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Sample records for amyloid fibrils inhibits

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

    Science.gov (United States)

    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.

  2. Amyloid Fibril Solubility

    OpenAIRE

    Rizzi, L. G.; Auer, S.

    2015-01-01

    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 interactio...

  3. Phenolsulfonphthalein, but not phenolphthalein, inhibits amyloid fibril formation: implications for the modulation of amyloid self-assembly.

    Science.gov (United States)

    Levy, Michal; Porat, Yair; Bacharach, Eran; Shalev, Deborah E; Gazit, Ehud

    2008-06-03

    The study of the mechanism of amyloid fibril formation and its inhibition is of key medical importance due to the lack of amyloid assembly inhibitors that are approved for clinical use. We have previously demonstrated the potent inhibitory potential of phenolsulfonphthalein, a nontoxic compound that was approved for diagnostic use in human subjects, on aggregation of islet amyloid polypeptide (IAPP) that is associated with type 2 diabetes. Here, we extend our studies on the mechanism of action of phenolsulfonphthalein by comparing its antiamyloidogenic effect to a very similar compound that is also approved for human use, phenolphthalein. While these compounds have very similar primary chemical structures, they significantly differ in their three-dimensional conformation. Our results clearly demonstrated that these two compounds had completely different inhibitory potencies: While phenolsulfonphthalein was a very potent inhibitor of amyloid fibril formation by IAPP, phenolphthalein did not show significant antiamyloidogenic activity. This behavior was observed with a short amyloid fragment of IAPP and also with the full-length polypeptide. The NMR spectrum of IAPP 20-29 in the presence of phenolsulfonphthalein showed chemical shift deviations that were different from the unbound or phenolphthalein-bound peptide. Differential activity was also observed in the inhibition of insulin amyloid formation by these two compounds, and density-gradient experiments clearly demonstrated the different inhibitory effect of the two compounds on the formation of prefibrillar assemblies. Taken together, our studies suggest that the three-dimensional arrangement of the polyphenol phenolsulfonphthalein has a central role in its amyloid formation inhibition activity.

  4. Identification of the primary peptide contaminant that inhibits fibrillation and toxicity in synthetic amyloid-β42.

    Science.gov (United States)

    Adams, Daniel J; Nemkov, Travis G; Mayer, John P; Old, William M; Stowell, Michael H B

    2017-01-01

    Understanding the pathophysiology of Alzheimer disease has relied upon the use of amyloid peptides from a variety of sources, but most predominantly synthetic peptides produced using t-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. These synthetic methods can lead to minor impurities which can have profound effects on the biological activity of amyloid peptides. Here we used a combination of cytotoxicity assays, fibrillation assays and high resolution mass spectrometry (MS) to identify impurities in synthetic amyloid preparations that inhibit both cytotoxicity and aggregation. We identify the Aβ42Δ39 species as the major peptide contaminant responsible for limiting both cytotoxicity and fibrillation of the amyloid peptide. In addition, we demonstrate that the presence of this minor impurity inhibits the formation of a stable Aβ42 dimer observable by MS in very pure peptide samples. These results highlight the critical importance of purity and provenance of amyloid peptides in Alzheimer's research in particular, and biological research in general.

  5. Synthesis of water-soluble curcumin derivatives and their inhibition on lysozyme amyloid fibrillation

    Science.gov (United States)

    Wang, Sujuan; Peng, Xixi; Cui, Liangliang; Li, Tongtong; Yu, Bei; Ma, Gang; Ba, Xinwu

    2018-02-01

    The potential application of curcumin was heavily limited in biomedicine because of its poor solubility in pure water. To circumvent the detracting feature, two novel water-soluble amino acid modified curcumin derivatives (MLC and DLC) have been synthesized through the condensation reaction between curcumin and Nα-Fmoc-Nε-Boc-L-lysine. Benefiting from the enhanced solubility of 3.32 × 10- 2 g/mL for MLC and 4.66 × 10- 2 g/mL for DLC, the inhibition effects of the as-prepared derivatives on the amyloid fibrillation of lysozyme (HEWL) were investigated detaily in water solution. The obtained results showed that the amyloid fibrillation of HEWL was inhibited to a great extent when the concentrations of MLC and DLC reach to 20.139 mM and 49.622 mM, respectively. The fluorescence quenching upon the addition of curcumin to HEWL provide a support for static and dynamic recombination quenching process. The binding driving force was assigned to classical hydrophobic interaction between curcumin derivatives and HEWL. In addition, UV-Vis absorption and circular dichroism (CD) spectra confirmed the change of the conformation of HEWL.

  6. Glucagon Amyloid-like Fibril Morphology Is Selected via Morphology-Dependent Growth Inhibition

    DEFF Research Database (Denmark)

    Andersen, C.B.; Otzen, D.; Christiansen, Gunna

    2007-01-01

    Protein Structure and Biophysics, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Malov, Denmark, Centre for Insoluble Protein Structures (inSPIN), Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark, and Institute of Medical Microbiology and Immunology...... twisted fibril seeds cannot grow at high concentrations. We conclude that there exists a morphology-dependent mechanism for inhibition of glucagon fibril growth. Light scattering experiments indicate that glucagon is mainly monomeric below 1 mg/mL and increasingly trimeric above this concentration. We...

  7. Nanomechanical properties of single amyloid fibrils

    International Nuclear Information System (INIS)

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

    2012-01-01

    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. (topical review)

  8. A nanobody binding to non-amyloidogenic regions of the protein human lysozyme enhances partial unfolding but inhibits amyloid fibril formation.

    Science.gov (United States)

    De Genst, Erwin; Chan, Pak-Ho; Pardon, Els; Hsu, Shang-Te D; Kumita, Janet R; Christodoulou, John; Menzer, Linda; Chirgadze, Dimitri Y; Robinson, Carol V; Muyldermans, Serge; Matagne, André; Wyns, Lode; Dobson, Christopher M; Dumoulin, Mireille

    2013-10-24

    We report the effects of the interaction of two camelid antibody fragments, generally called nanobodies, namely cAb-HuL5 and a stabilized and more aggregation-resistant variant cAb-HuL5G obtained by protein engineering, on the properties of two amyloidogenic variants of human lysozyme, I56T and D67H, whose deposition in vital organs including the liver, kidney, and spleen is associated with a familial non-neuropathic systemic amyloidosis. Both NMR spectroscopy and X-ray crystallographic studies reveal that cAb-HuL5 binds to the α-domain, one of the two lobes of the native lysozyme structure. The binding of cAb-HuL5/cAb-HuL5G strongly inhibits fibril formation by the amyloidogenic variants; it does not, however, suppress the locally transient cooperative unfolding transitions, characteristic of these variants, in which the β-domain and the C-helix unfold and which represents key early intermediate species in the formation of amyloid fibrils. Therefore, unlike two other nanobodies previously described, cAb-HuL5/cAb-HuL5G does not inhibit fibril formation via the restoration of the global cooperativity of the native structure of the lysozyme variants to that characteristic of the wild-type protein. Instead, it inhibits a subsequent step in the assembly of the fibrils, involving the unfolding and structural reorganization of the α-domain. These results show that nanobodies can protect against the formation of pathogenic aggregates at different stages in the structural transition of a protein from the soluble native state into amyloid fibrils, illustrating their value as structural probes to study the molecular mechanisms of amyloid fibril formation. Combined with their amenability to protein engineering techniques to improve their stability and solubility, these findings support the suggestion that nanobodies can potentially be developed as therapeutics to combat protein misfolding diseases.

  9. Energetics Underlying Twist Polymorphisms in Amyloid Fibrils

    NARCIS (Netherlands)

    Periole, Xavier; Huber, Thomas; Bonito-Oliva, Alessandra; Aberg, Karina C; van der Wel, Patrick C A; Sakmar, Thomas P; Marrink, Siewert J

    2018-01-01

    Amyloid fibrils are highly ordered protein aggregates associated with more than 40 human diseases. The exact conditions in which the fibrils are grown determine many types of reported fibril polymorphism, including different twist patterns. Twist-based polymorphs display unique mechanical properties

  10. Endocytosed 2-Microglobulin Amyloid Fibrils Induce Necrosis and Apoptosis of Rabbit Synovial Fibroblasts by Disrupting Endosomal/Lysosomal Membranes: A Novel Mechanism on the Cytotoxicity of Amyloid Fibrils.

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    Tadakazu Okoshi

    Full Text Available Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients. In dialysis-related amyloidosis, β2-microglobulin (β2-m amyloid fibrils deposit in the osteoarticular tissue, leading to carpal tunnel syndrome and destructive arthropathy with cystic bone lesions, but the mechanism by which these amyloid fibrils destruct bone and joint tissue is not fully understood. In this study, we assessed the cytotoxic effect of β2-m amyloid fibrils on the cultured rabbit synovial fibroblasts. Under light microscopy, the cells treated with amyloid fibrils exhibited both necrotic and apoptotic changes, while the cells treated with β2-m monomers and vehicle buffer exhibited no morphological changes. As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay. Interestingly, when the cells were incubated with amyloid fibrils for several hours, many endosomes/lysosomes filled with amyloid fibrils were observed under confocal laser microscopy and electron microscopy, Moreover, some endosomal/lysosomal membranes were disrupted by intravesicular fibrils, leading to the leakage of the fibrils into the cytosol and adjacent to mitochondria. Inhibition of actin-dependent endocytosis by cytochalasin D attenuated the toxicity of amyloid fibrils. These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid

  11. The contrasting effect of macromolecular crowding on amyloid fibril formation.

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    Qian Ma

    Full Text Available Amyloid fibrils associated with neurodegenerative diseases can be considered biologically relevant failures of cellular quality control mechanisms. It is known that in vivo human Tau protein, human prion protein, and human copper, zinc superoxide dismutase (SOD1 have the tendency to form fibril deposits in a variety of tissues and they are associated with different neurodegenerative diseases, while rabbit prion protein and hen egg white lysozyme do not readily form fibrils and are unlikely to cause neurodegenerative diseases. In this study, we have investigated the contrasting effect of macromolecular crowding on fibril formation of different proteins.As revealed by assays based on thioflavin T binding and turbidity, human Tau fragments, when phosphorylated by glycogen synthase kinase-3β, do not form filaments in the absence of a crowding agent but do form fibrils in the presence of a crowding agent, and the presence of a strong crowding agent dramatically promotes amyloid fibril formation of human prion protein and its two pathogenic mutants E196K and D178N. Such an enhancing effect of macromolecular crowding on fibril formation is also observed for a pathological human SOD1 mutant A4V. On the other hand, rabbit prion protein and hen lysozyme do not form amyloid fibrils when a crowding agent at 300 g/l is used but do form fibrils in the absence of a crowding agent. Furthermore, aggregation of these two proteins is remarkably inhibited by Ficoll 70 and dextran 70 at 200 g/l.We suggest that proteins associated with neurodegenerative diseases are more likely to form amyloid fibrils under crowded conditions than in dilute solutions. By contrast, some of the proteins that are not neurodegenerative disease-associated are unlikely to misfold in crowded physiological environments. A possible explanation for the contrasting effect of macromolecular crowding on these two sets of proteins (amyloidogenic proteins and non-amyloidogenic proteins has been

  12. Interaction of magnetic nanoparticles with lysozyme amyloid fibrils

    International Nuclear Information System (INIS)

    Gdovinová, Veronika; Tomašovičová, Natália; Batko, Ivan; Batková, Marianna; Balejčíková, Lucia; Garamus, Vasyl M.; Petrenko, Viktor I.; Avdeev, Mikhail V.; Kopčanský, Peter

    2017-01-01

    This work is devoted to the structural study of complex solutions of magnetic nanoparticles with lysozyme amyloid fibrils due to possible ordering of such system by applying the external magnetic field. The interaction of magnetic nanoparticles with amyloid fibrils has been followed by atomic force microscopy and small-angle X-ray scattering. It has been observed that magnetic nanoparticles (MNPs) adsorb to lysozyme amyloid fibrils. It was found that MNPs alter amyloids structures, namely the diameter of lysozyme amyloid fibrils is increased whereas the length of fibrils is decreased. In the same time MNPs do not change the helical pitch significantly. - Highlights: • Solution of MNPs with lysozyme amyloid fibrils was characterized by AFM and SAXS. • MNPs adsorb to lysozyme amyloid fibrils. • Diameter and size of lysozyme amyloid fibrils change due to doping with MNPs.

