratio, the isotropic (I) and nematic (N) phase coexistence expected from the first-order thermodynamic nature of the I-N phase transition, as predicted from the Onsager's theory, is vanishingly small and, in practice, challenging to experimentally observe. In this study we present a remarkable widening of the I + N biphasic region in β-lactoglobulin fibrils suspension via freeze-thaw (F-T) cycling. The demixing behavior can be induced and controlled by a slow growth of propagation front of the ice crystals, which grow by excluding the amyloid fibrils from the crystal phase and thus concentrating them in the liquid phase. The growth of the ice crystals is accompanied by the formation of concentrated and elongated tactoid-like structure in the liquid phase. During the subsequent thawing cycling, at large tactoid domains, the mismatch in density caused by the presence of amyloid fibrils is sufficient to generate a sedimentation of the N phase at the bottom of the vial, coexisting with an I phase on the top. We reason why, despite the remarkable stability of the coexisting I and N phases observed over several weeks after F-T cycling, the biphasic region is understood to be a nonequilibrium, metastable state. Yet, the results in this study suggest that the F-T treatment is an effective approach to stabilize multiphase coexistence of liquid crystalline phases in colloidal suspensions of anisotropic particles without the need of additives, such as depleting agents, needed to modify interaction potentials. PMID:26907697

  1. Role of sequence and structural polymorphism on the mechanical properties of amyloid fibrils.

    PubMed

    Yoon, Gwonchan; Lee, Myeongsang; Kim, Jae In; Na, Sungsoo; Eom, Kilho

    2014-01-01

    Amyloid fibrils playing a critical role in disease expression, have recently been found to exhibit the excellent mechanical properties such as elastic modulus in the order of 10 GPa, which is comparable to that of other mechanical proteins such as microtubule, actin filament, and spider silk. These remarkable mechanical properties of amyloid fibrils are correlated with their functional role in disease expression. This suggests the importance in understanding how these excellent mechanical properties are originated through self-assembly process that may depend on the amino acid sequence. However, the sequence-structure-property relationship of amyloid fibrils has not been fully understood yet. In this work, we characterize the mechanical properties of human islet amyloid polypeptide (hIAPP) fibrils with respect to their molecular structures as well as their amino acid sequence by using all-atom explicit water molecular dynamics (MD) simulation. The simulation result suggests that the remarkable bending rigidity of amyloid fibrils can be achieved through a specific self-aggregation pattern such as antiparallel stacking of β strands (peptide chain). Moreover, we have shown that a single point mutation of hIAPP chain constituting a hIAPP fibril significantly affects the thermodynamic stability of hIAPP fibril formed by parallel stacking of peptide chain, and that a single point mutation results in a significant change in the bending rigidity of hIAPP fibrils formed by antiparallel stacking of β strands. This clearly elucidates the role of amino acid sequence on not only the equilibrium conformations of amyloid fibrils but also their mechanical properties. Our study sheds light on sequence-structure-property relationships of amyloid fibrils, which suggests that the mechanical properties of amyloid fibrils are encoded in their sequence-dependent molecular architecture. PMID:24551113

  2. Morphology and mechanical properties of multi-stranded amyloid fibrils probed by atomistic and coarse-grained simulations

    NASA Astrophysics Data System (ADS)

    Yoon, Gwonchan; Lee, Myeongsang; Kim, Kyungwoo; In Kim, Jae; Chang, Hyun Joon; Baek, Inchul; Eom, Kilho; Na, Sungsoo

    2015-12-01

    Amyloid fibrils are responsible for pathogenesis of various diseases and exhibit the structural feature of an ordered, hierarchical structure such as multi-stranded helical structure. As the multi-strandedness of amyloid fibrils has recently been found to be highly correlated with their toxicity and infectivity, it is necessary to study how the hierarchical (i.e. multi-stranded) structure of amyloid fibril is formed. Moreover, although it has recently been reported that the nanomechanics of amyloid proteins plays a key role on the amyloid-induced pathogenesis, a critical role that the multi-stranded helical structure of the fibrils plays in their nanomechanical properties has not fully characterized. In this work, we characterize the morphology and mechanical properties of multi-stranded amyloid fibrils by using equilibrium molecular dynamics simulation and elastic network model. It is shown that the helical pitch of multi-stranded amyloid fibril is linearly proportional to the number of filaments comprising the amyloid fibril, and that multi-strandedness gives rise to improving the bending rigidity of the fibril. Moreover, we have also studied the morphology and mechanical properties of a single protofilament (filament) in order to understand the effect of cross-β structure and mutation on the structures and mechanical properties of amyloid fibrils. Our study sheds light on the underlying design principles showing how the multi-stranded amyloid fibril is formed and how the structure of amyloid fibrils governs their nanomechanical properties.

  3. Toward the Molecular Mechanism(s) by which EGCG Treatment Remodels Mature Amyloid Fibrils

    PubMed Central

    Palhano, Fernando L.; Lee, Jiyong; Grimster, Neil P.; Kelly, Jeffery W.

    2013-01-01

    Protein misfolding and/or aggregation has been implicated in several human diseases, such as Alzheimer’s and Parkinson’s diseases and familial amyloid polyneuropathy. These maladies are referred to as amyloid diseases, because they are named after the cross-β-sheet amyloid fibril aggregates or deposits common to these diseases. Epigallocatechin-3-gallate (EGCG), the principal polyphenol present in green tea, has been shown to be effective at preventing aggregation and is able to remodel amyloid fibrils comprising different amyloidogenic proteins, although the mechanistic underpinnings are unclear. Herein, we work towards an understanding of the molecular mechanism(s) by which EGCG remodels mature amyloid fibrils made up of Aβ1–40, IAPP8–24, or Sup35NM7–16. We show that EGCG amyloid remodeling activity in vitro is dependent on auto-oxidation of the EGCG. Oxidized and unoxidized EGCG binds to amyloid fibrils, preventing the binding of thioflavin T. This engagement of the hydrophobic binding sites in Aβ1–40, IAPP8–24, or Sup35NM7–16 amyloid fibrils seems to be sufficient to explain the majority of the amyloid remodeling observed by EGCG treatment, although how EGCG oxidation drives remodeling remains unclear. Oxidized EGCG molecules react with free amines within the amyloid fibril through the formation of Schiff bases, cross-linking the fibrils, which may prevent dissociation and toxicity, but these aberrant post-translational modifications do not appear to be the major driving force for amyloid remodeling by EGCG treatment. These insights into the molecular mechanism of action of EGCG provide boundary conditions for exploring amyloid remodeling in more detail. PMID:23611538

  4. Intrinsic Structural Heterogeneity and Long-Term Maturation of Amyloid β Peptide Fibrils

    PubMed Central

    2013-01-01

    Amyloid β peptides form fibrils that are commonly assumed to have a dry, homogeneous, and static internal structure. To examine these assumptions, fibrils under various conditions and different ages have been examined with multidimensional infrared spectroscopy. Each peptide in the fibril had a 13C=18O label in the backbone of one residue to disinguish the amide I′ absorption due to that residue from the amide I′ absorption of other residues. Fibrils examined soon after they formed, and reexamined after 1 year in the dry state, exhibited spectral changes confirming that structurally significant water molecules were present in the freshly formed fibrils. Results from fibrils incubated in solution for 4 years indicate that water molecules remained trapped within fibrils and mobile over the 4 year time span. These water molecules are structurally significant because they perturb the parallel β-sheet hydrogen bonding pattern at frequent intervals and at multiple points within individual fibrils, creating structural heterogeneity along the length of a fibril. These results show that the interface between β-sheets in an amyloid fibril is not a “dry zipper”, and that the internal structure of a fibril evolves while it remains in a fibrillar state. These features, water trapping, structural heterogeneity, and structural evolution within a fibril over time, must be accommodated in models of amyloid fibril structure and formation. PMID:23701594

  5. AFM-based force spectroscopy measurements of mature amyloid fibrils of the peptide glucagon

    NASA Astrophysics Data System (ADS)

    Dong, Mingdong; Bruun Hovgaard, Mads; Mamdouh, Wael; Xu, Sailong; Otzen, Daniel Erik; Besenbacher, Flemming

    2008-09-01

    We report on the mechanical characterization of individual mature amyloid fibrils by atomic force microscopy (AFM) and AFM-based single-molecule force spectroscopy (SMFS). These self-assembling materials, formed from the 29-residue amphiphatic peptide hormone glucagon, were found to display a reversible elastic behaviour. Based on AFM morphology and SMFS studies, we suggest that the observed elasticity is due to a force-induced conformational transition which is reversible due to the β-helical conformation of protofibrils, allowing a high degree of extension. The elastic properties of such mature fibrils contribute to their high stability, suggesting that the internal hydrophobic interactions of amyloid fibrils are likely to be of fundamental importance in the assembly of amyloid fibrils and therefore for the understanding of the progression of their associated pathogenic disorders. In addition, such biological amyloid fibril structures with highly stable mechanical properties can potentially be used to produce nanofibres (nanowires) that may be suitable for nanotechnological applications.

  6. Simultaneous Measurement of Amyloid Fibril Formation by Dynamic Light Scattering and Fluorescence Reveals Complex Aggregation Kinetics

    PubMed Central

    Streets, Aaron M.; Sourigues, Yannick; Kopito, Ron R.; Melki, Ronald; Quake, Stephen R.

    2013-01-01

    An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington's disease, in vitro. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation. PMID:23349924

  7. Human Hsp70 Disaggregase Reverses Parkinson's-Linked α-Synuclein Amyloid Fibrils.

    PubMed

    Gao, Xuechao; Carroni, Marta; Nussbaum-Krammer, Carmen; Mogk, Axel; Nillegoda, Nadinath B; Szlachcic, Anna; Guilbride, D Lys; Saibil, Helen R; Mayer, Matthias P; Bukau, Bernd

    2015-09-01

    Intracellular amyloid fibrils linked to neurodegenerative disease typically accumulate in an age-related manner, suggesting inherent cellular capacity for counteracting amyloid formation in early life. Metazoan molecular chaperones assist native folding and block polymerization of amyloidogenic proteins, preempting amyloid fibril formation. Chaperone capacity for amyloid disassembly, however, is unclear. Here, we show that a specific combination of human Hsp70 disaggregase-associated chaperone components efficiently disassembles α-synuclein amyloid fibrils characteristic of Parkinson's disease in vitro. Specifically, the Hsc70 chaperone, the class B J-protein DNAJB1, and an Hsp110 family nucleotide exchange factor (NEF) provide ATP-dependent activity that disassembles amyloids within minutes via combined fibril fragmentation and depolymerization. This ultimately generates non-toxic α-synuclein monomers. Concerted, rapid interaction cycles of all three chaperone components with fibrils generate the power stroke required for disassembly. This identifies a powerful human Hsp70 disaggregase activity that efficiently disassembles amyloid fibrils and points to crucial yet undefined biology underlying amyloid-based diseases. PMID:26300264

  8. Particle tracking microrheology of gel-forming amyloid fibril networks

    NASA Astrophysics Data System (ADS)

    Corrigan, A. M.; Donald, A. M.

    2009-04-01

    Microrheology is a technique that is increasingly used to investigate the local viscoelastic properties of complex fluids non-invasively, by tracking the motion of micron-sized probe spheres. In this work, passive Particle Tracking Microrheology (PTM) is used to study network formation in the milk protein β -lactoglobulin at 80 ° C and p H 2. In these conditions the protein aggregates to form thread-like structures known as amyloid fibrils, which can further aggregate into elastic networks. Using PTM, gels were observed to form at significantly lower concentrations than determined by bulk rheometry, where the oscillatory shear forces may disrupt either fibril or network formation. During incubation, the Mean Square Displacement (MSD) of the probe particles exhibited time-cure superposition, allowing the critical relaxation exponent to be calculated as ˜ 0.63 , consistent with other biopolymer gels. Combined with the gel-like appearance of the complex modulus at long incubation times, this confirms that a true gel is forming, with physical or chemical crosslinks forming between the fibrils, refining the conclusions of other workers in the field.

  9. Inhibition of amyloid fibrillation and cytotoxicity of lysozyme fibrillation products by polyphenols.

    PubMed

    Shariatizi, Sajad; Meratan, Ali Akbar; Ghasemi, Atiyeh; Nemat-Gorgani, Mohsen

    2015-09-01

    An increasing number of studies conducted under in vitro and in vivo conditions, have concluded that polyphenols, compounds frequently occurring in many herbs with antioxidant properties, prevent and reverse amyloid fibril formation. However, the mechanisms by which these natural products modulate the protein aggregation process are poorly understood. Herein, a range of techniques including thioflavin T (ThT) and ANS fluorescence assays, electron microscopy and circular dichroism have been employed to determine the efficacy of rosmarinic acid (RA) and resveratrol (Res) on the inhibition/reversion of fibrillogenesis and hindering cytotoxicity induced by protofibrils and amyloid fibrils of hen egg white lysozyme (HEWL). Results demonstrated that both polyphenols effectively inhibit fibrillogenesis and destabilize preformed fibrils of HEWL in a concentration-dependent manner. Cytotoxicity protection on PC12 cells was also observed using the MTT assay, ROS production assay, and phase-contrast microscopy. It is suggested that the mechanism underlying the inhibitory effects of RA and Res is to prevent hydrophobic interactions between HEWL amyloidogenic prefibrillar species, although additional studies is needed to elucidate the detailed mechanisms involved. A combination of antioxidative and anti-amyloidogenic properties of these molecules may provide them with the described neuroprotective capacities. PMID:26102331

  10. Microscale structural model of Alzheimer Aβ(1-40) amyloid fibril

    NASA Astrophysics Data System (ADS)

    Paparcone, Raffaella; Buehler, Markus J.

    2009-06-01

    Amyloid fibril formation and characterization are crucial due to their association with severe degenerative disorders such as Alzheimer's, type II diabetes, and Parkinson's disease. Here we present an atomistic-based multiscale analysis, utilized to predict the structure of Alzheimer Aβ(1-40) fibrils. Our study provides a structural model of amyloid fibers with lengths of hundreds of nanometers at atomistic resolution. We report a systematic analysis of the energies, structural changes and H-bonding for varying fibril lengths, elucidating their size dependent properties. Our model predicts the formation of twisted amyloid microfibers with a periodicity of ≈82 nm, in close agreement with experimental results.

  11. A setup for simultaneous measurement of infrared spectra and light scattering signals: Watching amyloid fibrils grow from intact proteins

    SciTech Connect

    Li, Yang; Maurer, Jürgen; Roth, Andreas; Vogel, Vitali; Winter, Ernst; Mäntele, Werner

    2014-08-15

    A setup for the simultaneous measurement of mid-infrared spectra and static light scattering is described that can be used for the analysis of the formation of nanoscale and microscopic aggregates from smaller molecules to biopolymers. It can be easily integrated into sample chambers of infrared spectrometers or combined with laser beams from tunable infrared lasers. Here, its use for the analysis of the formation of amyloid fibrils from intact proteins is demonstrated. The formation of amyloid fibrils or plaques from proteins is a widespread and pathogenetic relevant process, and a number of diseases are caused and correlated with the deposition of amyloid fibrils in cells and tissues. The molecular mechanisms of these transformations, however, are still unclear. We report here the simultaneous measurement of infrared spectra and static light scattering for the analysis of fibril formation from egg-white lysozyme. The transformation of the native form into non-native forms rich in β-sheet structure is measured by analysis of the amide I spectral region in the infrared spectra, which is sensitive for local structures. At the same time, light scattering signals at forward direction as well as the forward/backward ratio, which are sensitive for the number of scattering centers and their approximate sizes, respectively, are collected for the analysis of fibril growth. Thermodynamic and kinetic parameters as well as mechanistic information are deduced from the combination of the two complementary techniques.

  12. Nucleic acid-containing amyloid fibrils potently induce type I interferon and stimulate systemic autoimmunity

    PubMed Central

    Di Domizio, Jeremy; Dorta-Estremera, Stephanie; Gagea, Mihai; Ganguly, Dipyaman; Meller, Stephan; Li, Ping; Zhao, Bihong; Tan, Filemon K.; Bi, Liqi; Gilliet, Michel; Cao, Wei

    2012-01-01

    The immunopathophysiologic development of systemic autoimmunity involves numerous factors through complex mechanisms that are not fully understood. In systemic lupus erythematosus, type I IFN (IFN-I) produced by plasmacytoid dendritic cells (pDCs) critically promotes the autoimmunity through its pleiotropic effects on immune cells. However, the host-derived factors that enable abnormal IFN-I production and initial immune tolerance breakdown are largely unknown. Previously, we found that amyloid precursor proteins form amyloid fibrils in the presence of nucleic acids. Here we report that nucleic acid-containing amyloid fibrils can potently activate pDCs and enable IFN-I production in response to self-DNA, self-RNA, and dead cell debris. pDCs can take up DNA-containing amyloid fibrils, which are retained in the early endosomes to activate TLR9, leading to high IFNα/β production. In mice treated with DNA-containing amyloid fibrils, a rapid IFN response correlated with pDC infiltration and activation. Immunization of nonautoimmune mice with DNA-containing amyloid fibrils induced antinuclear serology against a panel of self-antigens. The mice exhibited positive proteinuria and deposited antibodies in their kidneys. Intriguingly, pDC depletion obstructed IFN-I response and selectively abolished autoantibody generation. Our study reveals an innate immune function of nucleic acid-containing amyloid fibrils and provides a potential link between compromised protein homeostasis and autoimmunity via a pDC-IFN axis. PMID:22904191

  13. The emergence of superstructural order in insulin amyloid fibrils upon multiple rounds of self-seeding

    PubMed Central

    Surmacz-Chwedoruk, Weronika; Babenko, Viktoria; Dec, Robert; Szymczak, Piotr; Dzwolak, Wojciech

    2016-01-01

    Typically, elongation of an amyloid fibril entails passing conformational details of the mother seed to daughter generations of fibrils with high fidelity. There are, however, several factors that can potentially prevent such transgenerational structural imprinting from perpetuating, for example heterogeneity of mother seeds or so-called conformational switching. Here, we examine phenotypic persistence of bovine insulin amyloid ([BI]) upon multiple rounds of self-seeding under quiescent conditions. According to infrared spectroscopy, with the following passages of homologous seeding, daughter fibrils gradually depart from the mother seed’s spectral characteristics. We note that this transgenerational structural drift in [BI] amyloid leads toward fibrils with infrared, chiroptical, and morphological traits similar to those of the superstructural variant of fibrils which normally forms upon strong agitation of insulin solutions. However, in contrast to agitation-induced insulin amyloid, the superstructural assemblies of daughter fibrils isolated through self-seeding are sonication-resistant. Our results suggest that formation of single amyloid fibrils is not a dead-end of the amyloidogenic self-assembly. Instead, the process appears to continue toward the self-assembly of higher-order structures although on longer time-scales. From this perspective, the fast agitation-induced aggregation of insulin appears to be a shortcut to amyloid superstructures whose formation under quiescent conditions is slow. PMID:27558445

  14. The emergence of superstructural order in insulin amyloid fibrils upon multiple rounds of self-seeding.

    PubMed

    Surmacz-Chwedoruk, Weronika; Babenko, Viktoria; Dec, Robert; Szymczak, Piotr; Dzwolak, Wojciech

    2016-01-01

    Typically, elongation of an amyloid fibril entails passing conformational details of the mother seed to daughter generations of fibrils with high fidelity. There are, however, several factors that can potentially prevent such transgenerational structural imprinting from perpetuating, for example heterogeneity of mother seeds or so-called conformational switching. Here, we examine phenotypic persistence of bovine insulin amyloid ([BI]) upon multiple rounds of self-seeding under quiescent conditions. According to infrared spectroscopy, with the following passages of homologous seeding, daughter fibrils gradually depart from the mother seed's spectral characteristics. We note that this transgenerational structural drift in [BI] amyloid leads toward fibrils with infrared, chiroptical, and morphological traits similar to those of the superstructural variant of fibrils which normally forms upon strong agitation of insulin solutions. However, in contrast to agitation-induced insulin amyloid, the superstructural assemblies of daughter fibrils isolated through self-seeding are sonication-resistant. Our results suggest that formation of single amyloid fibrils is not a dead-end of the amyloidogenic self-assembly. Instead, the process appears to continue toward the self-assembly of higher-order structures although on longer time-scales. From this perspective, the fast agitation-induced aggregation of insulin appears to be a shortcut to amyloid superstructures whose formation under quiescent conditions is slow. PMID:27558445

  15. Ultrasonication-dependent formation and degradation of α-synuclein amyloid fibrils.

    PubMed

    Yagi, Hisashi; Mizuno, Aiko; So, Masatomo; Hirano, Miki; Adachi, Masayuki; Akazawa-Ogawa, Yoko; Hagihara, Yoshihisa; Ikenoue, Tatsuya; Lee, Young-Ho; Kawata, Yasushi; Goto, Yuji

    2015-03-01

    Ultrasonication can be used to break the supersaturation of α-synuclein, a protein associated with Parkinson's disease, at pH7.4 above the critical concentration of fibrillation, thereby inducing the formation of amyloid fibrils. We speculated that ultrasonication could also be used to depolymerize preformed fibrils below the critical concentration. However, extensive ultrasonic irradiation transformed preformed fibrils into amorphous aggregates even above the critical concentration. Exposing preformed fibrils to the hydrophobic air-water interface of cavitation bubbles may have destabilized the fibrils and stabilized amorphous aggregates. Upon extensive ultrasonic irradiation, the accompanying decomposition of chemical structures was suggested when monitored by analytical ultracentrifugation. Amorphous aggregates produced by extensive ultrasonication showed higher cytotoxicity, suggesting that, although ultrasonication might be a useful approach for inactivating amyloid fibrils, potential cytotoxicity of amorphous aggregates should be considered. PMID:25528988

  16. Carnosine's Effect on Amyloid Fibril Formation and Induced Cytotoxicity of Lysozyme

    PubMed Central

    Wu, Josephine W.; Liu, Kuan-Nan; How, Su-Chun; Chen, Wei-An; Lai, Chia-Min; Liu, Hwai-Shen; Hu, Chaur-Jong; Wang, Steven S. -S.