  13. Interaction of magnetic nanoparticles with lysozyme amyloid fibrils

    Energy Technology Data Exchange (ETDEWEB)

    Gdovinová, Veronika [Institute of Experimental Physics SAS, Watsonova 47, 040 01 Košice (Slovakia); Frank Laboratory of Neutron Physics, Joint Institute for Nuclear Research, Joliot-Curie 6, 141980 Dubna, Moscow Region (Russian Federation); Tomašovičová, Natália, E-mail: nhudak@saske.sk [Institute of Experimental Physics SAS, Watsonova 47, 040 01 Košice (Slovakia); Frank Laboratory of Neutron Physics, Joint Institute for Nuclear Research, Joliot-Curie 6, 141980 Dubna, Moscow Region (Russian Federation); Batko, Ivan; Batková, Marianna; Balejčíková, Lucia [Institute of Experimental Physics SAS, Watsonova 47, 040 01 Košice (Slovakia); Garamus, Vasyl M. [Helmholtz-Zentrum Geesthacht: Zentrum fr Material, und Kstenforschung GmbH, Max-Plank-Strae 1, Geesthacht 216502 (Germany); Petrenko, Viktor I. [Frank Laboratory of Neutron Physics, Joint Institute for Nuclear Research, Joliot-Curie 6, 141980 Dubna, Moscow Region (Russian Federation); Physics Department, Taras Shevchenko Kyiv National University, Volodymyrska Street 64, 01601 Kyiv (Ukraine); Avdeev, Mikhail V. [Frank Laboratory of Neutron Physics, Joint Institute for Nuclear Research, Joliot-Curie 6, 141980 Dubna, Moscow Region (Russian Federation); Kopčanský, Peter [Institute of Experimental Physics SAS, Watsonova 47, 040 01 Košice (Slovakia)

    2017-06-01

    This work is devoted to the structural study of complex solutions of magnetic nanoparticles with lysozyme amyloid fibrils due to possible ordering of such system by applying the external magnetic field. The interaction of magnetic nanoparticles with amyloid fibrils has been followed by atomic force microscopy and small-angle X-ray scattering. It has been observed that magnetic nanoparticles (MNPs) adsorb to lysozyme amyloid fibrils. It was found that MNPs alter amyloids structures, namely the diameter of lysozyme amyloid fibrils is increased whereas the length of fibrils is decreased. In the same time MNPs do not change the helical pitch significantly. - Highlights: • Solution of MNPs with lysozyme amyloid fibrils was characterized by AFM and SAXS. • MNPs adsorb to lysozyme amyloid fibrils. • Diameter and size of lysozyme amyloid fibrils change due to doping with MNPs.

  14. Hsp40 interacts directly with the native state of the yeast prion protein Ure2 and inhibits formation of amyloid-like fibrils.

    Science.gov (United States)

    Lian, Hui-Yong; Zhang, Hong; Zhang, Zai-Rong; Loovers, Harriët M; Jones, Gary W; Rowling, Pamela J E; Itzhaki, Laura S; Zhou, Jun-Mei; Perrett, Sarah

    2007-04-20

    Ure2 is the protein determinant of the [URE3] prion phenotype in Saccharomyces cerevisiae and consists of a flexible N-terminal prion-determining domain and a globular C-terminal glutathione transferase-like domain. Overexpression of the type I Hsp40 member Ydj1 in yeast cells has been found to result in the loss of [URE3]. However, the mechanism of prion curing by Ydj1 remains unclear. Here we tested the effect of overexpression of Hsp40 members Ydj1, Sis1, and Apj1 and also Hsp70 co-chaperones Cpr7, Cns1, Sti1, and Fes1 in vivo and found that only Ydj1 showed a strong curing effect on [URE3]. We also investigated the interaction of Ydj1 with Ure2 in vitro. We found that Ydj1 was able to suppress formation of amyloid-like fibrils of Ure2 by delaying the process of fibril formation, as monitored by thioflavin T binding and atomic force microscopy imaging. Controls using bovine serum albumin, Sis1, or the human Hsp40 homologues Hdj1 or Hdj2 showed no significant inhibitory effect. Ydj1 was only effective when added during the lag phase of fibril formation, suggesting that it interacts with Ure2 at an early stage in fibril formation and delays the nucleation process. Using surface plasmon resonance and size exclusion chromatography, we demonstrated a direct interaction between Ydj1 and both wild type and N-terminally truncated Ure2. In contrast, Hdj2, which did not suppress fibril formation, did not show this interaction. The results suggest that Ydj1 inhibits Ure2 fibril formation by binding to the native state of Ure2, thus delaying the onset of oligomerization.

  15. AL amyloid imaging and therapy with a monoclonal antibody to a cryptic epitope on amyloid fibrils.

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    Jonathan S Wall

    Full Text Available The monoclonal antibody 2A4 binds an epitope derived from a cleavage site of serum amyloid protein A (sAA containing a -Glu-Asp- amino acid pairing. In addition to its reactivity with sAA amyloid deposits, the antibody was also found to bind amyloid fibrils composed of immunoglobulin light chains. The antibody binds to synthetic fibrils and human light chain (AL amyloid extracts with high affinity even in the presence of soluble light chain proteins. Immunohistochemistry with biotinylated 2A4 demonstrated positive reaction with ALκ and ALλ human amyloid deposits in various organs. Surface plasmon resonance analyses using synthetic AL fibrils as a substrate revealed that 2A4 bound with a K(D of ∼10 nM. Binding was inhibited in the presence of the -Glu-Asp- containing immunogen peptide. Radiolabeled 2A4 specifically localized with human AL amyloid extracts implanted in mice (amyloidomas as evidenced by single photon emission (SPECT imaging. Furthermore, co-localization of the radiolabeled mAb with amyloid was shown in biodistribution and micro-autoradiography studies. Treatment with 2A4 expedited regression of ALκ amyloidomas in mice, likely mediated by the action of macrophages and neutrophils, relative to animals that received a control antibody. These data indicate that the 2A4 mAb might be of interest for potential imaging and immunotherapy in patients with AL amyloidosis.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sievers, Stuart A.; Karanicolas, John; Chang, Howard W.; Zhao, Anni; Jiang, Lin; Zirafi, Onofrio; Stevens, Jason T.; Münch, Jan; Baker, David; Eisenberg, David (UCLA); (UWASH); (UL); (Kansas); (Ulm)

    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.

  17. Immunoprecipitation of amyloid fibrils by the use of an antibody that recognizes a generic epitope common to amyloid fibrils.

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    Erin R Greiner

    Full Text Available Amyloid fibrils are associated with many maladies, including Alzheimer's disease (AD. The isolation of amyloids from natural materials is very challenging because the extreme structural stability of amyloid fibrils makes it difficult to apply conventional protein science protocols to their purification. A protocol to isolate and detect amyloids is desired for the diagnosis of amyloid diseases and for the identification of new functional amyloids. Our aim was to develop a protocol to purify amyloid from organisms, based on the particular characteristics of the amyloid fold, such as its resistance to proteolysis and its capacity to be recognized by specific conformational antibodies. We used a two-step strategy with proteolytic digestion as the first step followed by immunoprecipitation using the amyloid conformational antibody LOC. We tested the efficacy of this method using as models amyloid fibrils produced in vitro, tissue extracts from C. elegans that overexpress Aβ peptide, and cerebrospinal fluid (CSF from patients diagnosed with AD. We were able to immunoprecipitate Aβ(1-40 amyloid fibrils, produced in vitro and then added to complex biological extracts, but not α-synuclein and gelsolin fibrils. This method was useful for isolating amyloid fibrils from tissue homogenates from a C. elegans AD model, especially from aged worms. Although we were able to capture picogram quantities of Aβ(1-40 amyloid fibrils produced in vitro when added to complex biological solutions, we could not detect any Aβ amyloid aggregates in CSF from AD patients. Our results show that although immunoprecipitation using the LOC antibody is useful for isolating Aβ(1-40 amyloid fibrils, it fails to capture fibrils of other amyloidogenic proteins, such as α-synuclein and gelsolin. Additional research might be needed to improve the affinity of these amyloid conformational antibodies for an array of amyloid fibrils without compromising their selectivity before

  18. How curcumin affords effective protection against amyloid fibrillation in insulin?

    DEFF Research Database (Denmark)

    Rabiee, Atefeh; Ebrahim Habibi, Azadeh; Ghasemi, Atiyeh Ghasemi

    2013-01-01

    Since the formation of amyloid structures from proteins was recognized in numerous diseases, many efforts have been devoted to the task of finding effective anti-amyloidogenic compounds. In a number of these investigations, the existence of “generic” compounds is implicitly acknowledged. Curcumin...... been shown effectively influenced by micro molar concentrations of curcumin. Under amyloidogenic conditions (pH 2.5 and 37°C), the compound was observed to inhibit fibril formation of insulin in a dose-dependent manner. Moreover, addition of curcumin to the protein incubated in such conditions...... at different time points resulted in reduced amounts of final fibrils. Disaggregation of pre-formed fibrils was also observed upon addition of curcumin, as well as reduction in final fibril amounts after seeding. Overall, this compound appears to be able to interact with native, intermediate and fibrillar...

  19. Solid-State-NMR-Structure-Based Inhibitor Design to Achieve Selective Inhibition of the Parallel-in-Register β-Sheet versus Antiparallel Iowa Mutant β-Amyloid Fibrils.

    Science.gov (United States)

    Cheng, Qinghui; Qiang, Wei

    2017-06-08

    Solid-state nuclear magnetic resonance (ssNMR) spectroscopy has been widely applied to characterize the high-resolution structures of β-amyloid (Aβ) fibrils. While these structures provide crucial molecular insights on the deposition of amyloid plaques in Alzheimer's diseases (AD), ssNMR structures have been rarely used so far as the basis for designing inhibitors. It remains a challenge because the ssNMR-based Aβ fibril structures were usually obtained with sparsely isotope-labeled peptides with limited experimental constraints, where the structural models, especially the side-chain coordinates, showed restricted precision. However, these structural models often possess a higher accuracy within the hydrophobic core regions with more well-defined experimental data, which provide potential targets for the molecular design. This work presents an ssNMR-based molecular design to achieve selective inhibition of a particular type of Aβ fibrillar structure, which was formed with the Iowa mutant of Aβ with parallel-in-register β-sheet hydrophobic core. The results show that short peptides that mimic the C-terminal β-strands of the fibril may have a preference in binding to the parallel Aβ fibrils rather than the antiparallel fibrils, mainly due to the differences in the high-resolution structures in the fibril elongation interfaces. The Iowa mutant Aβ fibrils are utilized in this work mainly as a model to demonstrate the feasibility of the strategy because it is relatively straightforward to distinguish the parallel and antiparallel fibril structures using ssNMR. Our results suggest that it is potentially feasible to design structure-selective inhibitors and/or diagnostic agents to Aβ fibrils using ssNMR-based structural models.

  20. Kinetically controlled thermal response of beta2-microglobulin amyloid fibrils.

    Science.gov (United States)

    Sasahara, Kenji; Naiki, Hironobu; Goto, Yuji

    2005-09-23

    Calorimetric measurements were carried out using a differential scanning calorimeter in the temperature range from 10 to 120 degrees C for characterizing the thermal response of beta2-microglobulin amyloid fibrils. The thermograms of amyloid fibril solution showed a remarkably large decrease in heat capacity that was essentially released upon the thermal unfolding of the fibrils, in which the magnitude of negative heat capacity change was not explicable in terms of the current accessible surface area model of protein structural thermodynamics. The heat capacity-temperature curve of amyloid fibrils prior to the fibril unfolding exhibited an unusual dependence on the fibril concentration and the heating rate. Particularly, the heat needed to induce the thermal response was found to be linearly dependent on the heating rate, indicating that its thermal response is under a kinetic control and precluding the interpretation in terms of equilibrium thermodynamics. Furthermore, amyloid fibrils of amyloid beta peptides also exhibited a heating rate-dependent exothermic process before the fibril unfolding, indicating that the kinetically controlled thermal response may be a common phenomenon to amyloid fibrils. We suggest that the heating rate-dependent negative change in heat capacity is coupled to the association of amyloid fibrils with characteristic hydration pattern.

  1. Influence of Aluminium and EGCG on Fibrillation and Aggregation of Human Islet Amyloid Polypeptide

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    Zhi-Xue Xu

    2016-01-01

    Full Text Available The abnormal fibrillation of human islet amyloid polypeptide (hIAPP has been implicated in the development of type II diabetes. Aluminum is known to trigger the structural transformation of many amyloid proteins and induce the formation of toxic aggregate species. The (−-epigallocatechin gallate (EGCG is considered capable of binding both metal ions and amyloid proteins with inhibitory effect on the fibrillation of amyloid proteins. However, the effect of Al(III/EGCG complex on hIAPP fibrillation is unclear. In the present work, we sought to view insight into the structures and properties of Al(III and EGCG complex by using spectroscopic experiments and quantum chemical calculations and also investigated the influence of Al(III and EGCG on hIAPP fibrillation and aggregation as well as their combined interference on this process. Our studies demonstrated that Al(III could promote fibrillation and aggregation of hIAPP, while EGCG could inhibit the fibrillation of hIAPP and lead to the formation of hIAPP amorphous aggregates instead of the ordered fibrils. Furthermore, we proved that the Al(III/EGCG complex in molar ratio of 1 : 1 as Al(EGCG(H2O2 could inhibit the hIAPP fibrillation more effectively than EGCG alone. The results provide the invaluable reference for the new drug development to treat type II diabetes.

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

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    Meenakshi Verma

    2015-01-01

    Full Text Available 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.

  3. The Surprising Role of Amyloid Fibrils in HIV Infection

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    James Shorter

    2012-05-01

    Full Text Available Despite its discovery over 30 years ago, human immunodeficiency virus (HIV continues to threaten public health worldwide. Semen is the principal vehicle for the transmission of this retrovirus and several endogenous peptides in semen, including fragments of prostatic acid phosphatase (PAP248-286 and PAP85-120 and semenogelins (SEM1 and SEM2, assemble into amyloid fibrils that promote HIV infection. For example, PAP248-286 fibrils, termed SEVI (Semen derived Enhancer of Viral Infection, potentiate HIV infection by up to 105-fold. Fibrils enhance infectivity by facilitating virion attachment and fusion to target cells, whereas soluble peptides have no effect. Importantly, the stimulatory effect is greatest at low viral titers, which mimics mucosal transmission of HIV, where relatively few virions traverse the mucosal barrier. Devising a method to rapidly reverse fibril formation (rather than simply inhibit it would provide an innovative and urgently needed preventative strategy for reducing HIV infection via the sexual route. Targeting a host-encoded protein conformer represents a departure from traditional microbicidal approaches that target the viral machinery, and could synergize with direct antiviral approaches. Here, we review the identification of these amyloidogenic peptides, their mechanism of action, and various strategies for inhibiting their HIV-enhancing effects.

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

    Directory of Open Access Journals (Sweden)

    Helen P McWilliams-Koeppen

    Full Text Available 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(PH-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.

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

    Directory of Open Access Journals (Sweden)

    Christin Bissig

    2016-08-01

    Full Text Available 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.

  6. Detection of amyloid fibrils in Parkinson's disease using plasmonic chirality.

    Science.gov (United States)

    Kumar, Jatish; Eraña, Hasier; López-Martínez, Elena; Claes, Nathalie; Martín, Víctor F; Solís, Diego M; Bals, Sara; Cortajarena, Aitziber L; Castilla, Joaquín; Liz-Marzán, Luis M

    2018-03-27

    Amyloid fibrils, which are closely associated with various neurodegenerative diseases, are the final products in many protein aggregation pathways. The identification of fibrils at low concentration is, therefore, pivotal in disease diagnosis and development of therapeutic strategies. We report a methodology for the specific identification of amyloid fibrils using chiroptical effects in plasmonic nanoparticles. The formation of amyloid fibrils based on α-synuclein was probed using gold nanorods, which showed no apparent interaction with monomeric proteins but effective adsorption onto fibril structures via noncovalent interactions. The amyloid structure drives a helical nanorod arrangement, resulting in intense optical activity at the surface plasmon resonance wavelengths. This sensing technique was successfully applied to human brain homogenates of patients affected by Parkinson's disease, wherein protein fibrils related to the disease were identified through chiral signals from Au nanorods in the visible and near IR, whereas healthy brain samples did not exhibit any meaningful optical activity. The technique was additionally extended to the specific detection of infectious amyloids formed by prion proteins, thereby confirming the wide potential of the technique. The intense chiral response driven by strong dipolar coupling in helical Au nanorod arrangements allowed us to detect amyloid fibrils down to nanomolar concentrations.

  7. Structural origin of polymorphism of Alzheimer's amyloid β-fibrils.

    Science.gov (United States)

    Agopian, Audrey; Guo, Zhefeng

    2012-10-01

    Formation of senile plaques containing amyloid fibrils of Aβ (amyloid β-peptide) is a pathological hallmark of Alzheimer's disease. Unlike globular proteins, which fold into unique structures, the fibrils of Aβ and other amyloid proteins often contain multiple polymorphs. Polymorphism of amyloid fibrils leads to different toxicity in amyloid diseases and may be the basis for prion strains, but the structural origin for fibril polymorphism is still elusive. In the present study we investigate the structural origin of two major fibril polymorphs of Aβ40: an untwisted polymorph formed under agitated conditions and a twisted polymorph formed under quiescent conditions. Using electron paramagnetic resonance spectroscopy, we studied the inter-strand side-chain interactions at 14 spin-labelled positions in the Aβ40 sequence. The results of the present study show that the agitated fibrils have stronger inter-strand spin-spin interactions at most of the residue positions investigated. The two hydrophobic regions at residues 17-20 and 31-36 have the strongest interactions in agitated fibrils. Distance estimates on the basis of the spin exchange frequencies suggest that inter-strand distances at residues 17, 20, 32, 34 and 36 in agitated fibrils are approximately 0.2 Å (1 Å=0.1 nm) closer than in quiescent fibrils. We propose that the strength of inter-strand side-chain interactions determines the degree of β-sheet twist, which then leads to the different association patterns between different cross β-units and thus distinct fibril morphologies. Therefore the inter-strand side-chain interaction may be a structural origin for fibril polymorphism in Aβ and other amyloid proteins.

  8. UV-light exposed prion protein fails to form amyloid fibrils.

    Directory of Open Access Journals (Sweden)

    Abhay Kumar Thakur

    Full Text Available Amyloid fibril formation involves three steps; structural perturbation, nucleation and elongation. We have investigated amyloidogenesis using prion protein as a model system and UV-light as a structural perturbant. We find that UV-exposed prion protein fails to form amyloid fibrils. Interestingly, if provided with pre-formed fibrils as seeds, UV-exposed prion protein formed amyloid fibrils albeit with slightly different morphology. Atomic force microscopy and electron microscopic studies clearly show the formation of fibrils under these conditions. Circular dichroism study shows loss in helicity in UV-exposed protein. UV-exposed prion protein fails to form amyloid fibrils. However, it remains competent for fibril extension, suggesting that UV-exposure results in loss of nucleating capability. This work opens up possibility of segregating nucleation and elongation step of amyloidogenesis, facilitating screening of new drug candidates for specifically inhibiting either of these processes. In addition, the work also highlights the importance of light-induced structural and functional alterations which are important in protein based therapeutics.

  9. Destroying activity of magnetoferritin on lysozyme amyloid fibrils

    Energy Technology Data Exchange (ETDEWEB)

    Kopcansky, Peter; Siposova, Katarina [Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Kosice (Slovakia); Melnikova, Lucia, E-mail: melnikova@saske.sk [Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Kosice (Slovakia); Bednarikova, Zuzana [Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Kosice (Slovakia); Institute of Chemical Sciences, Faculty of Sciences, Safarik University, Kosice (Slovakia); Timko, Milan; Mitroova, Zuzana; Antosova, Andrea [Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Kosice (Slovakia); Garamus, Vasil M. [Helmholtz-Zentrum Geesthacht: Centre for Materials and Coastal Research, Max-Planck-Street 1, 21502 Geesthacht (Germany); Petrenko, Viktor I. [Joint Institute for Nuclear Research, Joliot-Curie 6, Dubna, 141980 Moscow Region (Russian Federation); Kyiv Taras Shevchenko National University, Volodymyrska Street 64, Kyiv 01033 (Ukraine); Avdeev, Mikhail V. [Joint Institute for Nuclear Research, Joliot-Curie 6, Dubna, 141980 Moscow Region (Russian Federation); Gazova, Zuzana [Institute of Experimental Physics, SAS, Watsonova 47, 040 01 Kosice (Slovakia); Department of Medical and Clinical Biochemistry and LABMED, Tr. SNP 1, 040 11 Kosice (Slovakia)

    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. - Highlights: • 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.

  10. General amyloid inhibitors? A critical examination of the inhibition of IAPP amyloid formation by inositol stereoisomers.

    Directory of Open Access Journals (Sweden)

    Hui Wang

    Full Text Available Islet amyloid polypeptide (IAPP or amylin forms amyloid deposits in the islets of Langerhans; a process that is believed to contribute to the progression of type 2 diabetes and to the failure of islet transplants. An emerging theme in amyloid research is the hypothesis that the toxic species produced during amyloid formation by different polypeptides share common features and exert their effects by common mechanisms. If correct, this suggests that inhibitors of amyloid formation by one polypeptide might be effective against other amyloidogenic sequences. IAPP and Aβ, the peptide responsible for amyloid formation in Alzheimer's disease, are particularly interesting in this regard as they are both natively unfolded in their monomeric states and share some common characteristics. Comparatively little effort has been expended on the design of IAPP amyloid inhibitors, thus it is natural to inquire if Aβ inhibitors are effective against IAPP, especially since no IAPP inhibitors have been clinically approved. A range of compounds inhibitamyloid formation, including various stereoisomers of inositol. Myo-, scyllo-, and epi-inositol have been shown to induce conformational changes in Aβ and prevent Aβ amyloid fibril formation by stabilizing non-fibrillar β-sheet structures. We investigate the ability of inositol stereoisomers to inhibit amyloid formation by IAPP. The compounds do not induce a conformational change in IAPP and are ineffective inhibitors of IAPP amyloid formation, although some do lead to modest apparent changes in IAPP amyloid fibril morphology. Thus not all classes of Aβ inhibitors are effective against IAPP. This work provides a basis of comparison to work on polyphenol based inhibitors of IAPP amyloid formation and helps provide clues as to the features which render them effective. The study also helps provide information for further efforts in rational inhibitor design.

  11. General Amyloid Inhibitors? A Critical Examination of the Inhibition of IAPP Amyloid Formation by Inositol Stereoisomers

    Science.gov (United States)

    Wang, Hui; Raleigh, Daniel P.

    2014-01-01

    Islet amyloid polypeptide (IAPP or amylin) forms amyloid deposits in the islets of Langerhans; a process that is believed to contribute to the progression of type 2 diabetes and to the failure of islet transplants. An emerging theme in amyloid research is the hypothesis that the toxic species produced during amyloid formation by different polypeptides share common features and exert their effects by common mechanisms. If correct, this suggests that inhibitors of amyloid formation by one polypeptide might be effective against other amyloidogenic sequences. IAPP and Aβ, the peptide responsible for amyloid formation in Alzheimer's disease, are particularly interesting in this regard as they are both natively unfolded in their monomeric states and share some common characteristics. Comparatively little effort has been expended on the design of IAPP amyloid inhibitors, thus it is natural to inquire if Aβ inhibitors are effective against IAPP, especially since no IAPP inhibitors have been clinically approved. A range of compounds inhibitamyloid formation, including various stereoisomers of inositol. Myo-, scyllo-, and epi-inositol have been shown to induce conformational changes in Aβ and prevent Aβ amyloid fibril formation by stabilizing non-fibrillar β-sheet structures. We investigate the ability of inositol stereoisomers to inhibit amyloid formation by IAPP. The compounds do not induce a conformational change in IAPP and are ineffective inhibitors of IAPP amyloid formation, although some do lead to modest apparent changes in IAPP amyloid fibril morphology. Thus not all classes of Aβ inhibitors are effective against IAPP. This work provides a basis of comparison to work on polyphenol based inhibitors of IAPP amyloid formation and helps provide clues as to the features which render them effective. The study also helps provide information for further efforts in rational inhibitor design. PMID:25260075

  12. Analysis of amyloid fibrils in the cheetah (Acinonyx jubatus).

    Science.gov (United States)

    Bergström, Joakim; Ueda, Mitsuharu; Une, Yumi; Sun, Xuguo; Misumi, Shogo; Shoji, Shozo; Ando, Yukio

    2006-06-01

    Recently, a high prevalence of amyloid A (AA) amyloidosis has been documented among captive cheetahs worldwide. Biochemical analysis of amyloid fibrils extracted from the liver of a Japanese captive cheetah unequivocally showed that protein AA was the main fibril constituent. Further characterization of the AA fibril components by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis revealed three main protein AA bands with approximate molecular weights of 8, 10 and 12 kDa. Mass spectrometry analysis of the 12-kDa component observed in SDS-PAGE and Western blotting confirmed the molecular weight of a 12,381-Da peak. Our finding of a 12-kDa protein AA component provides evidence that the cheetah SAA sequence is longer than the previously reported 90 amino acid residues (approximately 10 kDa), and hence SAA is part of the amyloid fibril.