    2013-01-01

    Carnosine, a common dipeptide in mammals, has previously been shown to dissemble alpha-crystallin amyloid fibrils. To date, the dipeptide's anti-fibrillogensis effect has not been thoroughly characterized in other proteins. For a more complete understanding of carnosine's mechanism of action in amyloid fibril inhibition, we have investigated the effect of the dipeptide on lysozyme fibril formation and induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Our study demonstrates a positive correlation between the concentration and inhibitory effect of carnosine against lysozyme fibril formation. Molecular docking results show carnosine's mechanism of fibrillogenesis inhibition may be initiated by binding with the aggregation-prone region of the protein. The dipeptide attenuates the amyloid fibril-induced cytotoxicity of human neuronal cells by reducing both apoptotic and necrotic cell deaths. Our study provides solid support for carnosine's amyloid fibril inhibitory property and its effect against fibril-induced cytotoxicity in SH-SY5Y cells. The additional insights gained herein may pave way to the discovery of other small molecules that may exert similar effects against amyloid fibril formation and its associated neurodegenerative diseases. PMID:24349167

  17. Stretching and ordering of amyloid fibrils at liquid interfaces

    NASA Astrophysics Data System (ADS)

    Del Gado, Emanuela; Schwenke, Konrad; Jordens, Sophia; Usov, Ivan; Mezzenga, Raffaele; Georgetown University Team; ETH Zurich Team

    We investigate the formation of nematic domains, which might be precursor of plaque formation, in the adsorption of amyloid fibrils at liquid interfaces. Combining experiments and computer simulations we analyse spatial correlations in the nematic order and in apparent persistence length. Non-equilibrium numerical simulations provide new insight into the coupling between those quantities. The emerging scenario is that the out-of-equilibrium adsorption favors the formation of spatial heterogeneities due to the presence of local nematic order that tend to persist upon increasing the surface coverage. Such structural heterogeneities are directly coupled to the apparent straightening of the brils and might affect the density and the mechanical properties of the final self-assembled material.

  18. Isolation and characterization of the integral glycosaminoglycan constituents of human amyloid A and monoclonal light-chain amyloid fibrils.

    PubMed Central

    Nelson, S R; Lyon, M; Gallagher, J T; Johnson, E A; Pepys, M B

    1991-01-01

    Amyloid fibrils were isolated by extraction in water from the livers and spleens of four patients who had died of monoclonal, light-chain (AL)-type, systemic amyloidosis and one with reactive systemic, amyloid A protein (AA)-type amyloidosis. Each fibril preparation contained 1-2% by weight of glycosaminoglycan (GAG) which was tightly associated with the fibrils and not just co-isolated from the tissues with them. After exhaustive digestion of the fibrils with papain and Pronase, the GAGs were specifically precipitated with cetylpyridinium chloride and were identified by cellulose acetate electrophoresis and selective susceptibility to specific glycosidases. All the preparations contained approximately equal amounts of heparan sulphate and dermatan sulphate. There was no evidence for the presence of chondroitin sulphate or other GAGs. Fine structural analysis by oligosaccharide mapping in gradient polyacrylamide gels, following partial digestion with specific glycosidases, showed very similar structures among the heparan sulphates and the dermatan sulphates, respectively. GAGs were also extracted by solubilizing amyloid fibrils in 4 M-guanidinium chloride followed by CsCl density-gradient ultracentrifugation. Although a minor proportion of the GAG material obtained in this way was apparently in the form of proteoglycan molecules, most of it was free GAG chains. The presence in amyloid fibrils of different types, in different organs and from different patients of particular GAG classes with similar structures supports the view that these molecules may be of pathogenic significance. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1902087

  19. Is supramolecular filament chirality the underlying cause of major morphology differences in amyloid fibrils?

    PubMed

    Kurouski, Dmitry; Lu, Xuefang; Popova, Ludmila; Wan, William; Shanmugasundaram, Maruda; Stubbs, Gerald; Dukor, Rina K; Lednev, Igor K; Nafie, Laurence A

    2014-02-12

    The unique enhanced sensitivity of vibrational circular dichroism (VCD) to the formation and development of amyloid fibrils in solution is extended to four additional fibril-forming proteins or peptides where it is shown that the sign of the fibril VCD pattern correlates with the sense of supramolecular filament chirality and, without exception, to the dominant fibril morphology as observed in AFM or SEM images. Previously for insulin, it has been demonstrated that the sign of the VCD band pattern from filament chirality can be controlled by adjusting the pH of the incubating solution, above pH 2 for "normal" left-hand-helical filaments and below pH 2 for "reversed" right-hand-helical filaments. From AFM or SEM images, left-helical filaments form multifilament braids of left-twisted fibrils while the right-helical filaments form parallel filament rows of fibrils with a flat tape-like morphology, the two major classes of fibril morphology that from deep UV resonance Raman scattering exhibit the same cross-β-core secondary structure. Here we investigate whether fibril supramolecular chirality is the underlying cause of the major morphology differences in all amyloid fibrils by showing that the morphology (twisted versus flat) of fibrils of lysozyme, apo-α-lactalbumin, HET-s (218-289) prion, and a short polypeptide fragment of transthyretin, TTR (105-115), directly correlates to their supramolecular chirality as revealed by VCD. The result is strong evidence that the chiral supramolecular organization of filaments is the principal underlying cause of the morphological heterogeneity of amyloid fibrils. Because fibril morphology is linked to cell toxicity, the chirality of amyloid aggregates should be explored in the widely used in vitro models of amyloid-associated diseases. PMID:24484302

  20. Ferulic acid destabilizes preformed {beta}-amyloid fibrils in vitro

    SciTech Connect

    Ono, Kenjiro; Hirohata, Mie; Yamada, Masahito . E-mail: m-yamada@med.kanazawa-u.ac.jp

    2005-10-21

    Inhibition of the formation of {beta}-amyloid fibrils (fA{beta}), as well as the destabilization of preformed fA{beta} in the CNS, would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). We reported previously that curcumin (Cur) inhibits fA{beta} formation from A{beta} and destabilizes preformed fA{beta} in vitro. Using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies, we examined the effects of ferulic acid (FA) on the formation, extension, and destabilization of fA{beta} at pH 7.5 at 37 deg C in vitro. We next compared the anti-amyloidogenic activities of FA with Cur, rifampicin, and tetracycline. Ferulic acid dose-dependently inhibited fA{beta} formation from amyloid {beta}-peptide, as well as their extension. Moreover, it destabilized preformed fA{beta}s. The overall activity of the molecules examined was in the order of: Cur > FA > rifampicin = tetracycline. FA could be a key molecule for the development of therapeutics for AD.

  1. Energy interactions in amyloid-like fibrils from NNQQNY.

    PubMed

    Cuesta, Inmaculada García; Sánchez de Merás, Alfredo M J

    2014-03-01

    We use large-scale MP2 calculations to analyze the interactions appearing in amyloid fibers, which are difficult to determine experimentally. To this end, dimers and trimers of the hexapeptide NNQQNY from the yeast prion-like protein Sup35 were considered as model systems. We studied the energy interactions present in the three levels of organization in which the formation of amyloid fibrils is structured. The structural changes in the hydrogen bonds were studied too. It was found that the most energetic process is the formation of the β-sheet, which is equally due to both hydrogen bonds and van der Waals interactions. The aromatic rings help stabilize these aggregates through stacking of the aromatic rings of tyrosine, the stability produced by the aromatics residues increasing with their aromaticity. The formation of the basic unit of the assembled proto-fiber, the steric zipper, is less energetic and is associated to both dispersion forces and hydrogen bonds. The interactions between pair of β-sheets across the peptide-to-peptide contact through the tyrosine rings are cooperative and due to dispersion effects. Moreover, the strength of this interaction can rationalize the variation of mobility of the aromatic ring in the tyrosine units found in solid NMR experiments. PMID:24458317

  2. Mechanical properties of amyloid-like fibrils defined by secondary structures.

    PubMed

    Bortolini, C; Jones, N C; Hoffmann, S V; Wang, C; Besenbacher, F; Dong, M

    2015-05-01

    Amyloid and amyloid-like fibrils represent a generic class of highly ordered nanostructures that are implicated in some of the most fatal neurodegenerative diseases. On the other hand, amyloids, by possessing outstanding mechanical robustness, have also been successfully employed as functional biomaterials. For these reasons, physical and chemical factors driving fibril self-assembly and morphology are extensively studied - among these parameters, the secondary structures and the pH have been revealed to be crucial, since a variation in pH changes the fibril morphology and net chirality during protein aggregation. It is important to quantify the mechanical properties of these fibrils in order to help the design of effective strategies for treating diseases related to the presence of amyloid fibrils. In this work, we show that by changing pH the mechanical properties of amyloid-like fibrils vary as well. In particular, we reveal that these mechanical properties are strongly related to the content of secondary structures. We analysed and estimated the Young's modulus (E) by comparing the persistence length (Lp) - measured from the observation of TEM images by using statistical mechanics arguments - with the mechanical information provided by peak force quantitative nanomechanical property mapping (PF-QNM). The secondary structure content and the chirality are investigated by means of synchrotron radiation circular dichroism (SR-CD). Results arising from this study could be fruitfully used as a protocol to investigate other medical or engineering relevant peptide fibrils. PMID:25839069

  3. Thioflavin T-Silent Denaturation Intermediates Support the Main-Chain-Dominated Architecture of Amyloid Fibrils.

    PubMed

    Noda, Sayaka; So, Masatomo; Adachi, Masayuki; Kardos, József; Akazawa-Ogawa, Yoko; Hagihara, Yoshihisa; Goto, Yuji

    2016-07-19

    Ultrasonication is considered one of the most effective agitations for inducing the spontaneous formation of amyloid fibrils. When we induced the ultrasonication-dependent fibrillation of β2-microglobulin and insulin monitored by amyloid-specific thioflavin T (ThT) fluorescence, both proteins showed a significant decrease in ThT fluorescence after the burst-phase increase. The decrease in ThT fluorescence was accelerated when the ultrasonic power was stronger, suggesting that this decrease was caused by the partial denaturation of preformed fibrils. The possible intermediates of denaturation retained amyloid-like morphologies, secondary structures, and seeding potentials. Similar denaturation intermediates were also observed when fibrils were denatured by guanidine hydrochloride or sodium dodecyl sulfate. The presence of these denaturation intermediates is consistent with the main-chain-dominated architecture of amyloid fibrils. Moreover, in the three types of denaturation experiments conducted, insulin fibrils were more stable than β2-microglobulin fibrils, suggesting that the relative stability of various fibrils is independent of the method of denaturation. PMID:27345358

  4. Aβ(1–40) forms five distinct amyloid structures whose β-sheet contents and fibril stabilities are correlated

    PubMed Central

    Kodali, Ravindra; Williams, Angela D.; Chemuru, Saketh; Wetzel, Ronald

    2010-01-01

    The ability of a single polypeptide sequence to grow into multiple stable amyloid fibrils sets these aggregates apart from most native globular proteins. The existence of multiple amyloid forms is the basis for strain effects in yeast prion biology, and may also contribute to variations in Alzheimer’s disease pathology. However, the structural basis for amyloid polymorphism is poorly understood. We report here five structurally distinct fibrillar aggregates of the Alzheimer’s plaque peptide Aβ(1–40), as well as a non-fibrillar aggregate induced by Zn+2. Each of these conformational forms exhibits a unique profile of physical properties, and all the fibrillar forms “breed true” in elongation reactions at a common set of growth conditions. Consistent with their defining cross-β structure, we find that in this series the amyloid fibrils containing more extensive β-sheet exhibit greater stability. At the same time, side chain packing outside of the β-sheet regions also contributes to stability, and to stability differences between polymorphic forms. Stability comparison is facilitated by the unique feature that the free energy of the monomer (equivalent to the unfolded state in a protein folding reaction) does not vary, and hence can be ignored, in the comparison of ΔG° of elongation values for each polymorphic fibril obtained at a single set of conditions. PMID:20600131

  5. Time-lapse atomic force microscopy in the characterization of amyloid-like fibril assembly and oligomeric intermediates.

    PubMed

    Goldsbury, Claire; Green, Janelle

    2005-01-01

    The atomic force microscope (AFM) images the topography of biological structures adsorbed to surfaces with nanometer to angstrom scale resolution. Amyloid-like fibrils and oligomers can be imaged in buffer solutions, allowing the samples to retain physiological-like properties while temporal changes in structure are monitored, e.g., the elongation of fibrils or the growth of single oligomers. These qualities distinguish AFM from conventional imaging techniques of comparable resolution, i.e., electron microscopy (EM). However, AFM is limited in that the specimen must be firmly attached to a solid support for measurement and that time-lapse imaging of individual assemblies can thus only be achieved for fibrils and oligomers growing on this support. Nevertheless, AFM has provided several insights into the in vitro assembly mechanism and structures of amyloid-like fibrils. The first section of this chapter provides a methodological introduction to AFM, whilst the second details the application of this technique to the investigation of amyloidogenic proteins, specifically amylin and amyloid-beta (Abeta) peptides. PMID:15980598

  6. Amyloid fibril formation at a uniformly sheared air/water interface

    NASA Astrophysics Data System (ADS)

    Posada, David; Hirsa, Amir

    2013-11-01

    Amyloid fibril formation is a process by which protein molecules in solution form nuclei and aggregate into fibrils. Amyloid fibrils have long been associated with several common diseases such as Parkinson's disease and Alzheimer's. More recently, fibril protein deposition has been implicated in uncommon disorders leading to the failure of various organs including the kidneys, heart, and liver. Fibrillization can also play a detrimental role in biotherapeutic production. Results from previous studies show that a hydrophobic interface, such air/water, can accelerate fibrillization. Studies also show that agitation accelerates fibrillization. When attempting to elucidate fundamental mechanisms of fibrillization and distinguish the effects of interfaces and flow, it can be helpful to experiment with uniformly sheared interfaces. A new Taylor-Couette device is introduced for in situ, real-time high resolution microscopy. With a sub-millimeter annular gap, surface tension acts as the channel floor, permitting a stable meniscus to be placed arbitrarily close to a microscope to study amyloid fibril formation over long periods.

  7. An equilibrium model for linear and closed-loop amyloid fibril formation

    PubMed Central

    Yang, Shuo; Griffin, Michael D. W.; Binger, Katrina J.; Schuck, Peter; Howlett, Geoffrey J.

    2014-01-01

    Amyloid fibrils and their soluble oligomeric intermediates are implicated in several age-related diseases including Alzheimer’s and Parkinson’s disease. The distribution of oligomers and fibrils is related to toxicity and is dependent on the pathways for fibril assembly, generally considered to occur via a slow nucleation step that precedes fibril elongation. Human apolipoprotein (apo) C-II forms amyloid fibrils via a reversible self-assembly process accompanied by closed-loop formation and fibril breaking and joining. Our fluorescence quenching and sedimentation velocity experiments with Alexa488-labelled apoC-II indicated a time-dependent sub-unit interchange for both linear and closed-loop fibrils, while dilution experiments using mature fibrils indicated a shift to smaller size distributions consistent with a reversible assembly pathway. To account for this behaviour we developed an equilibrium self-association model that describes the final size distributions of apoC-II fibrils formed at different starting concentrations. The model proposes a reversible isomerisation of apoC-II monomer to form an active conformer that self-assembles into fibrils via an isodesmic self-association pathway coupled to fibril length-dependent closed-loop formation. The model adequately described fibril size distributions and the proportion of closed-loops as a function of total apoC-II concentration over the concentration range 0.1–0.5 mg/ml. Extension of the model to include the rates of isomerisation, self-association and fibril breaking and joining provided satisfactory global fits to kinetic data on fibril formation and changes in average fibril size at different apoC-II starting concentrations. The model provides a simple thermodynamic description of the processes governing the size distribution of apoC-II fibrils at equilibrium and the formation of discrete oligomeric intermediates. PMID:22370559

  8. An equilibrium model for linear and closed-loop amyloid fibril formation.

    PubMed

    Yang, Shuo; Griffin, Michael D W; Binger, Katrina J; Schuck, Peter; Howlett, Geoffrey J

    2012-08-10

    Amyloid fibrils and their soluble oligomeric intermediates are implicated in several age-related diseases including Alzheimer's and Parkinson's diseases. The distribution of oligomers and fibrils is related to toxicity and is dependent on the pathways for fibril assembly, generally considered to occur via a slow nucleation step that precedes fibril elongation. Human apolipoprotein (apo) C-II forms amyloid fibrils via a reversible self-assembly process accompanied by closed-loop formation and fibril breaking and joining. Our fluorescence quenching and sedimentation velocity experiments with Alexa488-labeled apoC-II indicated a time-dependent subunit interchange for both linear and closed-loop fibrils, while dilution experiments using mature fibrils indicated a shift to smaller size distributions consistent with a reversible assembly pathway. To account for this behavior, we developed an equilibrium self-association model that describes the final size distributions of apoC-II fibrils formed at different starting concentrations. The model proposes a reversible isomerization of apoC-II monomer to form an active conformer that self-assembles into fibrils via an isodesmic self-association pathway coupled to fibril length-dependent closed-loop formation. The model adequately described fibril size distributions and the proportion of closed loops as a function of total apoC-II concentration over the concentration range 0.1-0.5 mg/ml. Extension of the model to include the rates of isomerization, self-association and fibril breaking and joining provided satisfactory global fits to kinetic data on fibril formation and changes in average fibril size at different apoC-II starting concentrations. The model provides a simple thermodynamic description of the processes governing the size distribution of apoC-II fibrils at equilibrium and the formation of discrete oligomeric intermediates. PMID:22370559

  9. Measurement of intrinsic properties of amyloid fibrils by the peak force QNM method

    NASA Astrophysics Data System (ADS)

    Adamcik, Jozef; Lara, Cecile; Usov, Ivan; Jeong, Jae Sun; Ruggeri, Francesco S.; Dietler, Giovanni; Lashuel, Hilal A.; Hamley, Ian W.; Mezzenga, Raffaele

    2012-07-01

    We report the investigation of the mechanical properties of different types of amyloid fibrils by the peak force quantitative nanomechanical (PF-QNM) technique. We demonstrate that this technique correctly measures the Young's modulus independent of the polymorphic state and the cross-sectional structural details of the fibrils, and we show that values for amyloid fibrils assembled from heptapeptides, α-synuclein, Aβ(1-42), insulin, β-lactoglobulin, lysozyme, ovalbumin, Tau protein and bovine serum albumin all fall in the range of 2-4 GPa.

  10. Influence of dendrimer's structure on its activity against amyloid fibril formation

    SciTech Connect

    Klajnert, B. . E-mail: aklajn@biol.uni.lodz.pl; Cortijo-Arellano, M.; Cladera, J.; Bryszewska, M.

    2006-06-23

    Inhibition of fibril assembly is a potential therapeutic strategy in neurodegenerative disorders such as prion and Alzheimer's diseases. Highly branched, globular polymers-dendrimers-are novel promising inhibitors of fibril formation. In this study, the effect of polyamidoamine (PAMAM) dendrimers (generations 3rd, 4th, and 5th) on amyloid aggregation of the prion peptide PrP 185-208 and the Alzheimer's peptide A{beta} 1-28 was examined. Amyloid fibrils were produced in vitro and their formation was monitored using the dye thioflavin T (ThT). Fluorescence studies were complemented with electron microscopy. The results show that the higher the dendrimer generation, the larger the degree of inhibition of the amyloid aggregation process and the more effective are dendrimers in disrupting the already existing fibrils. A hypothesis on dendrimer-peptide interaction mechanism is presented based on the dendrimers' molecular structure.

  11. Comparisons with Amyloid-β Reveal an Aspartate Residue That Stabilizes Fibrils of the Aortic Amyloid Peptide Medin*

    PubMed Central

    Davies, Hannah A.; Madine, Jillian; Middleton, David A.

    2015-01-01

    Aortic medial amyloid (AMA) is the most common localized human amyloid, occurring in virtually all of the Caucasian population over the age of 50. The main protein component of AMA, medin, readily assembles into amyloid-like fibrils in vitro. Despite the prevalence of AMA, little is known about the self-assembly mechanism of medin or the molecular architecture of the fibrils. The amino acid sequence of medin is strikingly similar to the sequence of the Alzheimer disease (AD) amyloid-β (Aβ) polypeptides around the structural turn region of Aβ, where mutations associated with familial, early onset AD, have been identified. Asp25 and Lys30 of medin align with residues Asp23 and Lys28 of Aβ, which are known to form a stabilizing salt bridge in some fibril morphologies. Here we show that substituting Asp25 of medin with asparagine (D25N) impedes assembly into fibrils and stabilizes non-cytotoxic oligomers. Wild-type medin, by contrast, aggregates into β-sheet-rich amyloid-like fibrils within 50 h. A structural analysis of wild-type fibrils by solid-state NMR suggests a molecular repeat unit comprising at least two extended β-strands, separated by a turn stabilized by a Asp25-Lys30 salt bridge. We propose that Asp25 drives the assembly of medin by stabilizing the fibrillar conformation of the peptide and is thus reminiscent of the influence of Asp23 on the aggregation of Aβ. Pharmacological comparisons of wild-type medin and D25N will help to ascertain the pathological significance of this poorly understood protein. PMID:25614623

  12. Comparisons with amyloid-β reveal an aspartate residue that stabilizes fibrils of the aortic amyloid peptide medin.

    PubMed

    Davies, Hannah A; Madine, Jillian; Middleton, David A

    2015-03-20

    Aortic medial amyloid (AMA) is the most common localized human amyloid, occurring in virtually all of the Caucasian population over the age of 50. The main protein component of AMA, medin, readily assembles into amyloid-like fibrils in vitro. Despite the prevalence of AMA, little is known about the self-assembly mechanism of medin or the molecular architecture of the fibrils. The amino acid sequence of medin is strikingly similar to the sequence of the Alzheimer disease (AD) amyloid-β (Aβ) polypeptides around the structural turn region of Aβ, where mutations associated with familial, early onset AD, have been identified. Asp(25) and Lys(30) of medin align with residues Asp(23) and Lys(28) of Aβ, which are known to form a stabilizing salt bridge in some fibril morphologies. Here we show that substituting Asp(25) of medin with asparagine (D25N) impedes assembly into fibrils and stabilizes non-cytotoxic oligomers. Wild-type medin, by contrast, aggregates into β-sheet-rich amyloid-like fibrils within 50 h. A structural analysis of wild-type fibrils by solid-state NMR suggests a molecular repeat unit comprising at least two extended β-strands, separated by a turn stabilized by a Asp(25)-Lys(30) salt bridge. We propose that Asp(25) drives the assembly of medin by stabilizing the fibrillar conformation of the peptide and is thus reminiscent of the influence of Asp(23) on the aggregation of Aβ. Pharmacological comparisons of wild-type medin and D25N will help to ascertain the pathological significance of this poorly understood protein. PMID:25614623

  13. 99mTc-DPD uptake reflects amyloid fibril composition in hereditary transthyretin amyloidosis

    PubMed Central

    Pilebro, Björn; Suhr, Ole B.; Näslund, Ulf; Westermark, Per; Lindqvist, Per; Sundström, Torbjörn

    2016-01-01

    Aims In transthyretin amyloid (ATTR) amyloidosis various principal phenotypes have been described: cardiac, neuropathic, or a mixed cardiac and neuropathic. In addition, two different types of amyloid fibrils have been identified (type A and type B). Type B fibrils have thus far only been found in predominantly early-onset V30M and in patients carrying the Y114C mutation, whereas type A is noted in all other mutations currently examined as well as in wild-type ATTR amyloidosis. The fibril type is a determinant of the ATTR V30M disease phenotype. 99mTc-DPD scintigraphy is a highly sensitive method for diagnosing heart involvement in ATTR amyloidosis. The objective of this study was to determine the relationship between ATTR fibril composition and 99mTc-DPD scintigraphy outcome in patients with biopsy-proven ATTR amyloidosis. Methods Altogether 55 patients with biopsy-proven diagnosis of ATTR amyloidosis and amyloid fibril composition determined were examined by 99mTc-DPD scintigraphy. The patients were grouped and compared according to their type of amyloid fibrils. Cardiovascular evaluation included ECG, echocardiography, and cardiac biomarkers. The medical records were scrutinized to identify subjects with hypertension or other diseases that have an impact on cardiac dimensions. Results A total of 97% with type A and none of the patients with type B fibrils displayed 99mTc-DPD uptake at scintigraphy (p < 0.001). Findings from analyses of cardiac biomarkers, ECG, and echocardiography, though significantly different, could not differentiate between type A and B fibrils in individual patients. Conclusion In ATTR amyloidosis, the outcome of 99mTc-DPD scintigraphy is strongly related to the patients’ transthyretin amyloid fibril composition. PMID:26849806

  14. (99m)Tc-DPD uptake reflects amyloid fibril composition in hereditary transthyretin amyloidosis.