  13. Ferulic acid destabilizes preformed β-amyloid fibrils in vitro

    International Nuclear Information System (INIS)

    Ono, Kenjiro; Hirohata, Mie; Yamada, Masahito

    2005-01-01

    Inhibition of the formation of β-amyloid fibrils (fAβ), as well as the destabilization of preformed fAβ in the CNS, would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). We reported previously that curcumin (Cur) inhibits fAβ formation from Aβ and destabilizes preformed fAβ 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 fAβ 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 fAβ formation from amyloid β-peptide, as well as their extension. Moreover, it destabilized preformed fAβs. 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

  14. Cold denaturation of α-synuclein amyloid fibrils.

    Science.gov (United States)

    Ikenoue, Tatsuya; Lee, Young-Ho; Kardos, József; Saiki, Miyu; Yagi, Hisashi; Kawata, Yasushi; Goto, Yuji

    2014-07-21

    Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. α-Synuclein fibrils cold-denatured to monomers at 0-20 °C and heat-denatured at 60-110 °C. Meanwhile, the fibrils of β2-microglobulin, Alzheimer's Aβ1-40/Aβ1-42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in stability at low temperature. A comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in fibril cores contributed to the cold denaturation of α-synuclein fibrils. We propose that although cold-denaturation is common to both native proteins and misfolded fibrillar states, the main-chain dominated amyloid structures may explain amyloid-specific cold denaturation arising from the unfavorable burial of charged side-chains in fibril cores. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Human islet amyloid polypeptide fibril binding to catalase: a transmission electron microscopy and microplate study.

    Science.gov (United States)

    Milton, Nathaniel G N; Harris, J Robin

    2010-05-18

    The diabetes-associated human islet amyloid polypeptide (IAPP) is a 37-amino-acid peptide that forms fibrils in vitro and in vivo. Human IAPP fibrils are toxic in a similar manner to Alzheimer's amyloid-beta (A-beta) and prion protein (PrP) fibrils. Previous studies have shown that catalase binds to A-beta fibrils and appears to recognize a region containing the Gly-Ala-Ile-Ile sequence that is similar to the Gly-Ala-Ile-Leu sequence found in human IAPP residues 24-27. This study presents a transmission electron microscopy (TEM)-based analysis of fibril formation and the binding of human erythrocyte catalase to IAPP fibrils. The results show that human IAPP 1-37, 8-37, and 20-29 peptides form fibrils with diverse and polymorphic structures. All three forms of IAPP bound catalase, and complexes of IAPP 1-37 or 8-37 with catalase were identified by immunoassay. The binding of biotinylated IAPP to catalase was high affinity with a KD of 0.77 nM, and could be inhibited by either human or rat IAPP 1-37 and 8-37 forms. Fibrils formed by the PrP 118-135 peptide with a Gly-Ala-Val-Val sequence also bound catalase. These results suggest that catalase recognizes a Gly-Ala-Ile-Leu-like sequence in amyloid fibril-forming peptides. For IAPP 1-37 and 8-37, the catalase binding was primarily directed towards fibrillar rather than ribbon-like structures, suggesting differences in the accessibility of the human IAPP 24-27 Gly-Ala-Ile-Leu region. This suggests that catalase may be able to discriminate between different structural forms of IAPP fibrils. The ability of catalase to bind IAPP, A-beta, and PrP fibrils demonstrates the presence of similar accessible structural motifs that may be targets for antiamyloid therapeutic development.

  16. Human Islet Amyloid Polypeptide Fibril Binding to Catalase: A Transmission Electron Microscopy and Microplate Study

    Directory of Open Access Journals (Sweden)

    Nathaniel G. N. Milton

    2010-01-01

    Full Text Available The diabetes-associated human islet amyloid polypeptide (IAPP is a 37-amino-acid peptide that forms fibrils in vitro and in vivo. Human IAPP fibrils are toxic in a similar manner to Alzheimer's amyloid-β (Aβ and prion protein (PrP fibrils. Previous studies have shown that catalase binds to Aβ fibrils and appears to recognize a region containing the Gly-Ala-Ile-Ile sequence that is similar to the Gly-Ala-Ile-Leu sequence found in human IAPP residues 24-27. This study presents a transmission electron microscopy (TEM—based analysis of fibril formation and the binding of human erythrocyte catalase to IAPP fibrils. The results show that human IAPP 1-37, 8-37, and 20-29 peptides form fibrils with diverse and polymorphic structures. All three forms of IAPP bound catalase, and complexes of IAPP 1-37 or 8-37 with catalase were identified by immunoassay. The binding of biotinylated IAPP to catalase was high affinity with a KD of 0.77nM, and could be inhibited by either human or rat IAPP 1-37 and 8-37 forms. Fibrils formed by the PrP 118-135 peptide with a Gly-Ala-Val-Val sequence also bound catalase. These results suggest that catalase recognizes a Gly-Ala-Ile-Leu—like sequence in amyloid fibril-forming peptides. For IAPP 1-37 and 8-37, the catalase binding was primarily directed towards fibrillar rather than ribbon-like structures, suggesting differences in the accessibility of the human IAPP 24-27 Gly-Ala-Ile-Leu region. This suggests that catalase may be able to discriminate between different structural forms of IAPP fibrils. The ability of catalase to bind IAPP, Aβ, and PrP fibrils demonstrates the presence of similar accessible structural motifs that may be targets for antiamyloid therapeutic development.

  17. Catechins and procyanidins of Ginkgo biloba show potent activities towards the inhibition of β-amyloid peptide aggregation and destabilization of preformed fibrils.

    Science.gov (United States)

    Xie, Haiyan; Wang, Jing-Rong; Yau, Lee-Fong; Liu, Yong; Liu, Liang; Han, Quan-Bin; Zhao, Zhongzhen; Jiang, Zhi-Hong

    2014-04-22

    Catechins and procyanidins, together with flavonoid glycosides and terpene trilactones, are three important categories of components in the standard extract of Ginkgo biloba leaves (EGb761). In this research, catechins and proanthocyanidins were found to exist in both the extract of Ginkgo leaves and Ginkgo products. By comparing with reference compounds, six of them were identified as (+)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin and procyanidins B1 and B3. The activities of these polyphenols in the inhibition of Aβ42 aggregation and the destabilization of preformed fibrils were evaluated using biochemical assays, which showed that all six of the polyphenols, as well as a fraction of the extract of Ginkgo biloba leaves (EGb) containing catechins and procyanidins, exerted potent inhibitory activities towards Aβ42 aggregation and could also destabilize the performed fibrils. Catechins and procyanidins can therefore be regarded as the potent active constituents of EGb761 in terms of their inhibition of Aβ42 aggregation and destabilization of the fibrils. Although quantitative mass spectroscopic analysis revealed that the catechins and procyanidins are only present in low concentrations in EGb761, these components should be studied in greater detail because of their potent inhibitory effects towards Aβ42 aggregation and their ability to destabilize preformed fibrils, especially during the quality control of Ginkgo leaves and the manufacture of Ginkgo products.

  18. Inhibition of Toxic IAPP Amyloid by Extracts of Common Fruits.

    Science.gov (United States)

    Kao, Pei-Yu; Green, Evangeline; Pereira, Catalina; Ekimura, Shauna; Juarez, Dennis; Whyte, Travis; Arhar, Taylor; Malaspina, Bianca; Nogaj, Luiza A; Moffet, David A

    2015-01-01

    The aggregation of the 37-amino acid polypeptide islet amyloid polypeptide (IAPP, amylin), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of pancreatic β-islet cells in type 2 diabetes. It is believed that inhibiting the aggregation of IAPP may slow down, if not prevent entirely, the progression of this disease. Extracts of thirteen different common fruits were analyzed for their ability to prevent the aggregation of amyloidogenic IAPP. Thioflavin T binding, immuno-detection and circular dichroism assays were performed to test the in vitro inhibitory potential of each extract. Atomic force microscopy was used to visualize the formation of amyloid fibrils with and without each fruit extract. Finally, extracts were tested for their ability to protect living mammalian cells from the toxic effects of amyloid IAPP. Several fruits showed substantial ability to inhibit IAPP aggregation and protect living cells from toxic IAPP amyloid.

  19. Aggregation properties of a short peptide that mediates amyloid fibril ...

    Indian Academy of Sciences (India)

    Short peptides have been identified from amyloidogenic proteins that form amyloid fibrils in isolation. The hexapeptide stretch 21DIDLHL26 has been shown to be important in the self-assembly of the Src homology 3 (SH3) domain of p85 subunit of bovine phosphatidylinositol-3-kinase (PI3-SH3). The SH3 domain of ...

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Sandra Arvidsson

    Full Text Available 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.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.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.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.

  2. Gold Nanoparticles and Microwave Irradiation Inhibit Beta-Amyloid Amyloidogenesis

    Directory of Open Access Journals (Sweden)

    Bastus Neus

    2008-01-01

    Full Text Available Abstract Peptide-Gold nanoparticles selectively attached to β-amyloid protein (Aβ amyloidogenic aggregates were irradiated with microwave. This treatment produces dramatic effects on the Aβ aggregates, inhibiting both the amyloidogenesis and the restoration of the amyloidogenic potential. This novel approach offers a new strategy to inhibit, locally and remotely, the amyloidogenic process, which could have application in Alzheimer’s disease therapy. We have studied the irradiation effect on the amyloidogenic process in the presence of conjugates peptide-nanoparticle by transmission electronic microscopy observations and by Thioflavine T assays to quantify the amount of fibrils in suspension. The amyloidogenic aggregates rather than the amyloid fibrils seem to be better targets for the treatment of the disease. Our results could contribute to the development of a new therapeutic strategy to inhibit the amyloidogenic process in Alzheimer’s disease.

  3. Interaction of the molecular chaperone DNAJB6 with growing amyloid-beta 42 (Aβ42) aggregates leads to sub-stoichiometric inhibition of amyloid formation

    NARCIS (Netherlands)

    Månsson, Cecilia; Arosio, Paolo; Hussein, Rasha; Kampinga, Harm H; Hashem, Reem M; Boelens, Wilbert C; Dobson, Christopher M; Knowles, Tuomas P J; Linse, Sara; Emanuelsson, Cecilia

    2014-01-01

    The human molecular chaperone protein DNAJB6 was recently found to inhibit the formation of amyloid fibrils from polyglutamine peptides associated with neurodegenerative disorders such as Huntington's disease. We show in the present study that DNAJB6 also inhibits amyloid formation by an even more

  4. Identification of a Common Binding Mode for Imaging Agents to Amyloid Fibrils from Molecular Dynamics Simulations

    DEFF Research Database (Denmark)

    Skeby, Katrine Kirkeby; Sørensen, Jesper; Schiøtt, Birgit

    2013-01-01

    Amyloid diseases are characterized by the misfolding and deposition of proteins in the body in the form of insoluble amyloid fibrils. Alzheimer’s disease and type 2 diabetes mellitus are two examples of amyloid diseases which are closely related both with respect to the atomic structures of the a...... binding modes for imaging agents is proposed to originate from subtle differences in amino acid composition of the surface grooves on an amyloid fibril, resulting in fine tuning of the binding affinities for a specific amyloid fibril....... experimentally due to the insoluble nature of amyloid fibrils. This study uses molecular dynamics simulations to investigate the interactions between 13 aromatic amyloid imaging agents, entailing 4 different organic scaffolds, and a model of an amyloid fibril. Clustering analysis combined with free energy...

  5. The impact of binding of macrocyclic metal complexes on amyloid fibrillization of insulin and lysozyme.

    Science.gov (United States)

    Kovalska, Vladyslava; Chernii, Svitlana; Cherepanov, Vsevolod; Losytskyy, Mykhaylo; Chernii, Victor; Varzatskii, Oleg; Naumovets, Anton; Yarmoluk, Sergiy

    2017-08-01

    Amyloid fibrils are insoluble protein aggregates whose accumulation in cells and tissues is connected with a range of pathological diseases. We studied the impact of 2 metal complexes (axially coordinated Hf phthalocyanine and iron (II) clathrochelate) on aggregation of insulin and lysozyme. For both proteins, the host-guest interaction with these compounds changes the kinetics of fibrillization and affects the morphology of final aggregates. The Hf phthalocyanine is a very efficient inhibitor of insulin fibrillization; in its presence, only very low amounts of fibrils with the diameters of 0.8 to 5 nm and spherical aggregates were found. Effective concentration of fibrillization inhibition (IC 50 ) was estimated to be 0.11 ± 0.04 μM. The clathrochelate induced the formation of thin fibrils with the diameters of 0.8 to 2.5 nm; IC 50 was estimated as 20 ± 9 μM. The lysozyme fibrillization remained quite intensive in the presence of the studied compounds; they induced the formation of long filaments (the length up to 2.5 μm, the diameters of 1.5-3.5 nm). These fibrils noticeably differed from those of free lysozyme short linear species (the diameters of 3-5 nm, the length up to 0.6 μm). Thinning and elongation of fibrils suggest that the metal complexes bind mainly to the grooves of protofilaments; this hinders the stacking of early aggregates or protofilaments together but does not hinder their growth. The image of the fibril separated into 2 protofilaments allows suggesting that the fibril formation occurs via the growth of the parallel protofilaments with their subsequent twisting in the fibril. The changes of the lysozyme intrinsic fluorescence indicate that both metal complexes interact with the protein during the stage of the fibrillar seeds formation. Copyright © 2017 John Wiley & Sons, Ltd.

  6. Amyloid fibril systems reduce, stabilize and deliver bioavailable nanosized iron

    Science.gov (United States)

    Shen, Yi; Posavec, Lidija; Bolisetty, Sreenath; Hilty, Florentine M.; Nyström, Gustav; Kohlbrecher, Joachim; Hilbe, Monika; Rossi, Antonella; Baumgartner, Jeannine; Zimmermann, Michael B.; Mezzenga, Raffaele

    2017-07-01

    Iron-deficiency anaemia (IDA) is a major global public health problem. A sustainable and cost-effective strategy to reduce IDA is iron fortification of foods, but the most bioavailable fortificants cause adverse organoleptic changes in foods. Iron nanoparticles are a promising solution in food matrices, although their tendency to oxidize and rapidly aggregate in solution severely limits their use in fortification. Amyloid fibrils are protein aggregates initially known for their association with neurodegenerative disorders, but recently described in the context of biological functions in living organisms and emerging as unique biomaterial building blocks. Here, we show an original application for these protein fibrils as efficient carriers for iron fortification. We use biodegradable amyloid fibrils from β-lactoglobulin, an inexpensive milk protein with natural reducing effects, as anti-oxidizing nanocarriers and colloidal stabilizers for iron nanoparticles. The resulting hybrid material forms a stable protein-iron colloidal dispersion that undergoes rapid dissolution and releases iron ions during acidic and enzymatic in vitro digestion. Importantly, this hybrid shows high in vivo iron bioavailability, equivalent to ferrous sulfate in haemoglobin-repletion and stable-isotope studies in rats, but with reduced organoleptic changes in foods. Feeding the rats with these hybrid materials did not result in abnormal iron accumulation in any organs, or changes in whole blood glutathione concentrations, inferring their primary safety. Therefore, these iron-amyloid fibril hybrids emerge as novel, highly effective delivery systems for iron in both solid and liquid matrices.

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

    Energy Technology Data Exchange (ETDEWEB)

    Okumura, Hisashi [Research Center for Computational Science, Institute for Molecular Science, Okazaki, Aichi 444-8585 (Japan); Department of Structural Molecular Science, The Graduate University for Advanced Studies, Okazaki, Aichi 444-8585 (Japan)

    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.

  8. Ascorbic acid inhibits human insulin aggregation and protects against amyloid induced cytotoxicity.

    Science.gov (United States)

    Alam, Parvez; Beg, Ayesha Zainab; Siddiqi, Mohammad Khursheed; Chaturvedi, Sumit Kumar; Rajpoot, Ravi Kant; Ajmal, Mohd Rehan; Zaman, Masihuz; Abdelhameed, Ali S; Khan, Rizwan Hasan

    2017-05-01

    Protein aggregation into oligomers and fibrils are associated with many human pathophysiologies. Compounds that modulate protein aggregation and interact with preformed fibrils and convert them to less toxic species, expect to serve as promising drug candidates and aid to the drug development efforts against aggregation diseases. In present study, the kinetics of amyloid fibril formation by human insulin (HI) and the anti-amyloidogenic activity of ascorbic acid (AA) were investigated by employing various spectroscopic, imaging and computational approaches. We demonstrate that ascorbic acid significantly inhibits the fibrillation of HI in a dose-dependent manner. Interestingly ascorbic acid destabilise the preformed amyloid fibrils and protects human neuroblastoma cell line (SH- SY5Y) against amyloid induced cytotoxicity. The present data signifies the role of ascorbic acid that can serve as potential molecule in preventing human insulin aggregation and associated pathophysiologies. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Quantitative morphological analysis reveals ultrastructural diversity of amyloid fibrils from alpha-synuclein mutants

    NARCIS (Netherlands)

    van Raaij, Martijn E; Segers-Nolten, Ine M J; Subramaniam, Vinod

    2006-01-01

    High resolution atomic force microscopy is a powerful tool to characterize nanoscale morphological features of protein amyloid fibrils. Comparison of fibril morphological properties between studies has been hampered by differences in analysis procedures and measurement error determination used by

  10. DIAGNOSTIC TOOLS FOR STRUCTURAL CHARACTERIZATION AND ELUCIDATION OF FIBRILS AND THEIR PRECURSORS IN AMYLOID FIBRIL FORMATION PATHWAY

    Directory of Open Access Journals (Sweden)

    Nidhi Kaur Bhatia

    2013-12-01

    Full Text Available The amyloid fibrils and their precursors are supposed to be responsible for several neurodegenerative diseases. Advances in recent experimental techniques rationalized our understanding on the mechanism of the amyloid fibril formation. The goal of this review is to revisit the various techniques used to diagnose the structural features of amyloid fibrils and their precursors, for a comprehensive view of the available tools, their advantages and disadvantages. The review will serve as a stepping stone for detailed understanding of each technique and its use as per specific requirements of a biological problem.

  11. Characterizing Structural Stability of Amyloid Motif Fibrils Mediated by Water Molecules.

    Science.gov (United States)

    Choi, Hyunsung; Chang, Hyun Joon; Lee, Myeongsang; Na, Sungsoo

    2017-04-05

    In biological systems, structural confinements of amyloid fibrils can be mediated by the role of water molecules. However, the underlying effect of the dynamic behavior of water molecules on structural stabilities of amyloid fibrils is still unclear. By performing molecular dynamics simulations, we investigate the dynamic features and the effect of interior water molecules on conformations and mechanical characteristics of various amyloid fibrils. We find that a specific mechanism induced by the dynamic properties of interior water molecules can affect diffusion of water molecules inside amyloid fibrils, inducing their different structural stabilities. The conformation of amyloid fibrils induced by interior water molecules show the fibrils' different mechanical features. We elucidate the role of confined and movable interior water molecules in structural stabilities of various amyloid fibrils. Our results offer insights not only in further understanding of mechanical features of amyloids as mediated by water molecules, but also in the fine-tuning of the functional abilities of amyloid fibrils for applications. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Cross-seeding of fibrils from two types of insulin induces new amyloid strains.

    Science.gov (United States)

    Surmacz-Chwedoruk, Weronika; Nieznańska, Hanna; Wójcik, Sławomir; Dzwolak, Wojciech

    2012-11-27

    The irreversibility and autocatalytic character of amyloidogenesis and the polymorphism of amyloid fibrils underlie the phenomenon of self-propagating strains, wherein the mother seed, rather than the seeding environment, determines the properties of daughter fibrils. Here we study the formation of amyloid fibrils from bovine insulin and the recombinant Lys(B31)-Arg(B32) human insulin analog. The two polypeptides are similar enough to cross-seed but, upon spontaneous aggregation, form amyloid fibrils with distinct spectral features in the infrared amide I' band region. When bovine insulin is cross-seeded with the analog amyloid (and vice versa), the shape, absorption maximum, and even fine fingerprint features of the amide I' band are passed from the mother to daughter fibrils with a high degree of fidelity. Although the differences in primary structure between bovine insulin and the Lys(B31)-Arg(B32) analog of human insulin lie outside of the polypeptide's critical amyloidogenic regions, they affect the secondary structure of fibrils, possibly the formation of intermolecular salt bridges, and the susceptibility to dissection and denaturation with dimethyl sulfoxide (DMSO). All these phenotypic features of mother fibrils are imprinted in daughter amyloid upon cross-seeding. Analysis of noncooperative DMSO-induced denaturation of daughter fibrils suggests that the self-propagating polymorphism underlying the emergence of new amyloid strains is encoded on the level of secondary structure. Our findings have been discussed in the context of polymorphism of fibrils, amyloid strains, and possible implications for mechanisms of amyloidogenesis.

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

    Directory of Open Access Journals (Sweden)

    Gwonchan Yoon

    Full Text Available 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.

  14. Infrared Probe Technique Reveals a Millipede-like Structure for Aβ(8-28) Amyloid Fibril.

    Science.gov (United States)

    Gao, Yachao; Zou, Ye; Ma, Yan; Wang, Dan; Sun, Ying; Ma, Gang

    2016-02-02

    Amyloid fibrils are unique fibrous polypeptide aggregates. They have been associated with more than 20 serious human diseases including Alzheimer's disease and Parkinson's disease. Besides their pathological significance, amyloid fibrils are also gaining increasing attention as emerging nanomaterials with novel functions. Structural characterization of amyloid fibril is no doubt fundamentally important for the development of therapeutics for amyloid-related diseases and for the rational design of amyloid-based materials. In this study, we explored to use side-chain-based infrared (IR) probe to gain detailed structural insights into the amyloid fibril by a 21-residue model amyloidogenic peptide, Aβ(8-28). We first proposed an approach to incorporate thiocyanate (SCN) IR probe in a site-specific manner into amyloidogenic peptide using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate as cyanylating agent. Using this approach, we obtained three Aβ(8-28) variants, labeled with SCN probe at three different positions. We then showed with thioflavin T fluorescence assay, Congo red assay, and atomic force microscopy that the three labeled Aβ(8-28) peptides can quickly form amyloid fibrils under high concentration and high salt conditions. Finally, we performed a detailed IR spectral analysis of the Aβ(8-28) fibril in both amide I and probe regions and proposed a millipede-like structure for the Aβ(8-28) fibril.

  15. Disulfide bridges remain intact while native insulin converts into amyloid fibrils.

    Directory of Open Access Journals (Sweden)

    Dmitry Kurouski

    Full Text Available Amyloid fibrils are β-sheet-rich protein aggregates commonly found in the organs and tissues of patients with various amyloid-associated diseases. Understanding the structural organization of amyloid fibrils can be beneficial for the search of drugs to successfully treat diseases associated with protein misfolding. The structure of insulin fibrils was characterized by deep ultraviolet resonance Raman (DUVRR and Nuclear Magnetic Resonance (NMR spectroscopy combined with hydrogen-deuterium exchange. The compositions of the fibril core and unordered parts were determined at single amino acid residue resolution. All three disulfide bonds of native insulin remained intact during the aggregation process, withstanding scrambling. Three out of four tyrosine residues were packed into the fibril core, and another aromatic amino acid, phenylalanine, was located in the unordered parts of insulin fibrils. In addition, using all-atom MD simulations, the disulfide bonds were confirmed to remain intact in the insulin dimer, which mimics the fibrillar form of insulin.

  16. Spectroscopic study of Alzheimer's amyloid fibrils using terahertz time domain spectroscopy

    International Nuclear Information System (INIS)

    Jung, Euna; Kim, Jeonghoi; Han, Younho; Moon, Kiwon; Lim, Meehyun; Han, Haewook; Park, Joonhyuck; Kim, Sungjee

    2008-01-01

    Alzheimer's disease, one of the most common neurodegenerative diseases, is characterized by extensive amyloid deposition. Amyloid deposits contain the abundant fibrils formed by amyloid β protein (Aβ). Because amyloid fibrils are associated with amyloid diseases, including Alzheimer's disease, type 2 diabetes, prion disease, Parkinson's disease, senile systemic amyloidosis and Huntington's disease, there has been considerable interest within the biomedical and biochemical research communities. In transmission electron microscopic (TEM)images, amyloid firils are 0.1∼10μm long and approximately 10nm wide. Amyloid fibrils commonly exhibit self assembled filaments, often described as twisted or parallel assemblies of finer protofilaments. They are formed by the spontaneous aggregation of a wide variety of peptides and proteins. Structural studies of amyloid fibrils have revealed that the common structural motif of virtually all amyloid fibrils consists of cross β sheets in which the peptide strands are arranged perpendicular to the long axis of the fiber. But little was known until recently about the molecular level structures of amyloid fibils. Therefore, spectroscopic investigation of both amyloid fibrils and Aβ at the molecular level can provide the significant evidence for the molecular understanding of amyloidogenesis and for the development of innovative therapeutic and diagnostic approaches. We used terahertz time domain spectroscopy (THz TDS)to investigate both Aβ and amyloid fibril. THz TDS, developed over the last two decades, is a powerful tool to extract the properties of biomaterials and provides unique spectral signatures of biomolecules within 0.1∼10THz, which exists between microwave and infrared frequency range. Current interest in THz radiation arises from its capability of probing the delocalized collective vibrational modes in proteins. Studying the collective modes of proteins in THz frequency range can play an