    PubMed

    Pilebro, Björn; Suhr, Ole B; Näslund, Ulf; Westermark, Per; Lindqvist, Per; Sundström, Torbjörn

    2016-01-01

    Aims In transthyretin amyloid (ATTR) amyloidosis various principal phenotypes have been described: cardiac, neuropathic, or a mixed cardiac and neuropathic. In addition, two different types of amyloid fibrils have been identified (type A and type B). Type B fibrils have thus far only been found in predominantly early-onset V30M and in patients carrying the Y114C mutation, whereas type A is noted in all other mutations currently examined as well as in wild-type ATTR amyloidosis. The fibril type is a determinant of the ATTR V30M disease phenotype. (99m)Tc-DPD scintigraphy is a highly sensitive method for diagnosing heart involvement in ATTR amyloidosis. The objective of this study was to determine the relationship between ATTR fibril composition and (99m)Tc-DPD scintigraphy outcome in patients with biopsy-proven ATTR amyloidosis. Methods Altogether 55 patients with biopsy-proven diagnosis of ATTR amyloidosis and amyloid fibril composition determined were examined by (99m)Tc-DPD scintigraphy. The patients were grouped and compared according to their type of amyloid fibrils. Cardiovascular evaluation included ECG, echocardiography, and cardiac biomarkers. The medical records were scrutinized to identify subjects with hypertension or other diseases that have an impact on cardiac dimensions. Results A total of 97% with type A and none of the patients with type B fibrils displayed (99m)Tc-DPD uptake at scintigraphy (p < 0.001). Findings from analyses of cardiac biomarkers, ECG, and echocardiography, though significantly different, could not differentiate between type A and B fibrils in individual patients. Conclusion In ATTR amyloidosis, the outcome of (99m)Tc-DPD scintigraphy is strongly related to the patients' transthyretin amyloid fibril composition. PMID:26849806

  15. The Effect of Milk Constituents and Crowding Agents on Amyloid Fibril Formation by κ-Casein.

    PubMed

    Liu, Jihua; Dehle, Francis C; Liu, Yanqin; Bahraminejad, Elmira; Ecroyd, Heath; Thorn, David C; Carver, John A

    2016-02-17

    When not incorporated into the casein micelle, κ-casein, a major milk protein, rapidly forms amyloid fibrils at physiological pH and temperature. In this study, the effects of milk components (calcium, lactose, lipids, and heparan sulfate) and crowding agents on reduced and carboxymethylated (RCM) κ-casein fibril formation was investigated using far-UV circular dichroism spectroscopy, thioflavin T binding assays, and transmission electron microscopy. Longer-chain phosphatidylcholine lipids, which form the lining of milk ducts and milk fat globules, enhanced RCM κ-casein fibril formation irrespective of whether the lipids were in a monomeric or micellar state, whereas shorter-chain phospholipids and triglycerides had little effect. Heparan sulfate, a component of the milk fat globule membrane and catalyst of amyloid deposition in extracellular tissue, had little effect on the kinetics of RCM κ-casein fibril formation. Major nutritional components such as calcium and lactose also had no significant effect. Macromolecular crowding enhances protein-protein interactions, but in contrast to other fibril-forming species, the extent of RCM κ-casein fibril formation was reduced by the presence of a variety of crowding agents. These data are consistent with a mechanism of κ-casein fibril formation in which the rate-determining step is dissociation from the oligomer to give the highly amyloidogenic monomer. We conclude that the interaction of κ-casein with membrane-associated phospholipids along its secretory pathway may contribute to the development of amyloid deposits in mammary tissue. However, the formation of spherical oligomers such as casein micelles is favored over amyloid fibrils in the crowded environment of milk, within which the occurrence of amyloid fibrils is low. PMID:26807595

  16. Effect of initial stagger selection on the handedness of Amyloid beta helical fibrils

    SciTech Connect

    Ghattyvenkatakrishna, Pavan K; Cheng, Xiaolin; Uberbacher, Edward C

    2013-01-01

    Various structural models for Amyloid $\\beta$ fibrils derived from a variety of experimental techniques are currently available. However, this data cannot differentiate between the relative position of the two arms of the $\\beta$ hairpin called the stagger. Amyloid fibrils of various heirarchical levels form left--handed helices composed of $\\beta$ sheets. However it is unclear if positive, negative and neutral staggers all form the macroscopic left--handed helices. Studying this is important since the success of computational approaches to develop drugs for amyloidic diseases will depend on selecting the physiologically relevant structure of the sheets. To address this issue we have conducted extensive molecular dynamics simulations of Amyloid$\\beta$ sheets of various staggers and show that only negative staggers generate the experimentally observed left--handed helices while positive staggers generate the incorrect right--handed helices. The implications of this result extend in to all amyloidic--aggregation type diseases.

  17. Generating local amyloidosis in mice by the subcutaneous injection of human insulin amyloid fibrils.

    PubMed

    Chinisaz, Maryam; Ebrahim-Habibi, Azadeh; Yaghmaei, Parichehreh; Parivar, Kazem; Dehpour, Ahmad-Reza

    2014-08-01

    Localized deposits of amyloid structures are observed in various pathological conditions. One example of when local amyloidosis occurs is following repeated insulin injections in diabetic patients. The present study aimed to simulate the same condition in mice. To obtain the amyloid structures, regular insulin was incubated at 57°C for 24 h. The subsequently formed amyloid fibrils were analyzed using the Congo red absorbance test, as well as transmission electron microscopy images, and then injected into mice once per day for 21 consecutive days. Firm waxy masses were developed following this period, which were excised, prepared as thin sections and stained with hematoxylin and eosin, Congo red and Sudan black. Histological examination revealed that these masses contained adipose cells and connective tissue, in which amyloid deposition was visible. Thus, localized amyloidosis was obtained by the subcutaneous injection of insulin fibrils. The present results may be of further use in the development of models of amyloid tumors. PMID:25009591

  18. Generating local amyloidosis in mice by the subcutaneous injection of human insulin amyloid fibrils

    PubMed Central

    CHINISAZ, MARYAM; EBRAHIM-HABIBI, AZADEH; YAGHMAEI, PARICHEHREH; PARIVAR, KAZEM; DEHPOUR, AHMAD-REZA

    2014-01-01

    Localized deposits of amyloid structures are observed in various pathological conditions. One example of when local amyloidosis occurs is following repeated insulin injections in diabetic patients. The present study aimed to simulate the same condition in mice. To obtain the amyloid structures, regular insulin was incubated at 57°C for 24 h. The subsequently formed amyloid fibrils were analyzed using the Congo red absorbance test, as well as transmission electron microscopy images, and then injected into mice once per day for 21 consecutive days. Firm waxy masses were developed following this period, which were excised, prepared as thin sections and stained with hematoxylin and eosin, Congo red and Sudan black. Histological examination revealed that these masses contained adipose cells and connective tissue, in which amyloid deposition was visible. Thus, localized amyloidosis was obtained by the subcutaneous injection of insulin fibrils. The present results may be of further use in the development of models of amyloid tumors. PMID:25009591

  19. Experimentally Derived Structural Constraints for Amyloid Fibrils of Wild-Type Transthyretin

    PubMed Central

    Bateman, David A.; Tycko, Robert; Wickner, Reed B.

    2011-01-01

    Transthyretin (TTR) is a largely β-sheet serum protein responsible for transporting thyroxine and vitamin A. TTR is found in amyloid deposits of patients with senile systemic amyloidosis. TTR mutants lead to familial amyloidotic polyneuropathy and familial amyloid cardiomyopathy, with an earlier age of onset. Studies of amyloid fibrils of familial amyloidotic polyneuropathy mutant TTR suggest a structure similar to the native state with only a simple opening of a β-strand-loop-strand region exposing the two main β-sheets of the protein for fibril elongation. However, we find that the wild-type TTR sequence forms amyloid fibrils that are considerably different from the previously suggested amyloid structure. Using protease digestion with mass spectrometry, we observe the amyloid core to be primarily composed of the C-terminal region, starting around residue 50. Solid-state NMR measurements prove that TTR differs from other pathological amyloids in not having an in-register parallel β-sheet architecture. We also find that the TTR amyloid is incapable of binding thyroxine as monitored by either isothermal calorimetry or 1,8-anilinonaphthalene sulfonate competition. Taken together, our experiments are consistent with a significantly different configuration of the β-sheets compared to the previously suggested structure. PMID:22098747

  20. Experimentally derived structural constraints for amyloid fibrils of wild-type transthyretin.

    PubMed

    Bateman, David A; Tycko, Robert; Wickner, Reed B

    2011-11-16

    Transthyretin (TTR) is a largely β-sheet serum protein responsible for transporting thyroxine and vitamin A. TTR is found in amyloid deposits of patients with senile systemic amyloidosis. TTR mutants lead to familial amyloidotic polyneuropathy and familial amyloid cardiomyopathy, with an earlier age of onset. Studies of amyloid fibrils of familial amyloidotic polyneuropathy mutant TTR suggest a structure similar to the native state with only a simple opening of a β-strand-loop-strand region exposing the two main β-sheets of the protein for fibril elongation. However, we find that the wild-type TTR sequence forms amyloid fibrils that are considerably different from the previously suggested amyloid structure. Using protease digestion with mass spectrometry, we observe the amyloid core to be primarily composed of the C-terminal region, starting around residue 50. Solid-state NMR measurements prove that TTR differs from other pathological amyloids in not having an in-register parallel β-sheet architecture. We also find that the TTR amyloid is incapable of binding thyroxine as monitored by either isothermal calorimetry or 1,8-anilinonaphthalene sulfonate competition. Taken together, our experiments are consistent with a significantly different configuration of the β-sheets compared to the previously suggested structure. PMID:22098747

  1. The effects of ginsenosides to amyloid fibril formation by RCMκ-casein.

    PubMed

    Liu, Jihua; Chen, Fanbo; Yin, Jianyuan; Bu, Fengquan; Zheng, Baohua; Yang, Miao; Wang, Yunhua; Sun, Dandan; Meng, Qin

    2015-08-01

    When not incorporated into the casein micelle, isolated κ-casein spontaneously forms amyloid fibrils under physiological conditions, and is a convenient model for researching generic aspects of fibril formation. Ginsenosides have recently attracted much research interest because of the effects on aging diseases, which are always associated with amyloid fibril formation, for example, Alzheimer's, Parkinson's, and Huntington's diseases. In addition, the mechanism remains unclear that ginsenosides exert the effects against aging diseases. To address these aspects, we have investigated the ability of ginsenoside Rb1, Rc, Rg1, and Re influencing fibril formation by RCMκ-casein (reduced and carboxymethylated κ-casein), with the methods of Thioflavin T fluorescence assay, transmission electron microscopy (TEM), and intrinsic fluorescence spectroscopy. The results showed that ginsenoside Rb1 and Rg1 inhibited obviously RCMκ-CN fibrillation in both the initial rate and final level of ThT fluorescence. On the contrary, ginsenoside Re had a few effect on promoting RCMκ-CN fibril formation, proved by thick and larger fibrils observed frequently in TEM. While Rc did not influence RCMκ-CN fibrillation. It is demonstrated that Rg1 prevent RCMκ-CN fibril formation by stabilising RCMκ-CN in its native like state. Additional chemical structure difference of ginsenosides and the effects on fibril formation are also implicated. PMID:25934110

  2. Picosecond dissociation of amyloid fibrils with infrared laser: A nonequilibrium simulation study

    NASA Astrophysics Data System (ADS)

    Hoang Viet, Man; Derreumaux, Philippe; Li, Mai Suan; Roland, Christopher; Sagui, Celeste; Nguyen, Phuong H.

    2015-10-01

    Recently, mid-infrared free-electron laser technology has been developed to dissociate amyloid fibrils. Here, we present a theoretical framework for this type of experiment based on laser-induced nonequilibrium all-atom molecular dynamics simulations. We show that the fibril is destroyed due to the strong resonance between its amide I vibrational modes and the laser field. The effects of laser irradiation are determined by a balance between fibril formation and dissociation. While the overall rearrangements of the fibril finish over short time scales, the interaction between the peptides and the solvent continues over much longer times indicating that the waters play an important role in the dissociation process. Our results thus provide new insights into amyloid fibril dissociation by laser techniques and open up new venues to investigate the complex phenomena associated with amyloidogenesis.

  3. Picosecond dissociation of amyloid fibrils with infrared laser: A nonequilibrium simulation study

    SciTech Connect

    Hoang Viet, Man; Roland, Christopher Sagui, Celeste; Derreumaux, Philippe; Nguyen, Phuong H.; Li, Mai Suan

    2015-10-21

    Recently, mid-infrared free-electron laser technology has been developed to dissociate amyloid fibrils. Here, we present a theoretical framework for this type of experiment based on laser-induced nonequilibrium all-atom molecular dynamics simulations. We show that the fibril is destroyed due to the strong resonance between its amide I vibrational modes and the laser field. The effects of laser irradiation are determined by a balance between fibril formation and dissociation. While the overall rearrangements of the fibril finish over short time scales, the interaction between the peptides and the solvent continues over much longer times indicating that the waters play an important role in the dissociation process. Our results thus provide new insights into amyloid fibril dissociation by laser techniques and open up new venues to investigate the complex phenomena associated with amyloidogenesis.

  4. Supersaturation-limited and Unlimited Phase Transitions Compete to Produce the Pathway Complexity in Amyloid Fibrillation.

    PubMed

    Adachi, Masayuki; So, Masatomo; Sakurai, Kazumasa; Kardos, József; Goto, Yuji

    2015-07-17

    Although amyloid fibrils and amorphous aggregates are two types of aggregates formed by denatured proteins, their relationship currently remains unclear. We used β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, to clarify the mechanism by which proteins form either amyloid fibrils or amorphous aggregates. When ultrasonication was used to accelerate the spontaneous fibrillation of β2m at pH 2.0, the effects observed depended on ultrasonic power; although stronger ultrasonic power effectively accelerated fibrillation, excessively strong ultrasonic power decreased the amount of fibrils formed, as monitored by thioflavin T fluorescence. An analysis of the products formed indicated that excessively strong ultrasonic power generated fibrillar aggregates that retained β-structures but without high efficiency as seeds. On the other hand, when the spontaneous fibrillation of β2m was induced at higher concentrations of NaCl at pH 2.0 with stirring, amorphous aggregates became more dominant than amyloid fibrils. These apparent complexities in fibrillation were explained comprehensively by a competitive mechanism in which supersaturation-limited reactions competed with supersaturation-unlimited reactions. We link the kinetics of protein aggregation and a conformational phase diagram, in which supersaturation played important roles. PMID:26063798

  5. Fibrillation of β amyloid peptides in the presence of phospholipid bilayers and the consequent membrane disruption.

    PubMed

    Qiang, Wei; Yau, Wai-Ming; Schulte, Jürgen

    2015-01-01

    Fibrillation of β amyloid (Aβ) peptides and the accumulation of amyloid plaques are considered as an important clinical hallmark to identify Alzheimer's disease (AD). The physiological connection between Aβ plaques and the disruption of neuronal cells has not been clearly understood. One hypothesis to explain the Aβ neurotoxicity is that the fibrillation process induces disruption to the cellular membrane. We studied the Aβ fibrillation process in two biologically relevant conditions with the peptide either pre-incorporated into or externally added to the synthetic phospholipid bilayers. These two sample preparation conditions mimic the physiological membrane proximities of Aβ peptides before and after the enzymatic cleavage of amyloid precursor protein (APP). Using thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM), we were able to monitor the kinetics and morphological evolution of fibril formation, which was highly sensitive to the two sample preparation protocols. While the external addition protocol generates long and mature fibrils through normal fibrillation process, the pre-incubation protocol was found to stabilize the immature protofibrils. Fluorescence spectroscopy studies with doubly-labeled phospholipids indicated that there may be a lipid uptake process associated with the fibril formation. Solid state nuclear magnetic resonance (NMR) spectroscopy provided evidence for high resolution structural variations in fibrils formed with different protocols, and in particular the stabilization of long-range contact between N- and C-terminal β strands. In addition, disruption of phospholipid bilayers was supported by measurements with ³¹P chemical shifts and relaxation time constants. PMID:24769158

  6. Cryo-EM reveals the steric zipper structure of a light chain-derived amyloid fibril.

    PubMed

    Schmidt, Andreas; Annamalai, Karthikeyan; Schmidt, Matthias; Grigorieff, Nikolaus; Fändrich, Marcus

    2016-05-31

    Amyloid fibrils are proteinaceous aggregates associated with diseases in humans and animals. The fibrils are defined by intermolecular interactions between the fibril-forming polypeptide chains, but it has so far remained difficult to reveal the assembly of the peptide subunits in a full-scale fibril. Using electron cryomicroscopy (cryo-EM), we present a reconstruction of a fibril formed from the pathogenic core of an amyloidogenic immunoglobulin (Ig) light chain. The fibril density shows a lattice-like assembly of face-to-face packed peptide dimers that corresponds to the structure of steric zippers in peptide crystals. Interpretation of the density map with a molecular model enabled us to identify the intermolecular interactions between the peptides and rationalize the hierarchical structure of the fibril based on simple chemical principles. PMID:27185936

  7. Analysis of Toxic Amyloid Fibril Interactions at Natively Derived Membranes by Ellipsometry

    PubMed Central

    Smith, Rachel A. S.; Nabok, Aleksey; Blakeman, Ben J. F.; Xue, Wei-Feng; Abell, Benjamin; Smith, David P.

    2015-01-01

    There is an ongoing debate regarding the culprits of cytotoxicity associated with amyloid disorders. Although small pre-fibrillar amyloid oligomers have been implicated as the primary toxic species, the fibrillar amyloid material itself can also induce cytotoxicity. To investigate membrane disruption and cytotoxic effects associated with intact and fragmented fibrils, the novel in situ spectroscopic technique of Total Internal Reflection Ellipsometry (TIRE) was used. Fibril lipid interactions were monitored using natively derived whole cell membranes as a model of the in vivo environment. We show that fragmented fibrils have an increased ability to disrupt these natively derived membranes by causing a loss of material from the deposited surface when compared with unfragmented fibrils. This effect was corroborated by observations of membrane disruption in live cells, and by dye release assay using synthetic liposomes. Through these studies we demonstrate the use of TIRE for the analysis of protein-lipid interactions on natively derived lipid surfaces, and provide an explanation on how amyloid fibrils can cause a toxic gain of function, while entangled amyloid plaques exert minimal biological activity. PMID:26172440

  8. Insulin amyloid fibrillation studied by terahertz spectroscopy and other biophysical methods

    SciTech Connect

    Liu, Rui; He, Mingxia; Su, Rongxin; Yu, Yanjun; Qi, Wei; He, Zhimin

    2010-01-01

    Assembly and fibrillation of amyloid proteins are believed to play a key role in the etiology of various human diseases, including Alzheimer's, Parkinson's, Huntington's and type II diabetes. Insights into conformational changes and formation processes during amyloid fibrillation are essential for the clinical diagnosis and drug discovery. To study the changes in secondary, tertiary, quaternary structures, and the alteration in the collective vibrational mode density of states during the amyloid fibrillation, bovine insulin in 20% acetic acid was incubated at 60 {sup o}C, and its multi-level structures were followed by various biophysical techniques, including circular dichroism (CD), thioflavin T fluorescence (ThT), dynamic light scattering (DLS), electron microscopy, and terahertz (THz) absorption spectroscopy. The experimental data demonstrated a transformation of {alpha}-helix into {beta}-sheet starting at 26 h. This was followed by the aggregation of insulin, as shown by ThT binding, with a transition midpoint at 41 h, and by the bulk formation of mature aggregates after about 71 h. THz is a quick and non-invasive technique, which has the advantage of allowing the study of the conformational state of biomolecules and tissues. We first applied THz spectroscopy to study the amyloid fibrillation. At the terahertz frequency range of 0.2-2.0 THz, there was an apparent increase in both the absorbance and refractive index in THz spectra. Thus, THz is expected to provide a new way of looking into amyloid fibrillation.

  9. Effect of Sequence Variation on the Mechanical Response of Amyloid Fibrils Probed by Steered Molecular Dynamics Simulation

    PubMed Central

    Ndlovu, Hlengisizwe; Ashcroft, Alison E.; Radford, Sheena E.; Harris, Sarah A.

    2012-01-01

    The mechanical failure of mature amyloid fibers produces fragments that act as seeds for the growth of new fibrils. Fragmentation may also be correlated with cytotoxicity. We have used steered atomistic molecular dynamics simulations to study the mechanical failure of fibrils formed by the amyloidogenic fragment of human amylin hIAPP20-29 subjected to force applied in a variety of directions. By introducing systematic variations to this peptide sequence in silico, we have also investigated the role of the amino-acid sequence in determining the mechanical stability of amyloid fibrils. Our calculations show that the force required to induce mechanical failure depends on the direction of the applied stress and upon the degree of structural order present in the β-sheet assemblies, which in turn depends on the peptide sequence. The results have implications for the importance of sequence-dependent mechanical properties on seeding the growth of new fibrils and the role of breakage events in cytotoxicity. PMID:22325282

  10. Beta-amyloid fibrils of Alzheimer's disease: pathologically altered, basement membrane-associated microfibrils?

    PubMed

    Inoue, S; Kisilevsky, R

    2001-01-01

    Beta amyloid fibrils were examined in situ in the cerebral cortex of brains from patients with Alzheimer's disease using high resolution ultrastructural and immunohistochemical techniques. The main body of the fibril was identical with that of microfibrils and was made up of a core containing amyloid P component (AP), and a surface layer. Beta amyloid protein (Abeta) in the form of 1 nm wide flexible filaments was associated with the external surface of the microfibril. In cerebrovascular amyloid angiopathy the fibrils were formed at the outer surface of the vascular basement membrane. Overproduction of microfibrils has been reported at the basement membrane of "leaky" capillaries including the glomerular capillary in disease or leaky alveolar-capillary walls of normal lungs. Similarly, in Alzheimer's disease overproduction of microfibril-like beta amyloid fibrils in amyloid angiopathy coincided with breakdown of the blood-brain barrier of the cerebromicrovasculature. Thus, in the above three locations, the presence of abundant microfibrils, or microfibril-like structures, may be related to plasma which leaks out of the circulation into the adjoining vascular basement membrane. AP is an essential constituent of microfibrils and since the only site where AP is available in the cerebral cortex is in leaky microvasculature, a chronic, steady supply of AP into perivascular areas may be the cause of overproduction of microfibrils. Brain "microfibrils" may further be altered pathologically into beta amyloid fibrils by the addition of Abeta. The origin of the fibrils in senile plaques may also be the microvasculature since in the area of the plaques no source of AP is apparent. PMID:11730002

  11. Amyloid fibrils formed by the programmed cell death regulator Bcl-xL.

    PubMed

    Chenal, Alexandre; Vendrely, Charlotte; Vitrac, Heidi; Karst, Johanna C; Gonneaud, Alexis; Blanchet, Clément E; Pichard, Sylvain; Garcia, Elisabeth; Salin, Bénédicte; Catty, Patrice; Gillet, Daniel; Hussy, Nicolas; Marquette, Christel; Almunia, Christine; Forge, Vincent

    2012-01-20

    The accumulation of amyloid fibers due to protein misfolding is associated with numerous human diseases. For example, the formation of amyloid deposits in neurodegenerative pathologies is correlated with abnormal apoptosis. We report here the in vitro formation of various types of aggregates by Bcl-xL, a protein of the Bcl-2 family involved in the regulation of apoptosis. Bcl-xL forms aggregates in three states, micelles, native-like fibrils, and amyloid fibers, and their biophysical characterization has been performed in detail. Bcl-xL remains in its native state within micelles and native-like fibrils, and our results suggest that native-like fibrils are formed by the association of micelles. Formation of amyloid structures, that is, nonnative intermolecular β-sheets, is favored by the proximity of proteins within fibrils at the expense of the Bcl-xL native structure. Finally, we provide evidence of a direct relationship between the amyloid character of the fibers and the tertiary-structure stability of the native Bcl-xL. The potential causality between the accumulation of Bcl-xL into amyloid deposits and abnormal apoptosis during neurodegenerative diseases is discussed. PMID:22119486

  12. The Aβ peptide forms non-amyloid fibrils in the presence of carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Luo, Jinghui; Wärmländer, Sebastian K. T. S.; Yu, Chien-Hung; Muhammad, Kamran; Gräslund, Astrid; Pieter Abrahams, Jan

    2014-05-01

    Carbon nanotubes have specific properties that make them potentially useful in biomedicine and biotechnology. However, carbon nanotubes may themselves be toxic, making it imperative to understand how carbon nanotubes interact with biomolecules such as proteins. Here, we used NMR, CD, and ThT/fluorescence spectroscopy together with AFM imaging to study pH-dependent molecular interactions between single walled carbon nanotubes (SWNTs) and the amyloid-beta (Aβ) peptide. The aggregation of the Aβ peptide, first into oligomers and later into amyloid fibrils, is considered to be the toxic mechanism behind Alzheimer's disease. We found that SWNTs direct the Aβ peptides to form a new class of β-sheet-rich yet non-amyloid fibrils.Carbon nanotubes have specific properties that make them potentially useful in biomedicine and biotechnology. However, carbon nanotubes may themselves be toxic, making it imperative to understand how carbon nanotubes interact with biomolecules such as proteins. Here, we used NMR, CD, and ThT/fluorescence spectroscopy together with AFM imaging to study pH-dependent molecular interactions between single walled carbon nanotubes (SWNTs) and the amyloid-beta (Aβ) peptide. The aggregation of the Aβ peptide, first into oligomers and later into amyloid fibrils, is considered to be the toxic mechanism behind Alzheimer's disease. We found that SWNTs direct the Aβ peptides to form a new class of β-sheet-rich yet non-amyloid fibrils. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr00291a

  13. Mechanical properties of amyloid-like fibrils defined by secondary structures

    NASA Astrophysics Data System (ADS)

    Bortolini, C.; Jones, N. C.; Hoffmann, S. V.; Wang, C.; Besenbacher, F.; Dong, M.

    2015-04-01

    Amyloid and amyloid-like fibrils represent a generic class of highly ordered nanostructures that are implicated in some of the most fatal neurodegenerative diseases. On the other hand, amyloids, by possessing outstanding mechanical robustness, have also been successfully employed as functional biomaterials. For these reasons, physical and chemical factors driving fibril self-assembly and morphology are extensively studied - among these parameters, the secondary structures and the pH have been revealed to be crucial, since a variation in pH changes the fibril morphology and net chirality during protein aggregation. It is important to quantify the mechanical properties of these fibrils in order to help the design of effective strategies for treating diseases related to the presence of amyloid fibrils. In this work, we show that by changing pH the mechanical properties of amyloid-like fibrils vary as well. In particular, we reveal that these mechanical properties are strongly related to the content of secondary structures. We analysed and estimated the Young's modulus (E) by comparing the persistence length (Lp) - measured from the observation of TEM images by using statistical mechanics arguments - with the mechanical information provided by peak force quantitative nanomechanical property mapping (PF-QNM). The secondary structure content and the chirality are investigated by means of synchrotron radiation circular dichroism (SR-CD). Results arising from this study could be fruitfully used as a protocol to investigate other medical or engineering relevant peptide fibrils.Amyloid and amyloid-like fibrils represent a generic class of highly ordered nanostructures that are implicated in some of the most fatal neurodegenerative diseases. On the other hand, amyloids, by possessing outstanding mechanical robustness, have also been successfully employed as functional biomaterials. For these reasons, physical and chemical factors driving fibril self-assembly and morphology

  14. Atomic Details of the Interactions of Glycosaminoglycans with AmyloidFibrils.

    PubMed

    Stewart, Katie L; Hughes, Eleri; Yates, Edwin A; Akien, Geoffrey R; Huang, Teng-Yi; Lima, Marcelo A; Rudd, Timothy R; Guerrini, Marco; Hung, Shang-Cheng; Radford, Sheena E; Middleton, David A

    2016-07-13

    The amyloid plaques associated with Alzheimer's disease (AD) comprise fibrillar amyloid-β (Aβ) peptides as well as non-protein factors including glycosaminoglycan (GAG) polysaccharides. GAGs affect the kinetics and pathway of Aβ self-assembly and can impede fibril clearance; thus, they may be accessory molecules in AD. Here we report the first high-resolution details of GAG-Aβ fibril interactions from the perspective of the saccharide. Binding analysis indicated that the GAG proxy heparin has a remarkably high affinity for Aβ fibrils with 3-fold cross-sectional symmetry (3Q). Chemical synthesis of a uniformly (13)C-labeled octasaccharide heparin analogue enabled magic-angle spinning solid-state NMR of the GAG bound to 3Q fibrils, and measurements of dynamics revealed a tight complex in which all saccharide residues are restrained without undergoing substantial conformational changes. Intramolecular (13)C-(15)N dipolar dephasing is consistent with close (<5 Å) contact between GAG anomeric position(s) and one or more histidine residues in the fibrils. These data provide a detailed model for the interaction between 3Q-seeded Aβ40 fibrils and a major non-protein component of AD plaques, and they reveal that GAG-amyloid interactions display a range of affinities that critically depend on the precise details of the fibril architecture. PMID:27281497

  15. Small Liposomes Accelerate the Fibrillation of Amyloid β (1–40)*

    PubMed Central

    Terakawa, Mayu S.; Yagi, Hisashi; Adachi, Masayuki; Lee, Young-Ho; Goto, Yuji

    2015-01-01

    The deposition of amyloid β (Aβ) peptides is a pathological hallmark of Alzheimer disease. Aβ peptides were previously considered to interact specifically with ganglioside-containing membranes. Several studies have suggested that Aβ peptides also bind to phosphatidylcholine membranes, which lead to deformation of membranes and fibrillation of Aβ. Moreover, the role of membrane curvature, one type of deformation produced by binding of proteins to a membrane, in the binding and fibrillation of Aβ remains unclear. To clearly understand the relationship between the binding, consequent membrane deformation, and fibrillation of Aβ, we examined the amyloid fibrillation of Aβ-(1–40) in the presence of liposomes of various sizes. Membrane curvature increased with a decrease in the size of the liposomes. We used liposomes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine to eliminate electrostatic effects. The results obtained showed that liposomes of smaller sizes (≤50 nm) significantly accelerated the nucleation step, thereby shortening the lag time of fibrillation. On the other hand, liposomes of larger sizes decreased the amount of fibrils but did not notably affect the lag time. The morphologies of fibrils, which were monitored by total internal reflection fluorescence microscopy, atomic force microscopy, and transmission electron microscopy, revealed that the length of Aβ-(1–40) fibrils became shorter and the amount of amorphous aggregates became larger as liposomes increased in size. These results suggest that the curvature of membranes coupled with an increase in water-accessible hydrophobic regions is important for binding and concentrating Aβ monomers, leading to amyloid nucleation. Furthermore, amyloid fibrillation on membranes may compete with non-productive binding to produce amorphous aggregates. PMID:25406316

  16. Curcumin Reduces Amyloid Fibrillation of Prion Protein and Decreases Reactive Oxidative Stress

    PubMed Central

    Lin, Chi-Fen; Yu, Kun-Hua; Jheng, Cheng-Ping; Chung, Raymond; Lee, Cheng-I

    2013-01-01

    Misfolding and aggregation into amyloids of the prion protein (PrP) is responsible for the development of fatal transmissible neurodegenerative diseases. Various studies on curcumin demonstrate promise for the prevention of Alzheimer’s disease and inhibition of PrPres accumulation. To evaluate the effect of curcumin on amyloid fibrillation of prion protein, we first investigated the effect of curcumin on mouse prion protein (mPrP) in a cell-free system. Curcumin reduced the prion fibril formation significantly. Furthermore, we monitored the change in apoptosis and reactive oxygen species (ROS) level upon curcumin treatment in mouse neuroblastoma cells (N2a). Curcumin effectively rescues the cells from apoptosis and decreases the ROS level caused by subsequent co-incubation with prion amyloid fibrils. The assays in cell-free mPrP and in N2a cells of this work verified the promising effect of curcumin on the prevention of transmissible neurodegenerative diseases. PMID:25437204

  17. Destruction of amyloid fibrils by graphene through penetration and extraction of peptides

    NASA Astrophysics Data System (ADS)

    Yang, Zaixing; Ge, Cuicui; Liu, Jiajia; Chong, Yu; Gu, Zonglin; Jimenez-Cruz, Camilo A.; Chai, Zhifang; Zhou, Ruhong

    2015-11-01

    Current therapies for Alzheimer's disease (AD) can provide a moderate symptomatic reduction or delay progression at various stages of the disease, but such treatments ultimately do not arrest the advancement of AD. As such, novel approaches for AD treatment and prevention are urgently needed. We here provide both experimental and computational evidence that pristine graphene and graphene-oxide nanosheets can inhibit Aβ peptide monomer fibrillation and clear mature amyloid fibrils, thus impacting the central molecular superstructures correlated with AD pathogenesis. Our molecular dynamics simulations for the first time reveal that graphene nanosheets can penetrate and extract a large number of peptides from pre-formed amyloid fibrils; these effects seem to be related to exceptionally strong dispersion interactions between peptides and graphene that are further enhanced by strong π-π stacking between the aromatic residues of extracted Aβ peptides and the graphene surface. Atomic force microscopy images confirm these predictions by demonstrating that mature amyloid fibrils can be cut into pieces and cleared by graphene oxides. Thioflavin fluorescence assays further illustrate the detailed dynamic processes by which graphene induces inhibition of monomer aggregation and clearance of mature amyloid fibrils, respectively. Cell viability and ROS assays indicate that graphene oxide can indeed mitigate cytotoxicity of Aβ peptide amyloids. Our findings provide new insights into the underlying molecular mechanisms that define graphene-amyloid interaction and suggest that further research on nanotherapies for Alzheimer's and other protein aggregation-related diseases is warranted.Current therapies for Alzheimer's disease (AD) can provide a moderate symptomatic reduction or delay progression at various stages of the disease, but such treatments ultimately do not arrest the advancement of AD. As such, novel approaches for AD treatment and prevention are urgently needed. We

  18. Unwinding fibril formation of medin, the peptide of the most common form of human amyloid

    SciTech Connect

    Larsson, Annika; Soederberg, Linda; Westermark, Gunilla T.; Sletten, Knut; Engstroem, Ulla; Tjernberg, Lars O.; Naeslund, Jan; Westermark, Per

    2007-10-05

    Medin amyloid affects the medial layer of the thoracic aorta of most people above 50 years of age. The consequences of this amyloid are not completely known but the deposits may contribute to diseases such as thoracic aortic aneurysm and dissection or to the general diminished elasticity of blood vessels seen in elderly people. We show that the 50-amino acid residue peptide medin forms amyloid-like fibrils in vitro. With the use of Congo red staining, Thioflavin T fluorescence, electron microscopy, and a solid-phase binding assay on different synthetic peptides, we identified the last 18-19 amino acid residues to constitute the amyloid-promoting region of medin. We also demonstrate that the two C-terminal phenylalanines, previously suggested to be of importance for amyloid formation, are not required for medin amyloid formation.

  19. Conversion of non-fibrillar {beta}-sheet oligomers into amyloid fibrils in Alzheimer's disease amyloid peptide aggregation

    SciTech Connect

    Benseny-Cases, Nuria; Cocera, Mercedes; Cladera, Josep

    2007-10-05

    A{beta}(1-40) is one of the main components of the fibrils found in amyloid plaques, a hallmark of brains affected by Alzheimer's disease. It is known that prior to the formation of amyloid fibrils in which the peptide adopts a well-ordered intermolecular {beta}-sheet structure, peptide monomers associate forming low and high molecular weight oligomers. These oligomers have been previously described in electron microscopy, AFM, and exclusion chromatography studies. Their specific secondary structures however, have not yet been well established. A major problem when comparing aggregation and secondary structure determinations in concentration-dependent processes such as amyloid aggregation is the different concentration range required in each type of experiment. In the present study we used the dye Thioflavin T (ThT), Fourier-transform infrared spectroscopy, and electron microscopy in order to structurally characterize the different aggregated species which form during the A{beta}(1-40) fibril formation process. A unique sample containing 90 {mu}M peptide was used. The results show that oligomeric species which form during the lag phase of the aggregation kinetics are a mixture of unordered, helical, and intermolecular non-fibrillar {beta}-structures. The number of oligomers and the amount of non-fibrillar {beta}-structures grows throughout the lag phase and during the elongation phase these non-fibrillar {beta}-structures are transformed into fibrillar (amyloid) {beta}-structures, formed by association of high molecular weight intermediates.

  20. Surface adsorption considerations when working with amyloid fibrils in multiwell plates and Eppendorf tubes.

    PubMed

    Murray, Amber N; Palhano, Fernando L; Bieschke, Jan; Kelly, Jeffery W

    2013-11-01

    The accumulation of cross-β-sheet amyloid fibrils is the hallmark of amyloid diseases. Recently, we reported the discovery of amyloid disaggregase activities in extracts from mammalian cells and Caenorhabditis elegans. However, we have discovered a problem with the interpretation of our previous results as Aβ disaggregation in vitro. Here, we show that Aβ fibrils adsorb to the plastic surface of multiwell plates and Eppendorf tubes. This adsorption is markedly increased in the presence of complex biological mixtures subjected to a denaturing air-water interface. The time-dependent loss of thioflavin T fluorescence that we interpreted previously as disaggregation is due to increased adsorption of Aβ amyloid to the surfaces of multiwell plates and Eppendorf tubes in the presence of biological extracts. As the proteins in biological extracts denature over time at the air-water interface due to agitation/shaking, their adsorption increases, in turn promoting adsorption of amyloid fibrils. We delineate important control experiments that quantify the extent of amyloid adsorption to the surface of plastic and quartz containers. Based on the results described in this article, we conclude that our interpretation of the kinetic fibril disaggregation assay data previously reported in Bieschke et al., Protein Sci 2009;18:2231-2241 and Murray et al., Protein Sci 2010;19:836-846 is invalid when used as evidence for a disaggregase activity. Thus, we correct the two prior publications reporting that worm or mammalian cell extracts disaggregate Aβ amyloid fibrils in vitro at 37°C (see Corrigenda in this issue of Protein Science). We apologize for misinterpreting our previous data and for any confounding experimental efforts this may have caused. PMID:23963844

  1. Photo-induced inhibition of insulin amyloid fibrillation on online laser measurement

    SciTech Connect

    Liu, Rui; Su, Rongxin; Qi, Wei; He, Zhimin

    2011-06-03

    Highlights: {yields} We compare the structures of insulin upon heating with or without laser irradiation. {yields} Laser irradiation inhibits insulin fibrillation and may be of insert for mechanistic disease studies. {yields} Online laser measurements should be carefully used in the study of amyloid proteins. -- Abstract: Protein aggregation and amyloid fibrillation can lead to several serious diseases and protein drugs ineffectiveness; thus, the detection and inhibition of these processes have been of great interest. In the present study, the inhibition of insulin amyloid fibrillation by laser irradiation was investigated using dynamic light scattering (DLS), transmission electron microscopy (TEM), far-UV circular dichroism (far-UV CD), and thioflavin T (ThT) fluorescence. During heat-induced aggregation, the size distribution of two insulin solutions obtained by online and offline dynamic light scattering were different. The laser-on insulin in the presence of 0.1 M NaCl exhibited fewer fibrils than the laser-off insulin, whereas no insulin fibril under laser irradiation was observed in the absence of 0.1 M NaCl for 45 h incubation. Moreover, our CD results showed that the laser-irradiated insulin solution maintained mainly an {alpha}-helical conformation, but the laser-off insulin solution formed bulk fibrils followed by a significant increase in {beta}-sheet content for 106 h incubation. These findings provide an inhibition method for insulin amyloid fibrillation using the laser irradiation and demonstrate that the online long-time laser measurements should be carefully used in the study of amyloid proteins because they may change the original results.

  2. Destruction of {alpha}-synuclein based amyloid fibrils by a low temperature plasma jet

    SciTech Connect

    Karakas, Erdinc; Laroussi, Mounir; Munyanyi, Agatha; Greene, Lesley

    2010-10-04

    Amyloid fibrils are ordered beta-sheet aggregates that are associated with a number of neurodegenerative diseases such as Alzheimer and Parkinson. At present, there is no cure for these progressive and debilitating diseases. Here we report initial studies that indicate that low temperature atmospheric pressure plasma can break amyloid fibrils into smaller units in vitro. The plasma was generated by the 'plasma pencil', a device capable of emitting a long, low temperature plasma plume/jet. This avenue of research may facilitate the development of a plasma-based medical treatment.

  3. Depolymerization of insulin amyloid fibrils by albumin-modified magnetic fluid

    NASA Astrophysics Data System (ADS)

    Siposova, Katarina; Kubovcikova, Martina; Bednarikova, Zuzana; Koneracka, Martina; Zavisova, Vlasta; Antosova, Andrea; Kopcansky, Peter; Daxnerova, Zuzana; Gazova, Zuzana

    2012-02-01

    Pathogenesis of amyloid-related diseases is associated with the presence of protein amyloid deposits. Insulin amyloids have been reported in a patient with diabetes undergoing treatment by injection of insulin and causes problems in the production and storage of this drug and in application of insulin pumps. We have studied the interference of insulin amyloid fibrils with a series of 18 albumin magnetic fluids (MFBSAs) consisting of magnetite nanoparticles modified by different amounts of bovine serum albumin (w/w BSA/Fe3O4 from 0.005 up to 15). We have found that MFBSAs are able to destroy amyloid fibrils in vitro. The extent of fibril depolymerization was affected by nanoparticle physical-chemical properties (hydrodynamic diameter, zeta potential and isoelectric point) determined by the BSA amount present in MFBSAs. The most effective were MFBSAs with lower BSA/Fe3O4 ratios (from 0.005 to 0.1) characteristic of about 90% depolymerizing activity. For the most active magnetic fluids (ratios 0.01 and 0.02) the DC50 values were determined in the range of low concentrations, indicating their ability to interfere with insulin fibrils at stoichiometric concentrations. We assume that the present findings represent a starting point for the application of the active MFBSAs as therapeutic agents targeting insulin amyloidosis.

  4. The interplay between carbon nanomaterials and amyloid fibrils in bio-nanotechnology

    NASA Astrophysics Data System (ADS)

    Li, Chaoxu; Mezzenga, Raffaele

    2013-06-01

    Recent advances in bio-nanotechnology have not only rapidly broadened the applications and scope of hybrid nanomaterials in biological fields, but also greatly enriched the examples of ordered materials based on supramolecular self-assembly. Among eminent examples of functional nanostructured materials of undisputed impact in nanotechnology and biological environments, carbon nanomaterials (such as fullerenes, carbon nanotubes and graphene) and amyloid fibrils have attracted great attention because of their unique architectures and exceptional physical properties. Nonetheless, combination of these two classes of nanomaterials into functional hybrids is far from trivial. For example, the presence of carbon nanomaterials can offer either an inhibitory effect or promotion of amyloid fibrillation, depending on the structural architectures of carbon nanomaterials and the starting amyloid proteins/peptides considered. To date, numerous studies have been devoted to evaluating both the biological toxicity of carbon nanomaterials and their use in developing therapies for amyloidosis. At the same time, hybridization of these two classes of nanomaterials offers new possibilities for combining some of their desirable properties into nanocomposites of possible use in electronics, actuators, sensing, biomedicine and structural materials. This review describes recent developments in the hybridization of carbon nanomaterials and amyloid fibrils and discusses the current state of the art on the application of carbon nanomaterial-amyloid fibril hybrids in bio-nanotechnology.