  17. Spectroscopic study of Alzheimer's amyloid fibrils using terahertz time domain spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Euna; Kim, Jeonghoi; Han, Younho; Moon, Kiwon; Lim, Meehyun; Han, Haewook; Park, Joonhyuck; Kim, Sungjee [POSTECH, Pohang (Korea, Republic of)

    2008-11-15

    Alzheimer's disease, one of the most common neurodegenerative diseases, is characterized by extensive amyloid deposition. Amyloid deposits contain the abundant fibrils formed by amyloid β protein (Aβ). Because amyloid fibrils are associated with amyloid diseases, including Alzheimer's disease, type 2 diabetes, prion disease, Parkinson's disease, senile systemic amyloidosis and Huntington's disease, there has been considerable interest within the biomedical and biochemical research communities. In transmission electron microscopic (TEM)images, amyloid firils are 0.1∼10μm long and approximately 10nm wide. Amyloid fibrils commonly exhibit self assembled filaments, often described as twisted or parallel assemblies of finer protofilaments. They are formed by the spontaneous aggregation of a wide variety of peptides and proteins. Structural studies of amyloid fibrils have revealed that the common structural motif of virtually all amyloid fibrils consists of cross β sheets in which the peptide strands are arranged perpendicular to the long axis of the fiber. But little was known until recently about the molecular level structures of amyloid fibils. Therefore, spectroscopic investigation of both amyloid fibrils and Aβ at the molecular level can provide the significant evidence for the molecular understanding of amyloidogenesis and for the development of innovative therapeutic and diagnostic approaches. We used terahertz time domain spectroscopy (THz TDS)to investigate both Aβ and amyloid fibril. THz TDS, developed over the last two decades, is a powerful tool to extract the properties of biomaterials and provides unique spectral signatures of biomolecules within 0.1∼10THz, which exists between microwave and infrared frequency range. Current interest in THz radiation arises from its capability of probing the delocalized collective vibrational modes in proteins. Studying the collective modes of proteins in THz frequency range can play an

  18. Amyloid Fibril-Induced Structural and Spectral Modifications in the Thioflavin-T Optical Probe

    DEFF Research Database (Denmark)

    Murugan, N. Arul; Olsen, Jógvan Magnus Haugaard; Kongsted, Jacob

    2013-01-01

    Motivated by future possibilities to design target molecules for fibrils with diagnostic or therapeutic capability related to amyloidosis diseases, we investigate in this work the dielectric nature of amyloid fibril microenvironments in different binding sites using an optical probe, thioflavin-T...

  19. Master and slave relationship between two types of self-propagating insulin amyloid fibrils.

    Science.gov (United States)

    Surmacz-Chwedoruk, Weronika; Babenko, Viktoria; Dzwolak, Wojciech

    2014-11-26

    Cross-seeding of fibrils of bovine insulin (BI) and Lys(B31)-Arg(B32) human insulin analog (KR) induces self-propagating amyloid variants with infrared features inherited from mother seeds. Here we report that when native insulin (BI or KR) is simultaneously seeded with mixture of equal amounts of both templates (i.e., of separately grown fibrils of BI and KR), the phenotype of resulting daughter fibrils is as in the case of the purely homologous seeding: heterologous cotemplates accelerate the fibrillation but do not determine infrared traits of the daughter amyloid. This implies that fibrillation-promoting and structure-imprinting properties of heterologous seeds become uncoupled in the presence of homologous seeds. We argue that explanation of such behavior requires that insulin molecules partly transformed through interactions with heterologous fibrils are subsequently recruited by homologous seeds. The selection bias toward homologous daughter amyloid is exceptional: more than 200-fold excess of heterologous seed is required to imprint its structural phenotype upon mixed seeding. Our study captures a snapshot of elusive docking interactions in statu nascendi of elongation of amyloid fibril and suggests that different types of seeds may collaborate in sequential processing of soluble protein into fibrils.

  20. S14G-humanin inhibits Aβ1-42 fibril formation, disaggregates preformed fibrils, and protects against Aβ-induced cytotoxicity in vitro.

    Science.gov (United States)

    Zhang, Wei; Du, Ying; Bai, Miao; Xi, Ye; Li, Zhuyi; Miao, Jianting

    2013-03-01

    The aggregation of soluble amyloid-beta (Aβ) peptide into oligomers/fibrils is one of the key pathological features in Alzheimer's disease (AD). The Aβ aggregates are considered to play a pivotal role in the pathogenesis of AD. Therefore, inhibiting Aβ aggregation and destabilizing preformed Aβ fibrils would be an attractive therapeutic target for prevention and treatment of AD. S14G-humanin (HNG), a synthetic derivative of Humanin (HN), has been shown to be a strong neuroprotective agent against various AD-related insults. Recent studies have shown that HNG can significantly improve cognitive deficits and reduce insoluble Aβ levels as well as amyloid plaque burden without affecting amyloid precursor protein processing and Aβ production in transgenic AD models. However, the potential mechanisms by which HNG reduces Aβ-related pathology in vivo remain obscure. In the present study, we found that HNG could significantly inhibit monomeric Aβ1-42 aggregation into fibrils and destabilize preformed Aβ1-42 fibrils in a concentration-dependent manner by Thioflavin T fluorescence assay. In transmission electron microscope study, we observed that HNG was effective in inhibiting Aβ1-42 fibril formation and disrupting preformed Aβ1-42 fibrils, exhibiting various types of amorphous aggregates without identifiable Aβ fibrils. Furthermore, HNG-treated monomeric or fibrillar Aβ1-42 was found to significantly reduce Aβ1-42-mediated cytotoxic effects on PC12 cells in a dose-dependent manner by MTT assay. Collectively, our results demonstrate for the first time that HNG not only inhibits Aβ1-42 fibril formation but also disaggregates preformed Aβ1-42 fibrils, which provides the novel evidence that HNG may have anti-Aβ aggregation and fibrillogenesis, and fibril-destabilizing properties. Together with previous studies, we concluded that HNG may have promising therapeutic potential as a multitarget agent for the prevention and/or treatment of AD. Copyright © 2013

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Hoang Viet, Man; Roland, Christopher, E-mail: cmroland@ncsu.edu; Sagui, Celeste, E-mail: sagui@ncsu.edu [Department of Physics, North Carolina State University, Raleigh, North Carolina 27695-8202 (United States); Derreumaux, Philippe; Nguyen, Phuong H., E-mail: phuong.nguyen@ibpc.fr [Laboratoire de Biochimie Théorique, UPR 9080, CNRS Université Denis Diderot, Sorbonne Paris Cité IBPC, 13 rue Pierre et Marie Curie, 75005 Paris (France); Li, Mai Suan [Institute of Physics, Polish Academy of Sciences, Al. Lotnikow 32/46, 02-668 Warsaw (Poland); Institute for Computational Science and Technology, SBI Building, Quang Trung Software City, Tan Chanh Hiep Ward, District 12, Ho Chi Minh City (Viet Nam)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Rui [State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); He, Mingxia [College of Precision Instrument and Optoelectronics Engineering, Tianjin University, Tianjin 300072 (China); Su, Rongxin, E-mail: surx@tju.edu.cn [State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072 (China); Yu, Yanjun [State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Qi, Wei; He, Zhimin [State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072 (China)

    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.

  4. Exploiting oleuropein for inhibiting collagen fibril formation.

    Science.gov (United States)

    Bharathy, H; Fathima, N Nishad

    2017-08-01

    Collagen fibrils accumulate in excessive amounts and impair the normal functioning of the organ; therefore it stimulates the interest for identifying the compounds that could prevent the formation of fibrils. Herein, inhibition of self-assembly of collagen using oleuropein has been studied. The changes in the physico-chemical characteristics of collagen on interaction with increasing concentration of oleuropein has been studied using techniques like viscosity, UV-vis, CD and FT-IR. The inhibitory effect of oleuropein on fibril formation of collagen was proved using SEM. Circular dichroism and FT-IR spectra elucidates the alterations in the secondary structure of collagen suggesting non-covalent interactions between oleuropein and collagen. The decreased rate of collagen fibril formation also confirms the inhibition in the self-assembly of collagen. Hence, our study suggests that inhibition of the self-assembly process using oleuropein may unfold new avenues to treat fibrotic diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers

    Directory of Open Access Journals (Sweden)

    Rasool Suhail

    2007-09-01

    Full Text Available Abstract Background Amyloid-related degenerative diseases are associated with the accumulation of misfolded proteins as amyloid fibrils in tissue. In Alzheimer disease (AD, amyloid accumulates in several distinct types of insoluble plaque deposits, intracellular Aβ and as soluble oligomers and the relationships between these deposits and their pathological significance remains unclear. Conformation dependent antibodies have been reported that specifically recognize distinct assembly states of amyloids, including prefibrillar oligomers and fibrils. Results We immunized rabbits with a morphologically homogeneous population of Aβ42 fibrils. The resulting immune serum (OC specifically recognizes fibrils, but not random coil monomer or prefibrillar oligomers, indicating fibrils display a distinct conformation dependent epitope that is absent in prefibrillar oligomers. The fibril epitope is also displayed by fibrils of other types of amyloids, indicating that the epitope is a generic feature of the polypeptide backbone. The fibril specific antibody also recognizes 100,000 × G soluble fibrillar oligomers ranging in size from dimer to greater than 250 kDa on western blots. The fibrillar oligomers recognized by OC are immunologically distinct from prefibrillar oligomers recognized by A11, even though their sizes overlap broadly, indicating that size is not a reliable indicator of oligomer conformation. The immune response to prefibrillar oligomers and fibrils is not sequence specific and antisera of the same specificity are produced in response to immunization with islet amyloid polypeptide prefibrillar oligomer mimics and fibrils. The fibril specific antibodies stain all types of amyloid deposits in human AD brain. Diffuse amyloid deposits stain intensely with anti-fibril antibody although they are thioflavin S negative, suggesting that they are indeed fibrillar in conformation. OC also stains islet amyloid deposits in transgenic mouse models of type

  6. Polymorphism of amyloid-like fibrils can be defined by the concentration of seeds

    Directory of Open Access Journals (Sweden)

    Tomas Sneideris

    2015-08-01

    Full Text Available Prions are infectious proteins where the same protein may express distinct strains. The strains are enciphered by different misfolded conformations. Strain-like phenomena have also been reported in a number of other amyloid-forming proteins. One of the features of amyloid strains is the ability to self-propagate, maintaining a constant set of physical properties despite being propagated under conditions different from those that allowed initial formation of the strain. Here we report a cross-seeding experiment using strains formed under different conditions. Using high concentrations of seeds results in rapid elongation and new fibrils preserve the properties of the seeding fibrils. At low seed concentrations, secondary nucleation plays the major role and new fibrils gain properties predicted by the environment rather than the structure of the seeds. Our findings could explain conformational switching between amyloid strains observed in a wide variety of in vivo and in vitro experiments.

  7. Disaggregation of human islet amyloid polypeptide fibril formation by ruthenium polypyridyl complexes.

    Science.gov (United States)

    Zhu, Dengsen; Gong, Gehui; Wang, Wenji; Du, Weihong

    2017-05-01

    The toxicity of amyloid proteins is associated with many degenerative and systematic diseases. The aggregation of human islet amyloid polypeptide may induce pancreatic β-cell death, which is linked to type II diabetes. Ruthenium complexes are inhibitors of various proteins and potential anticancer metallodrugs, which can also be used to disaggregate amyloid proteins. This work reported that several ruthenium polypyridyl complexes remarkably affected the peptide aggregation by predominant hydrophobic interaction and metal coordination, as reflected by thermodynamic parameters and mass spectrometry analysis. Morphology and particle size analysis showed that the amyloid fibrils were disaggregated from long fibrils into small nano particles. Addition of these complexes also decreased the cytotoxicity induced by the peptide. The results indicated that ruthenium polypyridyl complexes may be potential metallodrugs to treat amyloidosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Destruction of α-synuclein based amyloid fibrils by a low temperature plasma jet

    Science.gov (United States)

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

    2010-10-01

    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.

  9. Destruction of α-synuclein based amyloid fibrils by a low temperature plasma jet

    International Nuclear Information System (INIS)

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

    2010-01-01

    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.

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

    Science.gov (United States)

    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.

  11. Michler’s Hydrol Blue: A Sensitive Probe for Amyloid Fibril Detection

    KAUST Repository

    Kitts, Catherine C.

    2011-05-03

    Michler\\'s hydrol blue (MHB) is investigated with respect to photophysical properties in varied solvent environment and when bound to insulin and lysozyme fibrils. The MHB chromophore is shown to act like a molecular rotor and bind well to amyloid fibrils, where it exhibits a characteristic red-shift in its excitation spectrum and an increase in the emission quantum yield upon binding. MHB is more sensitive to environmental changes than Thioflavin T (ThT) and furthermore, in contrast to the latter amyloid probe, can differentiate between insulin and lysozyme fibrils by a more red-shifted excitation spectrum for insulin fibrils. To support the experimental observations, time-dependent density functional theory (TDDFT) calculations were performed on MHB at several levels of theory. The predicted changes of spectral properties as a function of the environment are in good agreement with the experimental results. Linear dichroism (LD) is used to determine the orientation of the MHB within the fibrils. It was shown through LD and molecular modeling that MHB aligns itself preferentially parallel with the amyloid fiber at an angle of 14°-22° to the fibril axis and along the grooves of the β-sheet. © 2011 American Chemical Society.

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

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Emily B.; Williams, Angela [Department of Medicine, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37922 (United States); Heidel, Eric [Department of Surgery, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37922 (United States); Macy, Sallie [Department of Medicine, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37922 (United States); Kennel, Stephen J. [Department of Medicine, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37922 (United States); Department of Radiology, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37922 (United States); Wall, Jonathan S., E-mail: jwall@utmck.edu [Department of Medicine, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37922 (United States); Department of Radiology, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37922 (United States)

    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. MAK33 antibody light chain amyloid fibrils are similar to oligomeric precursors.

    Directory of Open Access Journals (Sweden)

    Manuel Hora

    Full Text Available Little structural information is available so far on amyloid fibrils consisting of immunoglobulin light chains. It is not understood which features of the primary sequence of the protein result in fibril formation. We report here MAS solid-state NMR studies to identify the structured core of κ-type variable domain light chain fibrils. The core contains residues of the CDR2 and the β-strands D, E, F and G of the native immunoglobulin fold. The assigned core region of the fibril is distinct in comparison to the core identified in a previous solid-state NMR study on AL-09 by Piehl at. al, suggesting that VL fibrils can adopt different topologies. In addition, we investigated a soluble oligomeric intermediate state, previously termed the alternatively folded state (AFS, using NMR and FTIR spectroscopy. The NMR oligomer spectra display a high degree of similarity when compared to the fibril spectra, indicating a high structural similarity of the two aggregation states. Based on comparison to the native state NMR chemical shifts, we suggest that fibril formation via domain-swapping seems unlikely. Moreover, we used our results to test the quality of different amyloid prediction algorithms.

  14. Large proteins have a great tendency to aggregate but a low propensity to form amyloid fibrils.

    Directory of Open Access Journals (Sweden)

    Hassan Ramshini

    Full Text Available 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.

  15. Benzofuranone derivatives as effective small molecules related to insulin amyloid fibrillation: a structure-function study

    DEFF Research Database (Denmark)

    Rabiee, Atefeh; Ebrahim-Habibi, Azadeh; Navidpour, Latifeh

    2011-01-01

    . In this study, the effects of five new synthetic benzofuranone derivatives were investigated on the insulin amyloid formation process. Protein fibrillation was analyzed by thioflavin-T fluorescence, Congo red binding, circular dichroism, and electron microscopy. Despite high structural similarity, one...

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

    Science.gov (United States)

    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.

  17. Dissection of conformational conversion events during prion amyloid fibril formation using hydrogen exchange and mass spectrometry.

    Science.gov (United States)

    Singh, Jogender; Udgaonkar, Jayant B

    2013-09-23

    A molecular understanding of prion diseases requires an understanding of the mechanism of amyloid fibril formation by the prion protein. In particular, it is necessary to define the sequence of the structural events describing the conformational conversion of monomeric PrP to aggregated PrP. In this study, the sequence of the structural events in the case of amyloid fibril formation by recombinant mouse prion protein at pH7 has been characterized by hydrogen-deuterium exchange and mass spectrometry. The observation that fibrils are substantially more stable to hydrogen-deuterium exchange than is native monomer allows both forms to be quantified during the course of the aggregation reaction. Under the aggregation conditions utilized, native monomeric protein and amyloid fibrils are the only forms of the protein detectable during the course of the fibril formation reaction, suggesting that monomer directly adds on to the fibril template. Conformational conversion is shown to occur in two steps after the binding of monomer to fibril, with helix 1 unfolding only after helices 2 and 3 transform into β-sheet. Local stability in the β-sheet core region (residues ~159-225) of the fibrils is shown to be sequence dependent in that it varies along the length of the core, and local stability in protein molecules that are ordered in the structurally heterogeneous sequence segment 109-132 is shown to be similar to that in the core. This new understanding of the structural events during prion protein aggregation has important bearing on our comprehension of the molecular basis of prion pathogenesis. © 2013 Elsevier Ltd. All rights reserved.

  18. Investigating the effects of erythrosine B on amyloid fibril formation derived from lysozyme.

    Science.gov (United States)

    Kuo, Chun-Tien; Chen, Yi-Lin; Hsu, Wei-Tse; How, Su-Chun; Cheng, Yu-Hong; Hsueh, Shu-Shun; Liu, Hwai-Shen; Lin, Ta-Hsien; Wu, Josephine W; Wang, Steven S-S

    2017-05-01

    Formation of amyloid fibrils has been associated with at least 30 different protein aggregation diseases. The 129-residue polypeptide hen lysozyme, which is structurally homologous to human lysozyme, has been demonstrated to exhibit amyloid fibril-forming propensity in vitro. This study is aimed at exploring the influence of erythrosine B on the in vitro amyloid fibril formation of hen lysozyme at pH 2.0 and 55°C using ThT binding assay, transmission electron microscopy, far-UV circular dichroism absorption spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and synchronous fluorescence study. We found that lysozyme fibrillogenesis was dose-dependently suppressed by erythrosine B. In addition, our far-UV CD and ANS fluorescence data showed that, as compared with the untreated lysozyme control, the α-to-ß transition and exposure of hydrophobic clusters in lysozyme were reduced upon treatment with erythrosine B. Moreover, it could be inferred that the binding of erythrosine B occurred in the vicinity of the tryptophan residues. Finally, molecular docking and molecular dynamics simulations were further employed to gain some insights into the possible binding site(s) and interactions between lysozyme and erythrosine B. We believe the results obtained here may contribute to the development of potential strategies/approaches for the suppression of amyloid fibrillogenesis, which is implicated in amyloid pathology. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Pyroglutamate-Modified Amyloid β (11- 40) Fibrils Are More Toxic than Wildtype Fibrils but Structurally Very Similar.

    Science.gov (United States)

    Scheidt, Holger A; Adler, Juliane; Zeitschel, Ulrike; Höfling, Corinna; Korn, Alexander; Krueger, Martin; Roßner, Steffen; Huster, Daniel

    2017-11-07

    The morphology, structure, and dynamics of mature amyloid β (Aβ) fibrils formed by the Aβ variant, which is truncated at residue 11 and chemically modified by enzymatic pyroglutamate formation (pGlu 11 -Aβ(11-40)), was studied along with the investigation of the toxicity of these Aβ variants to neurons and astrocytes. The fibrils of pGlu 11 -Aβ (11-40) were more toxic than wildtype Aβ (1-40) and the longer pGlu3-Aβ (3-40) especially at higher concentration, whereas the overall morphology was quite similar. The secondary structure of pGlu 11 -Aβ (11-40) fibrils shows the typical two β-strands connected by a short turn as known for mature fibrils of Aβ (1-40) and also pGlu 3 -Aβ (3-40). Further insights into tertiary contacts exhibit some similarities of pGlu 11 -Aβ (11-40) fibrils with wildtype Aβ (1-40), but also a so far not described contact between Gly 25 and Ile 31 . This highlights the biological importance of chemical modifications on the molecular structure of Aβ. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Immobilization of organophosphate hydrolase on an amyloid fibril nanoscaffold: towards bioremediation and chemical detoxification.

    Science.gov (United States)

    Raynes, Jared K; Pearce, F Grant; Meade, Susie J; Gerrard, Juliet A

    2011-01-01

    Organophosphate hydrolase has potential as a bioremediation and chemical detoxification enzyme, but the problems of reusability and stability need to be addressed to use this enzyme on an industrial scale. Immobilizing the enzyme to a nanoscaffold may help to solve these problems. Amyloid fibrils generated from insulin and crystallin provided a novel nanoscaffold for the immobilization of organophosphate hydrolase, using glutaraldehyde as the crosslinking reagent. Electrophoretic, centrifugation, and temperature stability experiments, together with transmission electron microscopy were undertaken to verify that crosslinking had successfully occurred. The resulting fibrils remained active towards the substrate paraoxon and when immobilized to the insulin amyloid fibrils, the enzyme exhibited a significant (∼ 300%) increase in the relative temperature stability at 40, 45, and 50°C (as measured by comparing the initial enzyme activity to the activity remaining after heating), compared to free enzyme. This confirms that amyloid fibrils could provide a new type of nanoscaffold for enzyme immobilization. Copyright © 2010 American Institute of Chemical Engineers (AIChE).

  1. Systematic examination of polymorphism in amyloid fibrils by molecular-dynamics simulation.

    Science.gov (United States)

    Berryman, Joshua T; Radford, Sheena E; Harris, Sarah A

    2011-05-04

    Amyloid fibrils often exhibit polymorphism. Polymorphs are formed when proteins or peptides with identical sequences self-assemble into fibrils containing substantially different arrangements of the β-strands. We used atomistic molecular-dynamics simulation to examine the thermodynamic stability of a amyloid fibrils in different polymorphic forms by performing a systematic investigation of sequence and symmetry space for a series of peptides with a range of physicochemical properties. We show that the stability of fibrils depends on both sequence and the symmetry because these factors determine the availability of favorable interactions between the peptide strands within a sheet and in intersheet packing. By performing a detailed analysis of these interactions as a function of symmetry, we obtained a series of simple design rules that can be used to determine which polymorphs of a given sequence are most likely to form thermodynamically stable fibrils. These rules can potentially be employed to design peptide sequences that aggregate into a preferred polymorphic form for nanotechnological purposes. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Cong; Sawaya, Michael R.; Eisenberg, David (UCLA)

    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.

  3. Small Amphipathic Molecules Modulate Secondary Structure and Amyloid Fibril-forming Kinetics of Alzheimer Disease Peptide Aβ1–42

    Science.gov (United States)

    Ryan, Timothy M.; Friedhuber, Anna; Lind, Monica; Howlett, Geoffrey J.; Masters, Colin; Roberts, Blaine R.

    2012-01-01

    Amyloid fibril formation is associated with a number of debilitating systemic and neurodegenerative diseases. One of the most prominent is Alzheimer disease in which aggregation and deposition of the Aβ peptide occur. Aβ is widely considered to mediate the extensive neuronal loss observed in this disease through the formation of soluble oligomeric species, with the final fibrillar end product of the aggregation process being relatively inert. Factors that influence the aggregation of these amyloid-forming proteins are therefore very important. We have screened a library of 96 amphipathic molecules for effects on Aβ1–42 aggregation and self-association. We find, using thioflavin T fluorescence and electron microscopy assays, that 30 of the molecules inhibit the aggregation process, whereas 36 activate fibril formation. Several activators and inhibitors were subjected to further analysis using analytical ultracentrifugation and circular dichroism. Activators typically display a 1:10 peptide:detergent stoichiometry for maximal activation, whereas the inhibitors are effective at a 1:1 stoichiometry. Analytical ultracentrifugation and circular dichroism experiments show that activators promote a mixture of unfolded and β-sheet structures and rapidly form large aggregates, whereas inhibitors induce α-helical structures that form stable dimeric/trimeric oligomers. The results suggest that Aβ1–42 contains at least one small molecule binding site, which modulates the secondary structure and aggregation processes. Further studies of the binding of these compounds to Aβ may provide insight for developing therapeutic strategies aimed at stabilizing Aβ in a favorable conformation. PMID:22461629

  4. Small amphipathic molecules modulate secondary structure and amyloid fibril-forming kinetics of Alzheimer disease peptide Aβ(1-42).

    Science.gov (United States)

    Ryan, Timothy M; Friedhuber, Anna; Lind, Monica; Howlett, Geoffrey J; Masters, Colin; Roberts, Blaine R

    2012-05-11

    Amyloid fibril formation is associated with a number of debilitating systemic and neurodegenerative diseases. One of the most prominent is Alzheimer disease in which aggregation and deposition of the Aβ peptide occur. Aβ is widely considered to mediate the extensive neuronal loss observed in this disease through the formation of soluble oligomeric species, with the final fibrillar end product of the aggregation process being relatively inert. Factors that influence the aggregation of these amyloid-forming proteins are therefore very important. We have screened a library of 96 amphipathic molecules for effects on Aβ(1-42) aggregation and self-association. We find, using thioflavin T fluorescence and electron microscopy assays, that 30 of the molecules inhibit the aggregation process, whereas 36 activate fibril formation. Several activators and inhibitors were subjected to further analysis using analytical ultracentrifugation and circular dichroism. Activators typically display a 1:10 peptide:detergent stoichiometry for maximal activation, whereas the inhibitors are effective at a 1:1 stoichiometry. Analytical ultracentrifugation and circular dichroism experiments show that activators promote a mixture of unfolded and β-sheet structures and rapidly form large aggregates, whereas inhibitors induce α-helical structures that form stable dimeric/trimeric oligomers. The results suggest that Aβ(1-42) contains at least one small molecule binding site, which modulates the secondary structure and aggregation processes. Further studies of the binding of these compounds to Aβ may provide insight for developing therapeutic strategies aimed at stabilizing Aβ in a favorable conformation.