  5. High resolution structural characterization of Aβ42 amyloid fibrils by magic angle spinning NMR.

    PubMed

    Colvin, Michael T; Silvers, Robert; Frohm, Birgitta; Su, Yongchao; Linse, Sara; Griffin, Robert G

    2015-06-17

    The presence of amyloid plaques composed of amyloid beta (Aβ) fibrils is a hallmark of Alzheimer's disease (AD). The Aβ peptide is present as several length variants with two common alloforms consisting of 40 and 42 amino acids, denoted Aβ1-40 and Aβ1-42, respectively. While there have been numerous reports that structurally characterize fibrils of Aβ1-40, very little is known about the structure of amyloid fibrils of Aβ1-42, which are considered the more toxic alloform involved in AD. We have prepared isotopically (13)C/(15)N labeled AβM01-42 fibrils in vitro from recombinant protein and examined their (13)C-(13)C and (13)C-(15)N magic angle spinning (MAS) NMR spectra. In contrast to several other studies of Aβ fibrils, we observe spectra with excellent resolution and a single set of chemical shifts, suggesting the presence of a single fibril morphology. We report the initial structural characterization of AβM01-42 fibrils utilizing (13)C and (15)N shift assignments of 38 of the 43 residues, including the backbone and side chains, obtained through a series of cross-polarization based 2D and 3D (13)C-(13)C, (13)C-(15)N MAS NMR experiments for rigid residues along with J-based 2D TOBSY experiments for dynamic residues. We find that the first ∼5 residues are dynamic and most efficiently detected in a J-based TOBSY spectrum. In contrast, residues 16-42 are easily observed in cross-polarization experiments and most likely form the amyloid core. Calculation of ψ and φ dihedral angles from the chemical shift assignments indicate that 4 β-strands are present in the fibril's secondary structure. PMID:26001057

  6. Inhibition by small-molecule ligands of formation of amyloid fibrils of an immunoglobulin light chain variable domain

    PubMed Central

    Brumshtein, Boris; Esswein, Shannon R; Salwinski, Lukasz; Phillips, Martin L; Ly, Alan T; Cascio, Duilio; Sawaya, Michael R; Eisenberg, David S

    2015-01-01

    Overproduction of immunoglobulin light chains leads to systemic amyloidosis, a lethal disease characterized by the formation of amyloid fibrils in patients' tissues. Excess light chains are in equilibrium between dimers and less stable monomers which can undergo irreversible aggregation to the amyloid state. The dimers therefore must disassociate into monomers prior to forming amyloid fibrils. Here we identify ligands that inhibit amyloid formation by stabilizing the Mcg light chain variable domain dimer and shifting the equilibrium away from the amyloid-prone monomer. DOI: http://dx.doi.org/10.7554/eLife.10935.001 PMID:26576950

  7. Nanoscale Heterogeneity of the Molecular Structure of Individual hIAPP Amyloid Fibrils Revealed with Tip-Enhanced Raman Spectroscopy.

    PubMed

    vandenAkker, Corianne C; Deckert-Gaudig, Tanja; Schleeger, Michael; Velikov, Krassimir P; Deckert, Volker; Bonn, Mischa; Koenderink, Gijsje H

    2015-09-01

    Type 2 diabetes mellitus is characterized by the pathological deposition of fibrillized protein, known as amyloids. It is thought that oligomers and/or amyloid fibrils formed from human islet amyloid polypeptide (hIAPP or amylin) cause cell death by membrane damage. The molecular structure of hIAPP amyloid fibrils is dominated by β-sheet structure, as probed with conventional infrared and Raman vibrational spectroscopy. However, with these techniques it is not possible to distinguish between the core and the surface structure of the fibrils. Since the fibril surface crucially affects amyloid toxicity, it is essential to know its structure. Here the surface molecular structure and amino acid residue composition of hIAPP fibrils are specifically probed with nanoscale resolution using tip-enhanced Raman spectroscopy (TERS). The fibril surface mainly contains unordered or α-helical structures, in contrast to the β-sheet-rich core. This experimentally validates recent models of hIAPP amyloids based on NMR measurements. Spatial mapping of the surface structure reveals a highly heterogeneous surface structure. Finally, TERS can probe fibrils formed on a lipid interface, which is more representative of amyloids in vivo. PMID:25952953

  8. β2-Microglobulin Amyloid Fibrils Are Nanoparticles That Disrupt Lysosomal Membrane Protein Trafficking and Inhibit Protein Degradation by Lysosomes*

    PubMed Central

    Jakhria, Toral; Hellewell, Andrew L.; Porter, Morwenna Y.; Jackson, Matthew P.; Tipping, Kevin W.; Xue, Wei-Feng; Radford, Sheena E.; Hewitt, Eric W.

    2014-01-01

    Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of β2-microglobulin (β2m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented β2m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented β2m fibrils did not, however, cause cell death. Instead, fragmented β2m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway. PMID:25378395

  9. What Can the Kinetics of Amyloid Fibril Formation Tell about Off-pathway Aggregation?

    PubMed

    Crespo, Rosa; Villar-Alvarez, Eva; Taboada, Pablo; Rocha, Fernando A; Damas, Ana M; Martins, Pedro M

    2016-01-22

    Some of the most prevalent neurodegenerative diseases are characterized by the accumulation of amyloid fibrils in organs and tissues. Although the pathogenic role of these fibrils has not been completely established, increasing evidence suggests off-pathway aggregation as a source of toxic/detoxicating deposits that still remains to be targeted. The present work is a step toward the development of off-pathway modulators using the same amyloid-specific dyes as those conventionally employed to screen amyloid inhibitors. We identified a series of kinetic signatures revealing the quantitative importance of off-pathway aggregation relative to amyloid fibrillization; these include non-linear semilog plots of amyloid progress curves, highly variable end point signals, and half-life coordinates weakly influenced by concentration. Molecules that attenuate/intensify the magnitude of these signals are considered promising off-pathway inhibitors/promoters. An illustrative example shows that amyloid deposits of lysozyme are only the tip of an iceberg hiding a crowd of insoluble aggregates. Thoroughly validated using advanced microscopy techniques and complementary measurements of dynamic light scattering, CD, and soluble protein depletion, the new analytical tools are compatible with the high-throughput methods currently employed in drug discovery. PMID:26601940

  10. Analysis of the length distribution of amyloid fibrils by centrifugal sedimentation.

    PubMed

    Arosio, Paolo; Cedervall, Tommy; Knowles, Tuomas P J; Linse, Sara

    2016-07-01

    The aggregation of normally soluble peptides and proteins into amyloid fibrils is a process associated with a wide range of pathological conditions, including Alzheimer's and Parkinson's diseases. It has become apparent that aggregates of different sizes possess markedly different biological effects, with aggregates of lower relative molecular weight being associated with stronger neurotoxicity. Yet, although many approaches exist to measure the total mass concentration of aggregates, the ability to probe the length distribution of growing aggregates in solution has remained more elusive. In this work, we applied a differential centrifugation technique to measure the sedimentation coefficients of amyloid fibrils produced during the aggregation process of the amyloid β (M1-42) peptide (Aβ42). The centrifugal method has the advantage of providing structural information on the fibril distribution directly in solution and affording a short analysis time with respect to alternative imaging and analytical centrifugation approaches. We show that under quiescent conditions interactions between Aβ42 fibrils lead to lateral association and to the formation of entangled clusters. By contrast, aggregation under shaking generates a population of filaments characterized by shorter lengths. The results, which have been validated by cryogenic transmission electron microscopy (cryo-TEM) analysis, highlight the important role that fibril-fibril assembly can play in the deposition of aggregation-prone peptides. PMID:27033008

  11. Simulation of Two Dimensional Ultraviolet (2DUV) Spectroscopy of Amyloid Fibrils

    PubMed Central

    Jiang, Jun; Abramavicius, Darius; Falvo, Cyril; Bulheller, Benjamin M.; Hirst, Jonathan D.; Mukamel, Shaul

    2010-01-01

    Revealing the structure and aggregation mechanism of amyloid fibrils is essential for the treatment of over 20 diseases related to protein misfolding. Coherent two dimensional (2D) infrared spectroscopy is a novel tool that provides a wealth of new insight into the structure and dynamics of biomolecular systems. Recently developed ultrafast laser sources are extending multidimensional spectroscopy into the ultraviolet (UV) region, and this opens up new opportunities for probing fibrils. In a simulation study, we show that 2DUV spectra of the backbone of a 32-residue β-amyloid (Aβ9–40) fibril associated with Alzheimer’s disease, and two intermediate prefibrillar structures carry characteristic signatures of fibril size and geometry that could be used to monitor its formation kinetics. The dependence of these signals on the fibril size and geometry is explored. We demonstrate that the dominant features of the β-amyloid fibril spectra are determined by intramolecular interactions within a single Aβ9–40, while intermolecular interactions at the “external interface” have clear signatures in the fine details of these signals. PMID:20795695

  12. Peptide p5 binds both heparinase-sensitive glycosaminoglycans and fibrils in patient-derived AL amyloid extracts

    SciTech Connect

    Martin, Emily B.; Williams, Angela; Heidel, Eric; Macy, Sallie; Kennel, Stephen J.; Wall, Jonathan S.

    2013-06-21

    Highlights: •Polybasic peptide p5 binds human light chain amyloid extracts. •The binding of p5 with amyloid involves both glycosaminoglycans and fibrils. •Heparinase treatment led to a correlation between p5 binding and fibril content. •p5 binding to AL amyloid requires electrostatic interactions. -- Abstract: In previously published work, we have described heparin-binding synthetic peptides that preferentially recognize amyloid deposits in a mouse model of reactive systemic (AA) amyloidosis and can be imaged by using positron and single photon emission tomographic imaging. We wanted to extend these findings to the most common form of visceral amyloidosis, namely light chain (AL); however, there are no robust experimental animal models of AL amyloidosis. To further define the binding of the lead peptide, p5, to AL amyloid, we characterized the reactivity in vitro of p5 with in situ and patient-derived AL amyloid extracts which contain both hypersulfated heparan sulfate proteoglycans as well as amyloid fibrils. Histochemical staining demonstrated that the peptide specifically localized with tissue-associated AL amyloid deposits. Although we anticipated that p5 would undergo electrostatic interactions with the amyloid-associated glycosaminoglycans expressing heparin-like side chains, no significant correlation between peptide binding and glycosaminoglycan content within amyloid extracts was observed. In contrast, following heparinase I treatment, although overall binding was reduced, a positive correlation between peptide binding and amyloid fibril content became evident. This interaction was further confirmed using synthetic light chain fibrils that contain no carbohydrates. These data suggest that p5 can bind to both the sulfated glycosaminoglycans and protein fibril components of AL amyloid. Understanding these complex electrostatic interactions will aid in the optimization of synthetic peptides for use as amyloid imaging agents and potentially as

  13. Evidence for Novel [beta]-Sheet Structures in Iowa Mutant [beta]-Amyloid Fibrils

    SciTech Connect

    Tycko, Robert; Sciarretta, Kimberly L.; Orgel, Joseph P.R.O.; Meredith, Stephen C.

    2009-07-24

    Asp23-to-Asn mutation within the coding sequence of {beta}-amyloid, called the Iowa mutation, is associated with early onset, familial Alzheimer's disease and cerebral amyloid angiopathy, in which patients develop neuritic plaques and massive vascular deposition predominantly of the mutant peptide. We examined the mutant peptide, D23N-A{beta}40, by electron microscopy, X-ray diffraction, and solid-state NMR spectroscopy. D23N-A{beta}40 forms fibrils considerably faster than the wild-type peptide (k = 3.77 x 10{sup -3} min{sup -1} and 1.07 x 10{sup -4} min{sup -1} for D23N-A{beta}40 and the wild-type peptide WT-A{beta}40, respectively) and without a lag phase. Electron microscopy shows that D23N-A{beta}40 forms fibrils with multiple morphologies. X-ray fiber diffraction shows a cross-{beta} pattern, with a sharp reflection at 4.7 {angstrom} and a broad reflection at 9.4 {angstrom}, which is notably smaller than the value for WT-A{beta}40 fibrils (10.4 {angstrom}). Solid-state NMR measurements indicate molecular level polymorphism of the fibrils, with only a minority of D23N-A{beta}40 fibrils containing the in-register, parallel {beta}-sheet structure commonly found in WT-A{beta}40 fibrils and most other amyloid fibrils. Antiparallel {beta}-sheet structures in the majority of fibrils are indicated by measurements of intermolecular distances through 13C-13C and 15N-13C dipole-dipole couplings. An intriguing possibility exists that there is a relationship between the aberrant structure of D23N-A{beta}40 fibrils and the unusual vasculotropic clinical picture in these patients.

  14. Molecular Structure of Amyloid Fibrils Controls the Relationship between Fibrillar Size and Toxicity

    PubMed Central

    Lee, Young Jin; Savtchenko, Regina; Ostapchenko, Valeriy G.; Makarava, Natallia; Baskakov, Ilia V.

    2011-01-01

    Background According to the prevailing view, soluble oligomers or small fibrillar fragments are considered to be the most toxic species in prion diseases. To test this hypothesis, two conformationally different amyloid states were produced from the same highly pure recombinant full-length prion protein (rPrP). The cytotoxic potential of intact fibrils and fibrillar fragments generated by sonication from these two states was tested using cultured cells. Methodology/Principal Findings For one amyloid state, fibril fragmentation was found to enhance its cytotoxic potential, whereas for another amyloid state formed within the same amino acid sequence, the fragmented fibrils were found to be substantially less toxic than the intact fibrils. Consistent with the previous studies, the toxic effects were more pronounced for cell cultures expressing normal isoform of the prion protein (PrPC) at high levels confirming that cytotoxicity was in part PrPC-dependent. Silencing of PrPC expression by small hairpin RNAs designed to silence expression of human PrPC (shRNA-PrPC) deminished the deleterious effects of the two amyloid states to a different extent, suggesting that the role of PrPC-mediated and PrPC-independent mechanisms depends on the structure of the aggregates. Conclusions/Significance This work provides a direct illustration that the relationship between an amyloid's physical dimension and its toxic potential is not unidirectional but is controlled by the molecular structure of prion protein (PrP) molecules within aggregated states. Depending on the structure, a decrease in size of amyloid fibrils can either enhance or abolish their cytotoxic effect. Regardless of the molecular structure or size of PrP aggregates, silencing of PrPC expression can be exploited to reduce their deleterious effects. PMID:21625461

  15. Protein corona composition of gold nanoparticles/nanorods affects amyloid beta fibrillation process

    NASA Astrophysics Data System (ADS)

    Mirsadeghi, Somayeh; Dinarvand, Rassoul; Ghahremani, Mohammad Hossein; Hormozi-Nezhad, Mohammad Reza; Mahmoudi, Zohreh; Hajipour, Mohammad Javad; Atyabi, Fatemeh; Ghavami, Mahdi; Mahmoudi, Morteza

    2015-03-01

    Protein fibrillation process (e.g., from amyloid beta (Aβ) and α-synuclein) is the main cause of several catastrophic neurodegenerative diseases such as Alzheimer's and Parkinson diseases. During the past few decades, nanoparticles (NPs) were recognized as one of the most promising tools for inhibiting the progress of the disease by controlling the fibrillation kinetic process; for instance, gold NPs have a strong capability to inhibit Aβ fibrillations. It is now well understood that a layer of biomolecules would cover the surface of NPs (so called ``protein corona'') upon the interaction of NPs with protein sources. Due to the fact that the biological species (e.g., cells and amyloidal proteins) ``see'' the protein corona coated NPs rather than the pristine coated particles, one should monitor the fibrillation process of amyloidal proteins in the presence of corona coated NPs (and not pristine coated ones). Therefore, the previously obtained data on NPs effects on the fibrillation process should be modified to achieve a more reliable and predictable in vivo results. Herein, we probed the effects of various gold NPs (with different sizes and shapes) on the fibrillation process of Aβ in the presence and absence of protein sources (i.e., serum and plasma). We found that the protein corona formed a shell at the surface of gold NPs, regardless of their size and shape, reducing the access of Aβ to the gold inhibitory surface and, therefore, affecting the rate of Aβ fibril formation. More specifically, the anti-fibrillation potencies of various corona coated gold NPs were strongly dependent on the protein source and their concentrations (10% serum/plasma (simulation of an in vitro milieu) and 100% serum/plasma (simulation of an in vivo milieu)).Protein fibrillation process (e.g., from amyloid beta (Aβ) and α-synuclein) is the main cause of several catastrophic neurodegenerative diseases such as Alzheimer's and Parkinson diseases. During the past few decades

  16. High Resolution Structural Characterization of Aβ42 Amyloid Fibrils by Magic Angle Spinning NMR

    PubMed Central

    2015-01-01

    The presence of amyloid plaques composed of amyloid beta (Aβ) fibrils is a hallmark of Alzheimer’s disease (AD). The Aβ peptide is present as several length variants with two common alloforms consisting of 40 and 42 amino acids, denoted Aβ1–40 and Aβ1–42, respectively. While there have been numerous reports that structurally characterize fibrils of Aβ1–40, very little is known about the structure of amyloid fibrils of Aβ1–42, which are considered the more toxic alloform involved in AD. We have prepared isotopically 13C/15N labeled AβM01–42 fibrils in vitro from recombinant protein and examined their 13C–13C and 13C–15N magic angle spinning (MAS) NMR spectra. In contrast to several other studies of Aβ fibrils, we observe spectra with excellent resolution and a single set of chemical shifts, suggesting the presence of a single fibril morphology. We report the initial structural characterization of AβM01–42 fibrils utilizing 13C and 15N shift assignments of 38 of the 43 residues, including the backbone and side chains, obtained through a series of cross-polarization based 2D and 3D 13C–13C, 13C–15N MAS NMR experiments for rigid residues along with J-based 2D TOBSY experiments for dynamic residues. We find that the first ∼5 residues are dynamic and most efficiently detected in a J-based TOBSY spectrum. In contrast, residues 16–42 are easily observed in cross-polarization experiments and most likely form the amyloid core. Calculation of ψ and φ dihedral angles from the chemical shift assignments indicate that 4 β-strands are present in the fibril’s secondary structure. PMID:26001057

  17. Large Proteins Have a Great Tendency to Aggregate but a Low Propensity to Form Amyloid Fibrils

    PubMed Central

    Ramshini, Hassan; Parrini, Claudia; Relini, Annalisa; Zampagni, Mariagioia; Mannini, Benedetta; Pesce, Alessandra; Saboury, Ali Akbar; Nemat-Gorgani, Mohsen; Chiti, Fabrizio

    2011-01-01

    The assembly of soluble proteins into ordered fibrillar aggregates with cross-β structure is an essential event of many human diseases. The polypeptides undergoing aggregation are generally small in size. To explore if the small size is a primary determinant for the formation of amyloids under pathological conditions we have created two databases of proteins, forming amyloid-related and non-amyloid deposits in human diseases, respectively. The size distributions of the two protein populations are well separated, with the systems forming non-amyloid deposits appearing significantly larger. We have then investigated the propensity of the 486-residue hexokinase-B from Saccharomyces cerevisiae (YHKB) to form amyloid-like fibrils in vitro. This size is intermediate between the size distributions of amyloid and non-amyloid forming proteins. Aggregation was induced under conditions known to be most effective for amyloid formation by normally globular proteins: (i) low pH with salts, (ii) pH 5.5 with trifluoroethanol. In both situations YHKB aggregated very rapidly into species with significant β-sheet structure, as detected using circular dichroism and X-ray diffraction, but a weak Thioflavin T and Congo red binding. Moreover, atomic force microscopy indicated a morphology distinct from typical amyloid fibrils. Both types of aggregates were cytotoxic to human neuroblastoma cells, as indicated by the MTT assay. This analysis indicates that large proteins have a high tendency to form toxic aggregates, but low propensity to form regular amyloid in vivo and that such a behavior is intrinsically determined by the size of the protein, as suggested by the in vitro analysis of our sample protein. PMID:21249193

  18. Mechanisms of transthyretin amyloidogenesis. Antigenic mapping of transthyretin purified from plasma and amyloid fibrils and within in situ tissue localizations.

    PubMed Central

    Gustavsson, A.; Engström, U.; Westermark, P.

    1994-01-01

    Transthyretin (TTR) is the major amyloid fibril protein in senile systemic amyloidosis and in several forms of familial amyloidoses. However, the internal organization of the fibrils is virtually unknown. It is not known whether the structure of the TTR molecules is substantially altered within the fibrils. In this study we used various antigenic mapping procedures to determine whether major antigenic sites differ between normal TTR, ATTR (TTR from amyloid fibrils), and in situ amyloid fibrils. Antigenic mapping was achieved using standard immunological procedures (ie, ELISA, Western blot, and immunohistochemistry), synthetic peptides of the TTR molecule, antisera against these synthetic peptides and against normal TTR, ATTR, and alkali-degraded amyloid fibrils. Our results show that the antigenic sites on normal plasma TTR include the AB loop and the CD loop. The amino acid sequences associated with these loops are present on the outside of the TTR molecule. Antiserum against beta-strand H reacted only with TTR in amyloid fibrils and ATTR but not with normal plasma TTR or TTR in the islets of Langerhans. Our results suggest that there is an altered configuration of TTR within amyloid fibrils when compared with plasma TTR. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:8203468

  19. Evaluating Nuclei Concentration in Amyloid Fibrillation Reactions Using Back-Calculation Approach

    PubMed Central

    Sorci, Mirco; Silkworth, Whitney; Gehan, Timothy; Belfort, Georges

    2011-01-01

    Background In spite of our extensive knowledge of the more than 20 proteins associated with different amyloid diseases, we do not know how amyloid toxicity occurs or how to block its action. Recent contradictory reports suggest that the fibrils and/or the oligomer precursors cause toxicity. An estimate of their temporal concentration may broaden understanding of the amyloid aggregation process. Methodology/Principal Findings Assuming that conversion of folded protein to fibril is initiated by a nucleation event, we back-calculate the distribution of nuclei concentration. The temporal in vitro concentration of nuclei for the model hormone, recombinant human insulin, is estimated to be in the picomolar range. This is a conservative estimate since the back-calculation method is likely to overestimate the nuclei concentration because it does not take into consideration fibril fragmentation, which would lower the amount of nuclei Conclusions Because of their propensity to form aggregates (non-ordered) and fibrils (ordered), this very low concentration could explain the difficulty in isolating and blocking oligomers or nuclei toxicity and the long onset time for amyloid diseases. PMID:21625464

  20. Self-assembly of Amyloid Fibrils in One, Two and Three Dimensions

    NASA Astrophysics Data System (ADS)

    Mezzenga, Raffaele

    2015-03-01

    Amyloid fibrils are protein aggregates, which occur in-vivo in the case of neurodegenerative diseases and in-vitro in the design of advanced functional materials of relevance in nanotechnology and nanosciences. At length scales above the well-established atomistic fingerprint of amyloid fibrils, these colloidal aggregates exhibit mesoscopic properties comparable to those of natural polyelectrolytes, yet with persistence lengths several orders of magnitude beyond the Debye length. This intrinsic rigidity, together with their chiral, polar and charged nature, provides these systems with some unique physical behavior in one, two and three dimensions. In this talk I will discuss our current understanding on the mesoscopic properties of amyloid fibrils at the single molecule level, the implication of their semiflexible nature on their liquid crystalline properties, and I will illustrate how this information proves useful in understanding their collective behavior in bulk and when adsorbed at liquid interfaces. By the careful exploitation of the physical properties of amyloid fibrils, the design of advanced materials with unprecedented physical properties becomes possible, and I will give a few examples on how these systems can ideally suit the design of biosensors and biomaterials.

  1. Inhibitory effects of β-ionone on amyloid fibril formation of β-lactoglobulin.

    PubMed

    Ma, Baoliang; You, Xiong; Lu, Fujiao

    2014-03-01

    β-Lactoglobulin (β-LG) is the major constituent of whey food, which has been shown to interact with a wide range of aroma compounds. In the present work, a model aroma compound, β-ionone, is used to investigate the influence of aroma compounds on the urea-induced unfolding of β-LG at pH 7.0. β-Ionone is observed to enhance the stability of β-LG at pH 7.0. Moreover, the amyloid fibrils are observed when β-LG at pH 7.0 is incubated for 12-20 days at 37 °C in the presence of 3-5M urea. However, the formation of amyloid fibrils is inhibited when β-ionone is added into the samples and the inhibitory effects follow a concentration-dependent fashion. There is a clear correlation between Cm and lag time. The correlation demonstrates that protein stability affects the amyloid fibril formation of β-LG. The results highlight the critical role of protein stability and provide an approach to prevent the formation of amyloid fibrils in vitro. PMID:24325860

  2. Quantum dots induce charge-specific amyloid-like fibrillation of insulin at physiological conditions

    NASA Astrophysics Data System (ADS)

    Sukhanova, Alyona; Poly, Simon; Shemetov, Anton; Nabiev, Igor R.

    2012-10-01

    Agglomeration of some proteins may give rise to aggregates that have been identified as the main cause of amyloid diseases. For example, fibrillation of insulin is related to diabetes mellitus. Quantum dots (QDs) are of special interest as tagging agents for diagnostic and therapeutic studies due to their broad absorption spectra, narrow emission spectra, and high photostability. In this study, PEGylated CdSe/ZnS QDs have been shown to induce the formation of amyloid-like fibrils of human insulin under physiological conditions, this process being dependent on the variation of the surface charge of the nanoparticles (NPs) used. Circular dichroism (CD), protein secondary structure analysis, thioflavin T (ThT) fluorescence assay, and the dynamic light scattering (DLS) technique have been used for comparative analysis of different stages of the fibrillation process. In particular, insulin secondary structure remodelling accompanied by a considerable increase in the rate of amyloid fiber formation have been observed after insulin was mixed with PEGylated QDs. Nanoparticles may significantly influence the rate of protein fibrillation and induce new mechanisms of amyloid diseases, as well as offer opportunities for their treatment.

  3. Amyloid fibril formation requires a chemically discriminating nucleation event: studies of an amyloidogenic sequence from the bacterial protein OsmB.

    PubMed

    Jarrett, J T; Lansbury, P T

    1992-12-15

    The sequence of the Escherichia coli OsmB protein was found to resemble that of the C-terminal region of the beta amyloid protein of Alzheimer's disease, which seems to be the major determinant of its unusual structural and solubility properties. A peptide corresponding to residues 28-44 of the OsmB protein was synthesized, and its conformational properties and aggregation behavior were analyzed. The peptide OsmB(28-44) was shown to form amyloid fibrils, as did two sequence analogs designed to test the sequence specificity of fibril formation. These fibrils bound Congo red, and two of the peptides showed birefringence. The peptide fibrils were analyzed by electron microscopy and Fourier transform infrared spectroscopy. Subtle differences were observed which were not interpretable at the molecular level. The rate of fibril formation by each peptide was followed by monitoring the turbidity of supersaturated aqueous solutions. The kinetics of aggregation were characterized by a delay period during which the solution remained clear, followed by a nucleation event which led to a growth phase, during which the solution became viscous and turbid due to the presence of insoluble fibrils. The observation of a kinetic barrier to aggregation is typical of a crystallization event. The delay period could be eliminated by seeding the supersaturated solution with previously formed fibrils. Each peptide could be nucleated by fibrils formed from that same peptide, but not by fibrils from closely related sequences, suggesting that fibril growth requires specific hydrophobic interactions. It appears likely that this repeated sequence motif, which comprises most of the OsmB protein sequence, dictates the structure and possibly the function of that protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1463722

  4. Fibrillar Oligomers Nucleate the Oligomerization of Monomeric Amyloid β but Do Not Seed Fibril Formation*

    PubMed Central

    Wu, Jessica W.; Breydo, Leonid; Isas, J. Mario; Lee, Jerome; Kuznetsov, Yurii G.; Langen, Ralf; Glabe, Charles

    2010-01-01

    Soluble amyloid oligomers are potent neurotoxins that are involved in a wide range of human degenerative diseases, including Alzheimer disease. In Alzheimer disease, amyloid β (Aβ) oligomers bind to neuronal synapses, inhibit long term potentiation, and induce cell death. Recent evidence indicates that several immunologically distinct structural variants exist as follows: prefibrillar oligomers (PFOs), fibrillar oligomers (FOs), and annular protofibrils. Despite widespread interest, amyloid oligomers are poorly characterized in terms of structural differences and pathological significance. FOs are immunologically related to fibrils because they react with OC, a conformation-dependent, fibril-specific antibody and do not react with antibodies specific for other types of oligomers. However, fibrillar oligomers are much smaller than fibrils. FOs are soluble at 100,000 × g, rich in β-sheet structures, but yet bind weakly to thioflavin T. EPR spectroscopy indicates that FOs display significantly more spin-spin interaction at multiple labeled sites than PFOs and are more structurally similar to fibrils. Atomic force microscopy indicates that FOs are approximately one-half to one-third the height of mature fibrils. We found that Aβ FOs do not seed the formation of thioflavin T-positive fibrils from Aβ monomers but instead seed the formation of FOs from Aβ monomers that are positive for the OC anti-fibril antibody. These results indicate that the lattice of FOs is distinct from the fibril lattice even though the polypeptide chains are organized in an immunologically identical conformation. The FOs resulting from seeded reactions have the same dimensions and morphology as the initial seeds, suggesting that the seeds replicate by growing to a limiting size and then splitting, indicating that their lattice is less stable than fibrils. We suggest that FOs may represent small pieces of single fibril protofilament and that the addition of monomers to the ends of FOs is

  5. Methionine oxidation of amyloid peptides by peroxovanadium complexes: inhibition of fibril formation through a distinct mechanism.

    PubMed

    He, Lei; Wang, Xuesong; Zhu, Dengsen; Zhao, Cong; Du, Weihong

    2015-12-01

    Fibril formation of amyloid peptides is linked to a number of pathological states. The prion protein (PrP) and amyloid-β (Aβ) are two remarkable examples that are correlated with prion disorders and Alzheimer's disease, respectively. Metal complexes, such as those formed by platinum and ruthenium compounds, can act as inhibitors against peptide aggregation primarily through metal coordination. This study revealed the inhibitory effect of two peroxovanadium complexes, (NH4)[VO(O2)2(bipy)]·4H2O (1) and (NH4)[VO(O2)2(phen)]·2H2O (2), on amyloid fibril formation of PrP106-126 and Aβ1-42via site-specific oxidation of methionine residues, besides direct binding of the complexes with the peptides. Complexes 1 and 2 showed higher anti-amyloidogenic activity on PrP106-126 aggregation than on Aβ1-42, though their regulation on the cytotoxicity induced by the two peptides could not be differentiated. The action efficacy may be attributed to the different molecular structures of the vanadium complex and the peptide sequence. Results reflected that methionine oxidation may be a crucial action mode in inhibiting amyloid fibril formation. This study offers a possible application value for peroxovanadium complexes against amyloid proteins. PMID:26444976

  6. Chiral recognition in amyloid fiber growth.

    PubMed

    Torbeev, Vladimir; Grogg, Marcel; Ruiz, Jérémy; Boehringer, Régis; Schirer, Alicia; Hellwig, Petra; Jeschke, Gunnar; Hilvert, Donald

    2016-05-01

    Insoluble amyloid fibers represent a pathological signature of many human diseases. To treat such diseases, inhibition of amyloid formation has been proposed as a possible therapeutic strategy. d-Peptides, which possess high proteolytic stability and lessened immunogenicity, are attractive candidates in this context. However, a molecular understanding of chiral recognition phenomena for d-peptides and l-amyloids is currently incomplete. Here we report experiments on amyloid growth of individual enantiomers and their mixtures for two distinct polypeptide systems of different length and structural organization: a 44-residue covalently-linked dimer derived from a peptide corresponding to the [20-41]-fragment of human β2-microglobulin (β2m) and the 99-residue full-length protein. For the dimeric [20-41]β2m construct, a combination of electron paramagnetic resonance of nitroxide-labeled constructs and (13) C-isotope edited FT-IR spectroscopy of (13) C-labeled preparations was used to show that racemic mixtures precipitate as intact homochiral fibers, i.e. undergo spontaneous Pasteur-like resolution into a mixture of left- and right-handed amyloids. In the case of full-length β2m, the presence of the mirror-image d-protein affords morphologically distinct amyloids that are composed largely of enantiopure domains. Removal of the l-component from hybrid amyloids by proteolytic digestion results in their rapid transformation into characteristic long straight d-β2m amyloids. Furthermore, the full-length d-enantiomer of β2m was found to be an efficient inhibitor of l-β2m amyloid growth. This observation highlights the potential of longer d-polypeptides for future development into inhibitors of amyloid propagation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:26929241

  7. Islet amyloid polypeptide and high hydrostatic pressure: towards an understanding of the fibrillization process

    NASA Astrophysics Data System (ADS)

    Lopes, D. H. J.; Smirnovas, V.; Winter, R.

    2008-07-01

    Type II Diabetes Mellitus is a disease which is characterized by peripheral insulin resistance coupled with a progressive loss of insulin secretion that is associated with a decrease in pancreatic islet β-cell mass and the deposition of amyloid in the extracellular matrix of β-cells, which lead to islet cell death. The principal component of the islet amyloid is a pancreatic hormone called islet amyloid polypeptide (IAPP). High-pressure coupled with FT-IR, CD, ThT fluorescence spectroscopic and AFM studies were carried out to reveal information on the aggregation pathway as well as the aggregate structure of IAPP. Our data indicate that IAPP pre-formed fibrils exhibit a strong polymorphism with heterogeneous structures very sensitive to high hydrostatic pressure, indicating a high percentage of ionic and hydrophobic interactions being responsible for the stability the IAPP fibrils.

  8. Measurements of 13C multiple-quantum coherences in amyloid fibrils under magic-angle spinning.

    PubMed

    Chou, Fang-Chieh; Tsai, Tim W T; Cheng, Hsin-Mei; Chan, Jerry C C

    2012-06-21

    The excitation and detection of high-order multiple quantum coherences among (13)C nuclear spins are demonstrated in the samples of [1-(13)C]-L-alanine and (13)C labeled amyloid fibrils at a spinning frequency of 20 kHz. The technique is based on the double-quantum average Hamiltonian prepared by the DRAMA-XY4 pulse sequence. Empirically, we find that multiple supercycles are required to suppress the higher-order effects for real applications. Measurements for the fibril samples formed by the polypeptides of PrP(113-127) provide the first solid-state NMR evidence for the stacking of multiple β-sheet layers at the structural core of amyloid fibrils. PMID:22632418

  9. A folded and functional protein domain in an amyloid-like fibril

    PubMed Central

    Sackewitz, Mirko; von Einem, Sabrina; Hause, Gerd; Wunderlich, Michael; Schmid, Franz-Xaver; Schwarz, Elisabeth

    2008-01-01

    The effect of the polypeptide environment on polyalanine-induced fibril formation was investigated with amyloidogenic fragments from PAPBN1, a nuclear protein controlling polyadenylation. Mutation-caused extensions of the natural 10 alanine sequence up to maximally 17 alanines result in fibril formation of PABPN1 and the development of the disease oculopharyngeal muscular dystrophy (OPMD). We explored the influence of fibril formation on the structure and function of a one-domain protein linked to the fibril-forming part of PABPN1. The well-characterized, stably folded, one-domain protein, cold-shock protein CspB from Bacillus subtilis, was fused either to the C terminus of the entire N-terminal domain of PABPN1 or directly to peptides consisting of 10 or 17 alanine residues. The fusion protein between the N-terminal domain of PABPN1 and CspB formed fibrils in which the structure and activity of CspB were retained. In the fibrils formed by fusions in which the polyalanine sequence was directly linked to CspB, CspB was unfolded. These results indicate that the folded conformation and the function of a protein domain can be maintained in amyloid-like fibrils, and that the distance between this domain and the fibril plays an important role. PMID:18424511

  10. Guanidine hydrochloride can induce amyloid fibril formation from hen egg-white lysozyme.

    PubMed

    Vernaglia, Brian A; Huang, Jia; Clark, Eliana D

    2004-01-01

    The formation of amyloid fibrils is an intractable problem in which normally soluble protein polymerizes and forms insoluble ordered aggregates. Such aggregates can range from being a nuisance in vitro to being toxic in vivo. The latter is true for lysozyme, which has been shown to form toxic deposits in humans. In the present study, the effects of partial denaturation of hen egg-white lysozyme via incubation in a concentrated solution of the denaturant guanidine hydrochloride are investigated. Results show that when lysozyme is incubated under moderate guanidine hydrochloride concentrations (i.e., 2-5 M), where lysozyme is partially unfolded, fibrils form rapidly. Thioflavin T, Congo red, X-ray diffraction, transmission electron microscopy, atomic force microscopy, and circular dichroism spectroscopy are all used to verify the production of fibrils under these conditions. Incubation at very low or very high guanidine hydrochloride concentrations fails to produce fibrils. At very low denaturant concentrations, the structure of lysozyme is fully native and very stable. On the other hand, at very high denaturant concentrations, guanidine hydrochloride is capable of dissolving and dis-aggregating fibrils that are formed. Raising the temperature and/or concentration of lysozyme accelerates fibril formation by further adding to the concentration of partially unfolded species. The addition of preformed fibrils also accelerates fibril formation but only under partially unfolding conditions. The results presented here provide further evidence that partial unfolding is a prerequisite to fibril formation. Partial denaturation can accelerate fibril formation in much the same way that mutations have been shown to accelerate fibril formation. PMID:15244452

  11. Rapid amyloid fibril formation by a winter flounder antifreeze protein requires specific interaction with ice.

    PubMed

    Dubé, André; Leggiadro, Cindy; Ewart, Kathryn Vanya

    2016-05-01

    A typically α-helical antifreeze protein (wflAFP-6) from winter flounder, Pseudopleuronectes americanus, forms amyloid fibrils during freezing. In this study, the effects of distinct components of the freezing process were examined. Freezing of wflAFP-6 in the presence of template ice was shown to be necessary for rapid conversion to an amyloid conformation. Neither subfreezing temperature nor phase change was sufficient. Thus, specific interaction with the ice surface was essential. The ice-induced formation of amyloid appeared to be unique to this helical antifreeze, it required high concentrations of protein and it occurred over a range of pH values. These results define a method for rapid formation of amyloid by wflAFP-6 on demand under physiological conditions. PMID:27086686

  12. Elucidating the Locking Mechanism of Peptides onto Growing Amyloid Fibrils through Transition Path Sampling

    PubMed Central

    Schor, Marieke; Vreede, Jocelyne; Bolhuis, Peter G.

    2012-01-01

    We investigate the molecular mechanism of monomer addition to a growing amyloid fibril composed of the main amyloidogenic region from the insulin peptide hormone, the LVEALYLLVEALYL heptapeptide. Applying transition path sampling in combination with reaction coordinate analysis reveals that the transition from a docked peptide to a locked, fully incorporated peptide can occur in two ways. Both routes involve the formation of backbone hydrogen bonds between the three central amino acids of the attaching peptide and the fibril, as well as a reorientation of the central Glu side chain of the locking peptide toward the interface between two β-sheets forming the fibril. The mechanisms differ in the sequence of events. We also conclude that proper docking is important for correct alignment of the peptide with the fibril, as alternative pathways result in misfolding. PMID:22995502

  13. pH-Driven Polymorphism of Insulin Amyloid-Like Fibrils

    PubMed Central

    Sneideris, Tomas; Darguzis, Domantas; Botyriute, Akvile; Grigaliunas, Martynas; Winter, Roland; Smirnovas, Vytautas

    2015-01-01

    Prions are infective proteins, which can self-assemble into different strain conformations, leading to different disease phenotypes. An increasing number of studies suggest that prion-like self-propagation may be a common feature of amyloid-like structures. Thus it is important to unravel every possible factor leading to the formation of different amyloid strains. Here we report on the formation of two types of insulin amyloid-like fibrils with distinct infrared spectroscopic features grown under slightly different pH conditions. Similar to prion strains, both insulin fibril types are able to self-propagate their conformational template under conditions, favoring spontaneous formation of different type fibrils. The low-pH-induced insulin amyloid strain is structurally very similar to previously reported strains formed either in the presence of 20% ethanol, or by modification of the amino acid sequence of insulin. A deeper analysis of literature data in the context of our current findings suggests a shift of the monomer-dimer equilibrium of insulin as a possible factor controlling the formation of different strains. PMID:26313643

  14. On the Heat Stability of Amyloid-Based Biological Activity: Insights from Thermal Degradation of Insulin Fibrils

    PubMed Central

    Surmacz-Chwedoruk, Weronika; Malka, Iwona; Bożycki, Łukasz; Nieznańska, Hanna; Dzwolak, Wojciech

    2014-01-01

    Formation of amyloid fibrils in vivo has been linked to disorders such as Alzheimer’s disease and prion-associated transmissible spongiform encephalopathies. One of the characteristic features of amyloid fibrils is the high thermodynamic stability relative both to native and disordered states which is also thought to underlie the perplexingly remarkable heat resistance of prion infectivity. Here, we are comparing high-temperature degradation of native and fibrillar forms of human insulin. Decomposition of insulin amyloid has been studied under helium atmosphere and in the temperature range from ambient conditions to 750°C using thermogravimetry and differential scanning calorimetry coupled to mass spectrometry. While converting native insulin into amyloid does upshift onset of thermal decomposition by ca. 75°C, fibrils remain vulnerable to covalent degradation at temperatures below 300°C, as reflected by mass spectra of gases released upon heating of amyloid samples, as well as morphology and infrared spectra of fibrils subjected to incubation at 250°C. Mass spectra profiles of released gases indicate that degradation of fibrils is much more cooperative than degradation of native insulin. The data show no evidence of water of crystallization trapped within insulin fibrils. We have also compared untreated and heated amyloid samples in terms of capacity to seed daughter fibrils. Kinetic traces of seed-induced insulin fibrillation have shown that the seeding potency of amyloid samples decreases significantly already after exposure to 200°C, even though corresponding electron micrographs indicated persisting fibrillar morphology. Our results suggest that amyloid-based biological activity may not survive extremely high temperature treatments, at least in the absence of other stabilizing factors. PMID:24466022

  15. Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma

    PubMed Central

    Purkey, Hans E.; Dorrell, Michael I.; Kelly, Jeffery W.

    2001-01-01

    Transthyretin (TTR) tetramer dissociation and misfolding facilitate assembly into amyloid fibrils that putatively cause senile systemic amyloidosis and familial amyloid polyneuropathy. We have previously discovered more than 50 small molecules that bind to and stabilize tetrameric TTR, inhibiting amyloid fibril formation in vitro. A method is presented here to evaluate the binding selectivity of these inhibitors to TTR in human plasma, a complex biological fluid composed of more than 60 proteins and numerous small molecules. Our immunoprecipitation approach isolates TTR and bound small molecules from a biological fluid such as plasma, and quantifies the amount of small molecules bound to the protein by HPLC analysis. This approach demonstrates that only a small subset of the inhibitors that saturate the TTR binding sites in vitro do so in plasma. These selective inhibitors can now be tested in animal models of TTR amyloid disease to probe the validity of the amyloid hypothesis. This method could be easily extended to evaluate small molecule binding selectivity to any protein in a given biological fluid without the necessity of determining or guessing which other protein components may be competitors. This is a central issue to understanding the distribution, metabolism, activity, and toxicity of potential drugs. PMID:11344299

  16. Structure-Based Design of Non-Natural Amino Acid Inhibitors of Amyloid Fibrillation

    PubMed Central

    Sievers, Stuart A.; Karanicolas, John; Chang, Howard W.; Zhao, Anni; Jiang, Lin; Zirafi, Onofrio; Stevens, Jason T.; Münch, Jan; Baker, David; Eisenberg, David

    2014-01-01

    Many globular and natively disordered proteins can convert into amyloid fibers. These fibers are associated with numerous pathologies1 as well as with normal cellular functions2,3, and frequently form during protein denaturation4,5. Inhibitors of pathological amyloid fibers could serve as leads for therapeutics, provided the inhibitors were specific enough to avoid interfering with normal processes. Here we show that computer-aided, structure-based design can yield highly specific peptide inhibitors of amyloid formation. Using known atomic structures of segments of amyloid fibers as templates, we have designed and characterized an all D-amino acid inhibitor of fibrillation of the tau protein found in Alzheimer’s disease, and a non-natural L-amino acid inhibitor of an amyloid fiber that enhances sexual transmission of HIV. Our results indicate that peptides from structure-based designs can disrupt the fibrillation of full-length proteins, including those like tau that lack fully ordered native structures. PMID:21677644

  17. Gallic Acid Is an Antagonist of Semen Amyloid Fibrils That Enhance HIV-1 Infection.

    PubMed

    LoRicco, Josephine G; Xu, Changmingzi Sherry; Neidleman, Jason; Bergkvist, Magnus; Greene, Warner C; Roan, Nadia R; Makhatadze, George I

    2016-07-01

    Recent in vitro studies have demonstrated that amyloid fibrils found in semen from healthy and HIV-infected men, as well as semen itself, can markedly enhance HIV infection rates. Semen fibrils are made up of multiple naturally occurring peptide fragments derived from semen. The best characterized of these fibrils are SEVI (semen-derived enhancer of viral infection), made up of residues 248-286 of prostatic acidic phosphatase, and the SEM1 fibrils, made up of residues 86-107 of semenogelin 1. A small molecule screen for antagonists of semen fibrils identified four compounds that lowered semen-mediated enhancement of HIV-1 infectivity. One of the four, gallic acid, was previously reported to antagonize other amyloids and to exert anti-inflammatory effects. To better understand the mechanism by which gallic acid modifies the properties of semen amyloids, we performed biophysical measurements (atomic force microscopy, electron microscopy, confocal microscopy, thioflavin T and Congo Red fluorescence assays, zeta potential measurements) and quantitative assays on the effects of gallic acid on semen-mediated enhancement of HIV infection and inflammation. Our results demonstrate that gallic acid binds to both SEVI and SEM1 fibrils and modifies their surface electrostatics to render them less cationic. In addition, gallic acid decreased semen-mediated enhancement of HIV infection but did not decrease the inflammatory response induced by semen. Together, these observations identify gallic acid as a non-polyanionic compound that inhibits semen-mediated enhancement of HIV infection and suggest the potential utility of incorporating gallic acid into a multicomponent microbicide targeting both the HIV virus and host components that promote viral infection. PMID:27226574

  18. Engineered polymer nanoparticles containing hydrophobic dipeptide for inhibition of amyloidfibrillation.

    PubMed

    Skaat, Hadas; Chen, Ravit; Grinberg, Igor; Margel, Shlomo

    2012-09-10

    Protein aggregation into amyloid fibrils is implicated in the pathogenesis of many neurodegenerative diseases. Engineered nanoparticles have emerged as a potential approach to alter the kinetics of protein fibrillation process. Yet, there are only a few reports describing the use of nanoparticles for inhibition of amyloid-β 40 (Aβ(40)) peptide aggregation, involved in Alzheimer's disease (AD). In the present study, we designed new uniform biocompatible amino-acid-based polymer nanoparticles containing hydrophobic dipeptides in the polymer side chains. The dipeptide residues were designed similarly to the hydrophobic core sequence of Aβ. Poly(N-acryloyl-L-phenylalanyl-L-phenylalanine methyl ester) (polyA-FF-ME) nanoparticles of 57 ± 6 nm were synthesized by dispersion polymerization of the monomer A-FF-ME in 2-methoxy ethanol, followed by precipitation of the obtained polymer in aqueous solution. Cell viability assay confirmed that no significant cytotoxic effect of the polyA-FF-ME nanoparticles on different human cell lines, e.g., PC-12 and SH-SY5Y, was observed. A significantly slow secondary structure transition from random coil to β-sheets during Aβ(40) fibril formation was observed in the presence of these nanoparticles, resulting in significant inhibition of Aβ(40) fibrillation kinetics. However, the polyA-FF-ME analogous nanoparticles containing the L-alanyl-L-alanine (AA) dipeptide in the polymer side groups, polyA-AA-ME nanoparticles, accelerate the Aβ(40) fibrillation kinetics. The polyA-FF-ME nanoparticles and the polyA-AA-ME nanoparticles may therefore contribute to a mechanistic understanding of the fibrillation process, leading to the development of therapeutic strategies against amyloid-related diseases. PMID:22897679

  19. Binding Modes of Thioflavin T on the Surface of Amyloid Fibrils Studied by NMR.

    PubMed

    Ivancic, Valerie A; Ekanayake, Oshini; Lazo, Noel D

    2016-08-18

    The mechanism for the interaction of thioflavin T (ThT) with amyloid fibrils at the molecular level is not known. Here, we used (1) H NMR spectroscopy to determine the binding mode of ThT on the surface of fibrils from lysozyme and insulin. Relayed rotating-frame Overhauser enhancements in ThT were observed, indicating that the orientation of ThT is orthogonal to the fibril surface. Importantly, the assembly state of ThT on both surfaces is different. On the surface of insulin fibrils, ThT is oligomeric, as indicated by rapid (1) H spin-lattice relaxation rate in the rotating frame (R1ρ ), presumably due to intermolecular dipole-dipole interactions between ThT molecules. In contrast, ThT on the surface of lysozyme fibrils is a monomer, as indicated by slower (1) H R1ρ . These results shed new light into the mechanism for the enhancement of ThT fluorescence and may lead to more efficient detectors of amyloid assemblies, which have escaped detection by ThT in monomer form. PMID:27165642

  20. Structural and Mechanical Properties of Amyloid Beta Fibrils: A Combined Experimental and Theoretical Approach.

    PubMed

    Paul, Thomas J; Hoffmann, Zachary; Wang, Congzhou; Shanmugasundaram, Maruda; DeJoannis, Jason; Shekhtman, Alexander; Lednev, Igor K; Yadavalli, Vamsi K; Prabhakar, Rajeev

    2016-07-21

    In this combined experimental (deep ultraviolet resonance Raman (DUVRR) spectroscopy and atomic force microscopy (AFM)) and theoretical (molecular dynamics (MD) simulations and stress-strain (SS)) study, the structural and mechanical properties of amyloid beta (Aβ40) fibrils have been investigated. The DUVRR spectroscopy and AFM experiments confirmed the formation of linear, unbranched and β-sheet rich fibrils. The fibrils (Aβ40)n, formed using n monomers, were equilibrated using all-atom MD simulations. The structural properties such as β-sheet character, twist, interstrand distance, and periodicity of these fibrils were found to be in agreement with experimental measurements. Furthermore, Young's modulus (Y) = 4.2 GPa computed using SS calculations was supported by measured values of 1.79 ± 0.41 and 3.2 ± 0.8 GPa provided by two separate AFM experiments. These results revealed size dependence of structural and material properties of amyloid fibrils and show the utility of such combined experimental and theoretical studies in the design of precisely engineered biomaterials. PMID:27387853

  1. [beta subsccript 2]-microglobulin forms three-dimensional domain-swapped amyloid fibrils with disulfide linkages

    SciTech Connect

    Liu, Cong; Sawaya, Michael R.; Eisenberg, David

    2011-08-09

    {beta}{sub 2}-microglobulin ({beta}{sub 2}-m) is the light chain of the type I major histocompatibility complex. It deposits as amyloid fibrils within joints during long-term hemodialysis treatment. Despite the devastating effects of dialysis-related amyloidosis, full understanding of how fibrils form from soluble {beta}{sub 2}-m remains elusive. Here we show that {beta}{sub 2}-m can oligomerize and fibrillize via three-dimensional domain swapping. Isolating a covalently bound, domain-swapped dimer from {beta}{sub 2}-m oligomers on the pathway to fibrils, we were able to determine its crystal structure. The hinge loop that connects the swapped domain to the core domain includes the fibrillizing segment LSFSKD, whose atomic structure we also determined. The LSFSKD structure reveals a class 5 steric zipper, akin to other amyloid spines. The structures of the dimer and the zipper spine fit well into an atomic model for this fibrillar form of {beta}{sub 2}-m, which assembles slowly under physiological conditions.

  2. Effect of PEGylated superparamagnetic iron oxide nanoparticles (SPIONs) under magnetic field on amyloid beta fibrillation process.

    PubMed

    Mirsadeghi, Somayeh; Shanehsazzadeh, Saeed; Atyabi, Fatemeh; Dinarvand, Rassoul

    2016-02-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) with specific surface coatings have been shown appropriate potential in the diagnosis and treatment of various brain diseases such as Alzheimer's. Comprehensive understanding of SPIONs interactions with amyloid beta (Aβ) and other amyloidogenic proteins is essential for their clinical application. SPIONs could be delivered to the target tissue under the magnetic field, while they might be influenced by the applied field. In this work, we exhibit the effect of different SPIONs (magnetized or non-magnetized with different surface charges) on the kinetics of Aβ fibrillation in aqueous solution by the aid of ThT assay. The results showed that applying of magnetic field to the SPIONs influences on the Aβ fibrillation because of its effect on the size due to surface charge. It was found that under magnetic field and high concentrations of nanoparticles (SPIONs-PEG-NH2), the Aβ fibrillation process accelerates, while at lower concentrations the fibrillation is inhibited. Furthermore, the coating charge has a considerable role in fibrillation process and the positively charged SPIONs/magnetized, at lower particle concentrations, accelerate the fibrillation compared with the negatively charged or uncharged SPIONs. This hints that SPIONs with a positive charge have dual effects on the Aβ fibrillation process. They influence on the concentration of monomeric protein in solution and thereby the nucleation time. Also, SPIONs have an effect on binding during the protein conformation. PMID:26652388

  3. Dynamics of water in the amphiphilic pore of amyloid β fibrils

    NASA Astrophysics Data System (ADS)

    GhattyVenkataKrishna, Pavan K.; Mostofian, Barmak

    2013-09-01

    Alzheimers disease related amyloid peptide, Aβ, forms a fibrillar structure through aggregation. The aggregate is stabilized by a salt bridge that is responsible for the formation of an amphiphilic pore that can accommodate water molecules. None of the reported structures of Aβ, however, contain water. We present results from molecular dynamics simulations on dimeric Aβ fibrils solvated in water. Water penetrates and fills the amphiphilic pore increasing its volume. We observe a thick wire of water that is translationally and rotationally stiff in comparison to bulk water and may be essential for the stabilization of the amyloid Aβ protein.

  4. Atomic-resolution 3D structure of amyloid β fibrils: The Osaka mutation

    DOE PAGESBeta

    Schutz, Anne K.; Wall, Joseph; Vagt, Toni; Huber, Matthias; Ovchinnikova, Oxana Y.; Cadalbert, Riccardo; Guntert, Peter; Bockmann, Anja; Glockshuber, Rudi; Meier, Beat H.

    2014-11-13

    Despite its central importance for understanding the molecular basis of Alzheimer's disease (AD), high-resolution structural information on amyloid β-peptide (Aβ) fibrils, which are intimately linked with AD, is scarce. We report an atomic-resolution fibril structure of the Aβ 1-40 peptide with the Osaka mutation (E22Δ), associated with early-onset AD. The structure, which differs substantially from all previously proposed models, is based on a large number of unambiguous intra- and intermolecular solid-state NMR distance restraints

  5. Amyloid formation of growth hormone in presence of zinc: Relevance to its storage in secretory granules.

    PubMed

    Jacob, Reeba S; Das, Subhadeep; Ghosh, Saikat; Anoop, Arunagiri; Jha, Narendra Nath; Khan, Tuhin; Singru, Praful; Kumar, Ashutosh; Maji, Samir K

    2016-01-01

    Amyloids are cross-β-sheet fibrillar aggregates, associated with various human diseases and native functions such as protein/peptide hormone storage inside secretory granules of neuroendocrine cells. In the current study, using amyloid detecting agents, we show that growth hormone (GH) could be stored as amyloid in the pituitary of rat. Moreover, to demonstrate the formation of GH amyloid in vitro, we studied various conditions (solvents, glycosaminoglycans, salts and metal ions) and found that in presence of zinc metal ions (Zn(II)), GH formed short curvy fibrils. The amyloidogenic nature of these fibrils was examined by Thioflavin T binding, Congo Red binding, transmission electron microscopy and X-ray diffraction. Our biophysical studies also suggest that Zn(II) initiates the early oligomerization of GH that eventually facilitates the fibrillation process. Furthermore, using immunofluorescence study of pituitary tissue, we show that GH in pituitary significantly co-localizes with Zn(II), suggesting the probable role of zinc in GH aggregation within secretory granules. We also found that GH amyloid formed in vitro is capable of releasing monomers. The study will help to understand the possible mechanism of GH storage, its regulation and monomer release from the somatotrophs of anterior pituitary. PMID:27004850

  6. Amyloid formation of growth hormone in presence of zinc: Relevance to its storage in secretory granules

    PubMed Central

    Jacob, Reeba S.; Das, Subhadeep; Ghosh, Saikat; Anoop, Arunagiri; Jha, Narendra Nath; Khan, Tuhin; Singru, Praful; Kumar, Ashutosh; Maji, Samir K.

    2016-01-01

    Amyloids are cross-β-sheet fibrillar aggregates, associated with various human diseases and native functions such as protein/peptide hormone storage inside secretory granules of neuroendocrine cells. In the current study, using amyloid detecting agents, we show that growth hormone (GH) could be stored as amyloid in the pituitary of rat. Moreover, to demonstrate the formation of GH amyloid in vitro, we studied various conditions (solvents, glycosaminoglycans, salts and metal ions) and found that in presence of zinc metal ions (Zn(II)), GH formed short curvy fibrils. The amyloidogenic nature of these fibrils was examined by Thioflavin T binding, Congo Red binding, transmission electron microscopy and X-ray diffraction. Our biophysical studies also suggest that Zn(II) initiates the early oligomerization of GH that eventually facilitates the fibrillation process. Furthermore, using immunofluorescence study of pituitary tissue, we show that GH in pituitary significantly co-localizes with Zn(II), suggesting the probable role of zinc in GH aggregation within secretory granules. We also found that GH amyloid formed in vitro is capable of releasing monomers. The study will help to understand the possible mechanism of GH storage, its regulation and monomer release from the somatotrophs of anterior pituitary. PMID:27004850

  7. Universality in the morphology and mechanics of coarsening amyloid fibril networks.

    PubMed

    Rizzi, L G; Head, D A; Auer, S

    2015-02-20

    Peptide hydrogels have important applications as biomaterials and in nanotechnology, but utilization often depends on their mechanical properties for which we currently have no predictive capability. Here we use a peptide model to simulate the formation of percolating amyloid fibril networks and couple these to the elastic network theory to determine their mechanical properties. We find that the time variation of network length scales can be collapsed onto master curves by using a time scaling function that depends on the peptide interaction anisotropy. The same scaling applies to network mechanics, revealing a nonmonotonic dependence of the shear modulus with time. Our structure-function relationship between the peptide building blocks, network morphology, and network mechanical properties can aid in the design of amyloid fibril networks with tailored mechanical properties. PMID:25763974

  8. Re-entrant isotropic-nematic phase behavior in polymer-depleted amyloid fibrils.

    PubMed

    Zhao, Jianguo; Li, Chaoxu; Mezzenga, Raffaele

    2014-11-19

    Amyloid fibrils dispersed in water exhibit both isotropic (I) and nematic (N) phases, depending on concentration, but their coexistence, expected from the first order nature of the I - N thermodynamic transition is seldom observed. By adding a non-absorbing polymer to an amyloid fibrils suspension, we report, for the first time, an unusual closed-loop phase behavior. The phase diagrams reveal that the I + N coexistence does emerge, but only when the depleting polymer is present at intermediate concentrations. We combine depletion potentials in the dilute and semi-dilute polymer regime with the DLVO theory and the principle of equivalent law of corresponding states to calculate variations of the second virial coefficient with increasing polymer concentrations. We conclude that the decrease of the depletion potential range in the semi-dilute regime plays a pivotal role in the observed re-stabilization, leading to a closure of the I + N coexistence region. PMID:25347270

  9. Re-entrant isotropic-nematic phase behavior in polymer-depleted amyloid fibrils

    NASA Astrophysics Data System (ADS)

    Zhao, Jianguo; Li, Chaoxu; Mezzenga, Raffaele

    2014-11-01

    Amyloid fibrils dispersed in water exhibit both isotropic (I) and nematic (N) phases, depending on concentration, but their coexistence, expected from the first order nature of the I - N thermodynamic transition is seldom observed. By adding a non-absorbing polymer to an amyloid fibrils suspension, we report, for the first time, an unusual closed-loop phase behavior. The phase diagrams reveal that the I + N coexistence does emerge, but only when the depleting polymer is present at intermediate concentrations. We combine depletion potentials in the dilute and semi-dilute polymer regime with the DLVO theory and the principle of equivalent law of corresponding states to calculate variations of the second virial coefficient with increasing polymer concentrations. We conclude that the decrease of the depletion potential range in the semi-dilute regime plays a pivotal role in the observed re-stabilization, leading to a closure of the I + N coexistence region.

  10. Universality in the Morphology and Mechanics of Coarsening Amyloid Fibril Networks

    NASA Astrophysics Data System (ADS)

    Rizzi, L. G.; Head, D. A.; Auer, S.

    2015-02-01

    Peptide hydrogels have important applications as biomaterials and in nanotechnology, but utilization often depends on their mechanical properties for which we currently have no predictive capability. Here we use a peptide model to simulate the formation of percolating amyloid fibril networks and couple these to the elastic network theory to determine their mechanical properties. We find that the time variation of network length scales can be collapsed onto master curves by using a time scaling function that depends on the peptide interaction anisotropy. The same scaling applies to network mechanics, revealing a nonmonotonic dependence of the shear modulus with time. Our structure-function relationship between the peptide building blocks, network morphology, and network mechanical properties can aid in the design of amyloid fibril networks with tailored mechanical properties.

  11. Anastellin, an FN3 Fragment with Fibronectin Polymerization Activity, Resembles Amyloid Fibril Precursors

    SciTech Connect

    Briknarova, Klara; Akermann, Maria; Hoyt, David W. ); Ruoslahti, Erkki; Ely, Kathryn R.

    2003-08-01

    Anastellin is a carboxy-terminal fragment of the 1st FN3 domain from human fibronectin. It is capable of polymerizing fibronectin in vitro, and it displays anti-tumor, antimetastatic and anti-angiogenic properties in vivo. We have determined the structure of anastellin using nuclear magnetic resonance spectroscopy and identified residues critical for its activity. Anastellin exhibits dynamic fluctuations and conformational exchange in solution. Its overall topology is very similar to the corresponding region of full-length FN3 domains. However, its hydrophobic core becomes solvent accessible and some of its -strands lose their protection against hydrogen bonding to -strands from other molecules. These features seem to be relevant for the fibronectin polymerization activity of anastellin and resemble the characteristics of amyloid fibril precursors. We suggest that this analogy is not random and may reflect similarities between fibronectin and amyloid fibril formation.

  12. Elucidating the Role of Disulfide Bond on Amyloid Formation and Fibril Reversibility of Somatostatin-14

    PubMed Central

    Anoop, Arunagiri; Ranganathan, Srivastav; Dhaked, Bhagwan Das; Jha, Narendra Nath; Pratihar, Supriya; Ghosh, Saikat; Sahay, Shruti; Kumar, Santosh; Das, Subhadeep; Kombrabail, Mamata; Agarwal, Kumud; Jacob, Reeba S.; Singru, Praful; Bhaumik, Prasenjit; Padinhateeri, Ranjith; Kumar, Ashutosh; Maji, Samir K.

    2014-01-01

    The storage of protein/peptide hormones within subcellular compartments and subsequent release are crucial for their native function, and hence these processes are intricately regulated in mammalian systems. Several peptide hormones were recently suggested to be stored as amyloids within endocrine secretory granules. This leads to an apparent paradox where storage requires formation of aggregates, and their function requires a supply of non-aggregated peptides on demand. The precise mechanism behind amyloid formation by these hormones and their subsequent release remain an open question. To address this, we examined aggregation and fibril reversibility of a cyclic peptide hormone somatostatin (SST)-14 using various techniques. After proving that SST gets stored as amyloid in vivo, we investigated the role of native structure in modulating its conformational dynamics and self-association by disrupting the disulfide bridge (Cys3–Cys14) in SST. Using two-dimensional NMR, we resolved the initial structure of somatostatin-14 leading to aggregation and further probed its conformational dynamics in silico. The perturbation in native structure (S-S cleavage) led to a significant increase in conformational flexibility and resulted in rapid amyloid formation. The fibrils formed by disulfide-reduced noncyclic SST possess greater resistance to denaturing conditions with decreased monomer releasing potency. MD simulations reveal marked differences in the intermolecular interactions in SST and noncyclic SST providing plausible explanation for differential aggregation and fibril reversibility observed experimentally in these structural variants. Our findings thus emphasize that subtle changes in the native structure of peptide hormone(s) could alter its conformational dynamics and amyloid formation, which might have significant implications on their reversible storage and secretion. PMID:24782311

  13. Higher Order Amyloid Fibril Structure by MAS NMR and DNP Spectroscopy

    PubMed Central

    Debelouchina, Galia T.; Bayro, Marvin J.; Fitzpatrick, Anthony W.; Ladizhansky, Vladimir; Colvin, Michael T.; Caporini, Marc A.; Jaroniec, Christopher P.; Bajaj, Vikram S.; Rosay, Melanie; MacPhee, Cait E.; Vendruscolo, Michele; Maas, Werner E.; Dobson, Christopher M.; Griffin, Robert G.

    2014-01-01

    Protein magic angle spinning (MAS) NMR spectroscopy has generated structural models of several amyloid fibril systems, thus providing valuable information regarding the forces and interactions that confer the extraordinary stability of the amyloid architecture. Despite these advances, however, obtaining atomic resolution information describing the higher levels of structural organization within the fibrils remains a significant challenge. Here, we detail MAS NMR experiments and sample labeling schemes designed specifically to probe such higher order amyloid structure and we have applied them to the fibrils formed by an eleven-residue segment of the amyloidogenic protein transthyretin (TTR(105-115)). These experiments have allowed us to define unambiguously not only the arrangement of the peptide β-strands into β-sheets but also the β-sheet interfaces within each protofilament, and in addition to identify the nature of the protofilament-to-protofilament contacts that lead to the formation of the complete fibril. Our efforts have resulted in 111 quantitative distance and torsion angle restraints (10 per residue) that describe the various levels of structure organization. The experiments benefited extensively from the use of dynamic nuclear polarization (DNP), which in some cases allowed us to shorten the data acquisition time from days to hours and to improve significantly the signal-to-noise ratios of the spectra. The β-sheet interface and protofilament interactions identified here revealed local variations in the structure that result in multiple peaks for the exposed N- and C-termini of the peptide and in inhomogeneous line-broadening for the side-chains buried within the interior of the fibrils. PMID:24304221

  14. Atomic-resolution structure of a disease-relevant Aβ(1-42) amyloid fibril.

    PubMed

    Wälti, Marielle Aulikki; Ravotti, Francesco; Arai, Hiromi; Glabe, Charles G; Wall, Joseph S; Böckmann, Anja; Güntert, Peter; Meier, Beat H; Riek, Roland

    2016-08-23

    Amyloid-β (Aβ) is present in humans as a 39- to 42-amino acid residue metabolic product of the amyloid precursor protein. Although the two predominant forms, Aβ(1-40) and Aβ(1-42), differ in only two residues, they display different biophysical, biological, and clinical behavior. Aβ(1-42) is the more neurotoxic species, aggregates much faster, and dominates in senile plaque of Alzheimer's disease (AD) patients. Although small Aβ oligomers are believed to be the neurotoxic species, Aβ amyloid fibrils are, because of their presence in plaques, a pathological hallmark of AD and appear to play an important role in disease progression through cell-to-cell transmissibility. Here, we solved the 3D structure of a disease-relevant Aβ(1-42) fibril polymorph, combining data from solid-state NMR spectroscopy and mass-per-length measurements from EM. The 3D structure is composed of two molecules per fibril layer, with residues 15-42 forming a double-horseshoe-like cross-β-sheet entity with maximally buried hydrophobic side chains. Residues 1-14 are partially ordered and in a β-strand conformation, but do not display unambiguous distance restraints to the remainder of the core structure. PMID:27469165

  15. Atomic-resolution structure of a disease-relevant Aβ(1–42) amyloid fibril

    PubMed Central

    Wälti, Marielle Aulikki; Ravotti, Francesco; Arai, Hiromi; Glabe, Charles G.; Wall, Joseph S.; Böckmann, Anja; Güntert, Peter; Meier, Beat H.; Riek, Roland

    2016-01-01

    Amyloid-β (Aβ) is present in humans as a 39- to 42-amino acid residue metabolic product of the amyloid precursor protein. Although the two predominant forms, Aβ(1–40) and Aβ(1–42), differ in only two residues, they display different biophysical, biological, and clinical behavior. Aβ(1–42) is the more neurotoxic species, aggregates much faster, and dominates in senile plaque of Alzheimer’s disease (AD) patients. Although small Aβ oligomers are believed to be the neurotoxic species, Aβ amyloid fibrils are, because of their presence in plaques, a pathological hallmark of AD and appear to play an important role in disease progression through cell-to-cell transmissibility. Here, we solved the 3D structure of a disease-relevant Aβ(1–42) fibril polymorph, combining data from solid-state NMR spectroscopy and mass-per-length measurements from EM. The 3D structure is composed of two molecules per fibril layer, with residues 15–42 forming a double-horseshoe–like cross–β-sheet entity with maximally buried hydrophobic side chains. Residues 1–14 are partially ordered and in a β-strand conformation, but do not display unambiguous distance restraints to the remainder of the core structure. PMID:27469165

  16. Annealing prion protein amyloid fibrils at high temperature results in extension of a proteinase K-resistant core.

    PubMed

    Bocharova, Olga V; Makarava, Natallia; Breydo, Leonid; Anderson, Maighdlin; Salnikov, Vadim V; Baskakov, Ilia V

    2006-01-27

    Amyloids are highly ordered, rigid beta-sheet-rich structures that appear to have minimal dynamic flexibility in individual polypeptide chains. Here, we demonstrate that substantial conformational rearrangements occur within mature amyloid fibrils produced from full-length mammalian prion protein. The rearrangement results in a substantial extension of a proteinase K-resistant core and is accompanied by an increase in the beta-sheet-rich conformation. The conformational rearrangement was induced in the presence of low concentrations of Triton X-100 either by brief exposure to 80 degrees C or, with less efficacy, by prolonged incubation at 37 degrees C at pH 7.5 and is referred to here as "annealing." Upon annealing, amyloid fibrils acquired a proteinase K-resistant core identical to that found in bovine spongiform encephalopathy-specific scrapie-associated prion protein. Annealing was also observed when amyloid fibrils were exposed to high temperatures in the absence of detergent but in the presence of brain homogenate. These findings suggest that the amyloid fibrils exist in two conformationally distinct states that are separated by a high energy barrier and that yet unknown cellular cofactors may facilitate transition of the fibrils into thermodynamically more stable state. Our studies provide new insight into the complex behavior of prion polymerization and highlight the annealing process, a previously unknown step in the evolution of amyloid structures. PMID:16314415

  17. Self Assembly of Short Aromatic Peptides into Amyloid Fibrils and Related Nanostructures

    PubMed Central

    2007-01-01

    The formation of amyloid fibrils is the hallmark of more than twenty human disorders of unrelated etiology. In all these cases, ordered fibrillar protein assemblies with a diameter of 7–10 nm are being observed. In spite of the great clinical important of amyloidassociated diseases, the molecular recognition and self-assembly processes that lead to the formation of the fibrils are not fully understood. One direction to decipher the mechanism of amyloid formation is the use of short peptides fragments as model systems. Short peptide fragments, as short as pentapeptides, were shown to form typical amyloid assemblies in vitro that have ultrastructural, biophysical, and cytotoxic properties, as those of assemblies that are being formed by full length polypeptides. When we analyzed such short fragments, we identified the central role of aromatic moieties in the ability to aggregate into ordered nano-fibrillar structures. This notion allowed us to discover additional very short amyloidogenic peptides as well as other aromatic peptide motifs, which can form various assemblies at the nano-scale (including nanotubes, nanospheres, and macroscopic hydrogels with nano-scale order). Other practical utilization of this concept, together with novel β breakage methods, is their use for the development of novel classes of amyloid formation inhibitors. PMID:19164892

  18. Structural motif of polyglutamine amyloid fibrils discerned with mixed-isotope infrared spectroscopy.

    PubMed

    Buchanan, Lauren E; Carr, Joshua K; Fluitt, Aaron M; Hoganson, Andrew J; Moran, Sean D; de Pablo, Juan J; Skinner, James L; Zanni, Martin T

    2014-04-22

    Polyglutamine (polyQ) sequences are found in a variety of proteins, and mutational expansion of the polyQ tract is associated with many neurodegenerative diseases. We study the amyloid fibril structure and aggregation kinetics of K2Q24K2W, a model polyQ sequence. Two structures have been proposed for amyloid fibrils formed by polyQ peptides. By forming fibrils composed of both (12)C and (13)C monomers, made possible by protein expression in Escherichia coli, we can restrict vibrational delocalization to measure 2D IR spectra of individual monomers within the fibrils. The spectra are consistent with a β-turn structure in which each monomer forms an antiparallel hairpin and donates two strands to a single β-sheet. Calculated spectra from atomistic molecular-dynamics simulations of the two proposed structures confirm the assignment. No spectroscopically distinct intermediates are observed in rapid-scan 2D IR kinetics measurements, suggesting that aggregation is highly cooperative. Although 2D IR spectroscopy has advantages over linear techniques, the isotope-mixing strategy will also be useful with standard Fourier transform IR spectroscopy. PMID:24550484

  19. Structural stability of amyloid fibrils depends on the existence of the peripheral sequence near the core cross-β region.

    PubMed

    Saiki, Masatoshi; Shiba, Kohei; Okumura, Masaki

    2015-11-30

    Amyloid fibrils are fibrous protein assemblies with distinctive cross-β structures. For amyloidosis, there are disease-associated mutations outside of the cross-β structures. Thus, it is necessary to elucidate the role of peripheral sequences outside the cross-β structure. Amyloid fibrils are generally 10nm in width; however, the amyloid fibrils of truncated barnase M1 peptides missing the C-terminal sequence outside the cross-β structure are 20 nm in width. In this study, we performed comparative analysis of the structural stability of amyloids formed by the respective peptides. We found that the C-terminal amino acids dramatically affect the conformational instability in the presence of a denaturing reagent. PMID:26497084

  20. Is the serum amyloid A protein in acute phase plasma high density lipoprotein the precursor of AA amyloid fibrils?

    PubMed Central

    Baltz, M L; Rowe, I F; Caspi, D; Turnell, W G; Pepys, M B

    1986-01-01

    Serum amyloid A protein (SAA), an apolipoprotein of high density lipoprotein (HDL), is generally considered to be the precursor of AA protein, which forms the fibrils in reactive systemic amyloidosis in man and animals. This view is based on amino acid sequence identity between AA and the amino-terminal portion of SAA. However, in extensive and well-controlled studies of experimentally induced murine AA amyloidosis, we were unable to demonstrate a direct precursor-product relationship between SAA, in SAA-rich HDL preparations from acute phase or amyloidotic mouse or human serum, and AA protein in the amyloid deposits. This raises the possibility that SAA in its usual form, as an apolipoprotein of HDL synthesized during the acute phase response, may not be the major precursor of AA fibrils. The amyloidogenic forms of circulating SAA molecules may not be isolated during the preparation of HDL. Alternatively, particularly in the light of recent evidence that SAA mRNA is expressed in many different tissues throughout the body of appropriately stimulated animals, amyloidogenic SAA may be derived from sources other than the liver cells in which SAA-rich HDL is synthesized. PMID:3105937

  1. Assembly of the fungal SC3 hydrophobin into functional amyloid fibrils depends on its concentration and is promoted by cell wall polysaccharides.

    PubMed

    Scholtmeijer, Karin; de Vocht, Marcel L; Rink, Rick; Robillard, George T; Wösten, Han A B

    2009-09-25

    Class I hydrophobins function in fungal growth and development by self-assembling at hydrophobic-hydrophilic interfaces into amyloid-like fibrils. SC3 of the mushroom-forming fungus Schizophyllum commune is the best studied class I hydrophobin. This protein spontaneously adopts the amyloid state at the water-air interface. In contrast, SC3 is arrested in an intermediate conformation at the interface between water and a hydrophobic solid such as polytetrafluoroethylene (PTFE; Teflon). This finding prompted us to study conditions that promote assembly of SC3 into amyloid fibrils. Here, we show that SC3 adopts the amyloid state at the water-PTFE interface at high concentration (300 microg ml(-1)) and prolonged incubation (16 h). Moreover, we show that amyloid formation at both the water-air and water-PTFE interfaces is promoted by the cell wall components schizophyllan (beta(1-3),beta(1-6)-glucan) and beta(1-3)-glucan. Hydrophobin concentration and cell wall polysaccharides thus contribute to the role of SC3 in formation of aerial hyphae and in hyphal attachment. PMID:19654326

  2. Amyloid growth: combining experiment and kinetic theory

    NASA Astrophysics Data System (ADS)

    Knowles, Tuomas; Cohen, Samuel; Vendruscolo, Michele; Dobson, Christopher

    2012-02-01

    The conversion of proteins from their soluble forms into fibrillar amyloid nanostructures is a general type of behaviour encountered for many different proteins in the context of disease as well as for the generation of a select class of functional materials in nature. This talk focuses on the problem of defining the rates of the individual molecular level processes involved in the overall conversion reaction. A master equation approach is discussedootnotetextCohen et al, J Chem Phys 2011, 135, 065106 ootnotetextKnowles et al, Science, 2009, 326, 1533-1537 and used in combination with kinetic measurements to yield mechanistic insights into the amyloid growth phenomenon.

  3. Understanding amyloid fibril nucleation and aβ oligomer/drug interactions from computer simulations.

    PubMed

    Nguyen, Phuong; Derreumaux, Philippe

    2014-02-18

    Evolution has fine-tuned proteins to accomplish a variety of tasks. Yet, with aging, some proteins assemble into harmful amyloid aggregates associated with neurodegenerative diseases, such as Alzheimer's disease (AD), which presents a complex and costly challenge to our society. Thus, far, drug after drug has failed to slow the progression of AD, characterized by the self-assembly of the 39-43 amino acid β-amyloid (Aβ) protein into extracellular senile plaques that form a cross-β structure. While there is experimental evidence that the Aβ small oligomers are the primary toxic species, standard tools of biology have failed to provide structures of these transient, inhomogeneous assemblies. Despite extensive experimental studies, researchers have not successfully characterized the nucleus ensemble, the starting point for rapid fibril formation. Similarly scientists do not have atomic data to show how the compounds that reduce both fibril formation and toxicity in cells bind to Aβ42 oligomers. In this context, computer simulations are important tools for gaining insights into the self-assembly of amyloid peptides and the molecular mechanism of inhibitors. This Account reviews what analytical models and simulations at different time and length scales tell us about the dynamics, kinetics, and thermodynamics of amyloid fibril formation and, notably, the nucleation process. Though coarse-grained and mesoscopic protein models approximate atomistic details by averaging out unimportant degrees of freedom, they provide generic features of amyloid formation and insights into mechanistic details of the self-assembly process. The thermodynamics and kinetics vary from linear peptides adopting straight β-strands in fibrils to longer peptides adopting in parallel U shaped conformations in fibrils. In addition, these properties change with the balance between electrostatic and hydrophobic interactions and the intrinsic disorder of the system. However, simulations suggest that

  4. A carboxylated Zn-phthalocyanine inhibits fibril formation of Alzheimer's amyloid β peptide.

    PubMed

    Tabassum, Shatera; Sheikh, Abdullah M; Yano, Shozo; Ikeue, Takafumi; Handa, Makoto; Nagai, Atsushi

    2015-02-01

    Amyloid β (Aβ), a 39-42 amino acid peptide derived from amyloid precursor protein, is deposited as fibrils in Alzheimer's disease brains, and is considered to play a major role in the pathogenesis of the disease. We have investigated the effects of a water-soluble Zn-phthalocyanine, ZnPc(COONa)₈, a macrocyclic compound with near-infrared optical properties, on Aβ fibril formation in vitro. A thioflavin T fluorescence assay showed that ZnPc(COONa)₈ significantly inhibited Aβ fibril formation, increasing the lag time and dose-dependently decreasing the plateau level of fibril formation. Moreover, it destabilized pre-formed Aβ fibrils, resulting in an increase in low-molecular-weight species. After fibril formation in the presence of ZnPc(COONa)₈, immunoprecipitation of Aβ₁₋₄₂ using Aβ-specific antibody followed by near-infrared scanning demonstrated binding of ZnPc(COONa)₈ to Aβ₁₋₄₂. A study using the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonic acid showed that ZnPc(COONa)8 decreased the hydrophobicity during Aβ₁₋₄₂ fibril formation. CD spectroscopy showed an increase in the α helix structure and a decrease in the β sheet structure of Aβ₁₋₄₀ in fibril-forming buffer containing ZnPc(COONa)₈. SDS/PAGE and a dot-blot immunoassay showed that ZnPc(COONa)₈ delayed the disappearance of low-molecular-weight species and the appearance of higher-molecular-weight oligomeric species of Aβ₁₋₄₂. A cell viability assay showed that ZnPc(COONa)₈ was not toxic to a neuronal cell line (A1), but instead protected A1 cells against Aβ₁₋₄₂-induced toxicity. Overall, our results indicate that ZnPc(COONa)₈ binds to Aβ and decreases the hydrophobicity, and this change is unfavorable for Aβ oligomerization and fibril formation. PMID:25404240

  5. Structural Polymorphism of Alzheimer's β-Amyloid Fibrils as Controlled by an E22 Switch: A Solid-State NMR Study.

    PubMed

    Elkins, Matthew R; Wang, Tuo; Nick, Mimi; Jo, Hyunil; Lemmin, Thomas; Prusiner, Stanley B; DeGrado, William F; Stöhr, Jan; Hong, Mei

    2016-08-10

    The amyloid-β (Aβ) peptide of Alzheimer's disease (AD) forms polymorphic fibrils on the micrometer and molecular scales. Various fibril growth conditions have been identified to cause polymorphism, but the intrinsic amino acid sequence basis for this polymorphism has been unclear. Several single-site mutations in the center of the Aβ sequence cause different disease phenotypes and fibrillization properties. The E22G (Arctic) mutant is found in familial AD and forms protofibrils more rapidly than wild-type Aβ. Here, we use solid-state NMR spectroscopy to investigate the structure, dynamics, hydration and morphology of Arctic E22G Aβ40 fibrils. (13)C, (15)N-labeled synthetic E22G Aβ40 peptides are studied and compared with wild-type and Osaka E22Δ Aβ40 fibrils. Under the same fibrillization conditions, Arctic Aβ40 exhibits a high degree of polymorphism, showing at least four sets of NMR chemical shifts for various residues, while the Osaka and wild-type Aβ40 fibrils show a single or a predominant set of chemical shifts. Thus, structural polymorphism is intrinsic to the Arctic E22G Aβ40 sequence. Chemical shifts and inter-residue contacts obtained from 2D correlation spectra indicate that one of the major Arctic conformers has surprisingly high structural similarity with wild-type Aβ42. (13)C-(1)H dipolar order parameters, (1)H rotating-frame spin-lattice relaxation times and water-to-protein spin diffusion experiments reveal substantial differences in the dynamics and hydration of Arctic, Osaka and wild-type Aβ40 fibrils. Together, these results strongly suggest that electrostatic interactions in the center of the Aβ peptide sequence play a crucial role in the three-dimensional fold of the fibrils, and by inference, fibril-induced neuronal toxicity and AD pathogenesis. PMID:27414264

  6. Evaluation of protease resistance and toxicity of amyloid-like food fibrils from whey, soy, kidney bean, and egg white.

    PubMed

    Lassé, Moritz; Ulluwishewa, Dulantha; Healy, Jackie; Thompson, Dion; Miller, Antonia; Roy, Nicole; Chitcholtan, Kenny; Gerrard, Juliet A

    2016-02-01

    The structural properties of amyloid fibrils combined with their highly functional surface chemistry make them an attractive new food ingredient, for example as highly effective gelling agents. However, the toxic role of amyloid fibrils in disease may cause some concern about their food safety because it has not been established unequivocally if consumption of food fibrils poses a health risk to consumers. Here we present a study of amyloid-like fibrils from whey, kidney bean, soy bean, and egg white to partially address this concern. Fibrils showed varied resistance to proteolytic digestion in vitro by either Proteinase K, pepsin or pancreatin. The toxicity of mature fibrils was measured in vitro and compared to native protein, early-stage-fibrillar protein, and sonicated fibrils in two immortalised human cancer cell lines, Caco-2 and Hec-1a. There was no reduction in the viability of either Caco-2 or Hec-1a cells after treatment with a fibril concentration of up to 0.25 mg/mL. PMID:26304377

  7. Early aggregation preceding the nucleation of insulin amyloid fibrils as monitored by small angle X-ray scattering

    PubMed Central

    Chatani, Eri; Inoue, Rintaro; Imamura, Hiroshi; Sugiyama, Masaaki; Kato, Minoru; Yamamoto, Masahide; Nishida, Koji; Kanaya, Toshiji

    2015-01-01

    The nucleation event of amyloid fibrils is one of the most crucial processes that dictate the timing and rate of the pathology of diseases; however, information regarding how protein molecules associate to produce fibril nuclei is currently limited. In order to explore this issue in more detail, we performed time-resolved small angle X-ray scattering (SAXS) measurements on insulin fibrillation, in combination with additional multidirectional analyses of thioflavin T fluorescence, FTIR spectroscopy, light scattering, and light transmittance, during the fibrillation process of bovine insulin. SAXS monitoring revealed that insulin molecules associated into rod-like prefibrillar aggregates in the very early stage of the reaction. After the formation of these early aggregates, they appeared to further coalesce mutually to form larger clusters, and the SAXS profiles subsequently showed the further time evolution of conformational development towards mature amyloid fibrils. Distinct types of structural units in terms of shape in a nano-scale order, cross-β content, and thioflavin T fluorescence intensity were observed in a manner that was dependent on the fibrillation pathways. These results suggest the presence of diverse substructures that characterize various fibrillation pathways, and eventually, manifest polymorphisms in mature amyloid fibrils. PMID:26503463

  8. Early aggregation preceding the nucleation of insulin amyloid fibrils as monitored by small angle X-ray scattering.

    PubMed

    Chatani, Eri; Inoue, Rintaro; Imamura, Hiroshi; Sugiyama, Masaaki; Kato, Minoru; Yamamoto, Masahide; Nishida, Koji; Kanaya, Toshiji

    2015-01-01

    The nucleation event of amyloid fibrils is one of the most crucial processes that dictate the timing and rate of the pathology of diseases; however, information regarding how protein molecules associate to produce fibril nuclei is currently limited. In order to explore this issue in more detail, we performed time-resolved small angle X-ray scattering (SAXS) measurements on insulin fibrillation, in combination with additional multidirectional analyses of thioflavin T fluorescence, FTIR spectroscopy, light scattering, and light transmittance, during the fibrillation process of bovine insulin. SAXS monitoring revealed that insulin molecules associated into rod-like prefibrillar aggregates in the very early stage of the reaction. After the formation of these early aggregates, they appeared to further coalesce mutually to form larger clusters, and the SAXS profiles subsequently showed the further time evolution of conformational development towards mature amyloid fibrils. Distinct types of structural units in terms of shape in a nano-scale order, cross-β content, and thioflavin T fluorescence intensity were observed in a manner that was dependent on the fibrillation pathways. These results suggest the presence of diverse substructures that characterize various fibrillation pathways, and eventually, manifest polymorphisms in mature amyloid fibrils. PMID:26503463

  9. Flavone Derivatives as Inhibitors of Insulin Amyloid-Like Fibril Formation

    PubMed Central

    Malisauskas, Ricardas; Botyriute, Akvile; Cannon, Jonathan G.; Smirnovas, Vytautas

    2015-01-01

    Several natural and synthetic flavone derivatives have been reported to inhibit formation of amyloid fibrils or to remodel existing fibrils. These studies suggest that the numbers and positions of hydroxyl groups on the flavone rings determine their effectiveness as amyloid inhibitors. In many studies the primary method for determining the effectiveness of inhibition is measuring Thioflavin T (ThT) fluorescence. This method demonstrably results in a number of false positives for inhibition. We studied the effects of 265 commercially available flavone derivatives on insulin fibril formation. We enhanced the effectiveness of ThT fluorescence measurements by fitting kinetic curves to obtain halftime of aggregation (t50). Maximal values of ThT fluorescence varied two fold or more in one third of all cases, but this did not correlate with changes in t50. Changes in t50 values were more accurate measures of inhibition of amyloid formation. We showed that without a change in an assay, but just by observing complete kinetic curves it is possible to eliminate numbers of false positive and sometimes even false negative results. Examining the data from all 265 flavones we confirmed previous observations that identified the importance of hydroxyl groups for inhibition. Our evidence suggests the importance of hydroxyl groups at locations 5, 6, 7, and 4’, and the absence of a hydroxyl group at location 3, for inhibiting amyloid formation. However, the main conclusion is that the positions are not additive. The structures and their effects must be thought of in the context of the whole molecule. PMID:25799281

  10. Methionine oxidation induces amyloid fibril formation by full-length apolipoprotein A-I

    PubMed Central

    Wong, Yuan Qi; Binger, Katrina J.; Howlett, Geoffrey J.; Griffin, Michael D. W.

    2010-01-01

    Apolipoprotein A-I (apoA-I) is the major protein component of HDL, where it plays an important role in cholesterol transport. The deposition of apoA-I derived amyloid is associated with various hereditary systemic amyloidoses and atherosclerosis; however, very little is known about the mechanism of apoA-I amyloid formation. Methionine residues in apoA-I are oxidized via several mechanisms in vivo to form methionine sulfoxide (MetO), and significant levels of methionine oxidized apoA-I (MetO-apoA-I) are present in normal human serum. We investigated the effect of methionine oxidation on the structure, stability, and aggregation of full-length, lipid-free apoA-I. Circular dichrosim spectroscopy showed that oxidation of all three methionine residues in apoA-I caused partial unfolding of the protein and decreased its thermal stability, reducing the melting temperature (Tm) from 58.7 °C for native apoA-I to 48.2 °C for MetO-apoA-I. Analytical ultracentrifugation revealed that methionine oxidation inhibited the native self association of apoA-I to form dimers and tetramers. Incubation of MetO-apoA-I for extended periods resulted in aggregation of the protein, and these aggregates bound Thioflavin T and Congo Red. Inspection of the aggregates by electron microscopy revealed fibrillar structures with a ribbon-like morphology, widths of approximately 11 nm, and lengths of up to several microns. X-ray fibre diffraction studies of the fibrils revealed a diffraction pattern with orthogonal peaks at spacings of 4.64 Å and 9.92 Å, indicating a cross-β amyloid structure. This systematic study of fibril formation by full-length apoA-I represents the first demonstration that methionine oxidation can induce amyloid fibril formation. PMID:20133843