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

    Energy Technology Data Exchange (ETDEWEB)

    Briknarova, Klara (The Burnham Institute); Akermann, Maria (The Burnham Institute); Hoyt, David W.(BATTELLE (PACIFIC NW LAB)); Ruoslahti, Erkki (The Burnham Institute); Ely, Kathryn R.(The Burnham Institute)

    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.

  6. A disulfide-linked amyloid-beta peptide dimer forms a protofibril-like oligomer through a distinct pathway from amyloid fibril formation.

    Science.gov (United States)

    Yamaguchi, Takahiro; Yagi, Hisashi; Goto, Yuji; Matsuzaki, Katsumi; Hoshino, Masaru

    2010-08-24

    The conversion of the soluble, nontoxic amyloid-beta (Abeta) peptide into an aggregated, toxic form rich in beta-sheets is considered a key step in the development of Alzheimer's disease. Whereas growing evidence indicates that the Abeta amyloid fibrils consist of in-register parallel beta-sheets, little is known about the structure of soluble oligomeric intermediates because of their transient nature. To understand the mechanism by which amyloid fibrils form, especially the initial development of the "nucleus" oligomeric intermediates, we prepared covalently linked dimeric Abeta peptides and analyzed the kinetics of the fibril-forming process. A covalent bond introduced between two Abeta molecules dramatically facilitated the spontaneous formation of aggregates with a beta-sheet structure and affinity for thioflavin T. Transmission electron microscopy revealed, however, that these aggregates differed in morphology from amyloid fibrils, more closely resembling protofibrils. The protofibril-like aggregates were not the most thermodynamically stable state but were a kinetically trapped state. The results emphasize the importance of the conformational flexibility of the Abeta molecule and a balance in the association and dissociation rate for the formation of rigid amyloid fibrils.

  7. Effect of copper (II) ion against elongation behavior of amyloid {beta} fibrils on liposome membranes

    Energy Technology Data Exchange (ETDEWEB)

    Shimanouchi, T.; Onishi, R.; Kitaura, N.; Umakoshi, H.; Kuboi, R. [Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka (Japan)

    2012-01-15

    The fibril growth behavior of amyloid {beta} protein (A{beta}) on cell membranes is relating to the progression of Alzheimer's disease. This growth behavior of A{beta} fibrils is sensitively affected by the metal ions, neurotransmitters, or bioreactive substrate. The inhibitory effect of those materials was quantitatively estimated from the viewpoints of ''crystal growth''. In a bulk aqueous solution, copper (II) ion showed the strong inhibitory effect on the growth of A{beta} fibrils. Meanwhile, the addition of a closed-phospholipid bilayer membrane (liposome) could reduce the above inhibitory effect of copper (II) ion. (copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  8. Sucrose modulates insulin amyloid-like fibril formation: effect on the aggregation mechanism and fibril morphology

    DEFF Research Database (Denmark)

    Marasini, Carlotta; Foderà, Vito; Vestergaard, Bente

    2017-01-01

    Co-solutes, such as sugars, are used in in vitro protein aggregation experiments to mimic crowding and, in general, complex environments. Sugars often increase the stability of the native protein structure by affecting inter- and intramolecular protein–protein interactions. This, in turn, modifies...... in a delay of the onset of fibrillation. Moreover, it leads to a dramatic change in both the morphology and overall amount of fibrils. Our results emphasize that the detailed composition of protein surroundings likely influences not only the fibrillation kinetics but also the balance between different...

  9. Acidic pH retards the fibrillization of human islet amyloid polypeptide due to electrostatic repulsion of histidines

    Science.gov (United States)

    Li, Yang; Xu, Weixin; Mu, Yuguang; Zhang, John Z. H.

    2013-08-01

    The human Islet Amyloid Polypeptide (hIAPP) is the major constituent of amyloid deposits in pancreatic islets of type-II diabetes. IAPP is secreted together with insulin from the acidic secretory granules at a low pH of approximately 5.5 to the extracellular environment at a neutral pH. The increased accumulation of extracellular hIAPP in diabetes indicates that changes in pH may promote amyloid formation. To gain insights and underlying mechanisms of the pH effect on hIAPP fibrillogenesis, all-atom molecular dynamics simulations in explicit solvent model were performed to study the structural properties of five hIAPP protofibrillar oligomers, under acidic and neutral pH, respectively. In consistent with experimental findings, simulation results show that acidic pH is not conducive to the structural stability of these oligomers. This provides a direct evidence for a recent experiment [L. Khemtemourian, E. Domenech, J. P. F. Doux, M. C. Koorengevel, and J. A. Killian, J. Am. Chem. Soc. 133, 15598 (2011)], 10.1021/ja205007j, which suggests that acidic pH inhibits the fibril formation of hIAPP. In addition, a complementary coarse-grained simulation shows the repulsive electrostatic interactions among charged His18 residues slow down the dimerization process of hIAPP by twofold. Besides, our all-atom simulations reveal acidic pH mainly affects the local structure around residue His18 by destroying the surrounding hydrogen-bonding network, due to the repulsive interactions between protonated interchain His18 residues at acidic pH. It is also disclosed that the local interactions nearby His18 operating between adjacent β-strands trigger the structural transition, which gives hints to the experimental findings that the rate of hIAPP fibril formation and the morphologies of the fibrillar structures are strongly pH-dependent.

  10. Polymorphism of Protein Aggregation: From Amyloid Fibrils to Crystallization

    Science.gov (United States)

    Safari, Mohammad S.; Conrad, Jacinta C.; Vekilov, Peter G.

    Protein aggregation is commonly observed in neurological diseases and in different types of cancer. Despite the established mechanism of amyloid formation, the polymorphism of aggregation is not very well understood; improved knowledge of mechanisms for aggregation that operate in vivo or under physiological conditions is likely to inform therapeutic design. Here we show that reduction of disulfide bonds in lysozyme can lead to formation of gel-like oligomers that are precursors for protein crystal nucleation events. The growth in size of oligomers follows slow first-order kinetics, suggesting that monomers with free thiol contribute to formation of clusters. Free thiol concentration measurements showed that the thiol concentration was relatively stable over 12 hr, confirming the slow kinetics was due to gelation inside the clusters. We probed the hydrophobicity of the clusters using ANS and ThT assays, and showed that the protein conformation in these clusters differs from that of thermally denatured aggregates. Although partial unfolding aids the formation of precursors to both amyloids and crystals, our results suggest that these pathways exhibit distinct signatures even at the earliest stages. NASA.

  11. End-to-end Structural Restriction of α-Synuclein and Its Influence on Amyloid Fibril Formation

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Chul Suk; Park, Jae Hyung; Choe, Young Jun; Paik, Seung R. [Seoul National University, Seoul (Korea, Republic of)

    2014-09-15

    Relationship between molecular freedom of amyloidogenic protein and its self-assembly into amyloid fibrils has been evaluated with α-synuclein, an intrinsically unfolded protein related to Parkinson's disease, by restricting its structural plasticity through an end-to-end disulfide bond formation between two newly introduced cysteine residues on the N- and C-termini. Although the resulting circular form of α-synuclein exhibited an impaired fibrillation propensity, the restriction did not completely block the protein's interactive core since co-incubation with wild-type α-synuclein dramatically facilitated the fibrillation by producing distinctive forms of amyloid fibrils. The suppressed fibrillation propensity was instantly restored as the structural restriction was unleashed with β-mercaptoethanol. Conformational flexibility of the accreting amyloidogenic protein to pre-existing seeds has been demonstrated to be critical for fibrillar extension process by exerting structural adjustment to a complementary structure for the assembly.

  12. End-to-end Structural Restriction of α-Synuclein and Its Influence on Amyloid Fibril Formation

    International Nuclear Information System (INIS)

    Hong, Chul Suk; Park, Jae Hyung; Choe, Young Jun; Paik, Seung R.

    2014-01-01

    Relationship between molecular freedom of amyloidogenic protein and its self-assembly into amyloid fibrils has been evaluated with α-synuclein, an intrinsically unfolded protein related to Parkinson's disease, by restricting its structural plasticity through an end-to-end disulfide bond formation between two newly introduced cysteine residues on the N- and C-termini. Although the resulting circular form of α-synuclein exhibited an impaired fibrillation propensity, the restriction did not completely block the protein's interactive core since co-incubation with wild-type α-synuclein dramatically facilitated the fibrillation by producing distinctive forms of amyloid fibrils. The suppressed fibrillation propensity was instantly restored as the structural restriction was unleashed with β-mercaptoethanol. Conformational flexibility of the accreting amyloidogenic protein to pre-existing seeds has been demonstrated to be critical for fibrillar extension process by exerting structural adjustment to a complementary structure for the assembly

  13. Synthesis of Self-assembled Noble Metal Nanoparticle Chains Using Amyloid Fibrils of Lysozyme as Templates

    Directory of Open Access Journals (Sweden)

    Ziming Xu

    2016-01-01

    Full Text Available We reported a facile method for preparing self-assembled noble metal nanoparticle chains by using lysozyme amyloid fibrils as a biotemplate in an aqueous environ‐ ment. The nanoparticle chains of gold (AuNPCs, palladi‐ um (PdNPCs, platinum (PtNPCs and rhodium (RhNPCs, which are lysozyme fibrils coated by gold, palladium, platinum and rhodium nanoparticles, can be fabricated by simply reducing the corresponding metal salt precursors using NaBH4. Under the same molar ratio between salt precursors and fibrils, two types of morphologies of high- yield AuNPCs (thin- and thick- AuNPCs were synthesized as a result of adjusting the fibrosis time and temperature in the final stage. Abundant PdNPCs with a length of several micrometres intertwisted with each other to form PdNPC networks. The growth of RhNPCs started from the inner surface of the fibrils and gradually spread to the whole fibre as superabundant rhodium nanoparticles (RhNPs bound to the fibrils. Finally, PtNPCs at different growing periods were presented. The nanostructures were investigated by transmission electron microscope, UV-visible spectrosco‐ py, fluorescence spectroscopy, energy-dispersive X-ray spectroscopy and atomic force microscope.

  14. Fibrils from designed non-amyloid-related synthetic peptides induce AA-amyloidosis during inflammation in an animal model.

    Directory of Open Access Journals (Sweden)

    Per Westermark

    Full Text Available BACKGROUND: Mouse AA-amyloidosis is a transmissible disease by a prion-like mechanism where amyloid fibrils act by seeding. Synthetic peptides with no amyloid relationship can assemble into amyloid-like fibrils and these may have seeding capacity for amyloid proteins. PRINCIPAL FINDINGS: Several synthetic peptides, designed for nanotechnology, have been examined for their ability to produce fibrils with Congo red affinity and concomitant green birefringence, affinity for thioflavin S and to accelerate AA-amyloidosis in mice. It is shown that some amphiphilic fibril-forming peptides not only produced Congo red birefringence and showed affinity for thioflavin S, but they also shortened the lag phase for systemic AA-amyloidosis in mice when they were given intravenously at the time of inflammatory induction with silver nitride. Peptides, not forming amyloid-like fibrils, did not have such properties. CONCLUSIONS: These observations should caution researchers and those who work with synthetic peptides and their derivatives to be aware of the potential health concerns.

  15. Dewetting transition assisted clearance of (NFGAILS) amyloid fibrils from cell membranes by graphene

    International Nuclear Information System (INIS)

    Liu, Jiajia; Yang, Zaixing; Gu, Zonglin; Li, Haotian; Garate, Jose Antonio; Zhou, Ruhong

    2014-01-01

    Clearance of partially ordered oligomers and monomers deposited on cell membrane surfaces is believed to be an effective route to alleviate many potential protein conformational diseases (PCDs). With large-scale all-atom molecular dynamics simulations, here we show that graphene nanosheets can easily and quickly win a competitive adsorption of human islet amyloid polypeptides (hIAPP 22-28 ) NFGAILS and associated fibrils against cell membrane, due to graphene's unique two-dimensional, highly hydrophobic surface with its all-sp 2 hybrid structure. A nanoscale dewetting transition was observed at the interfacial region between the fibril (originally deposited on the membrane) and the graphene nanosheet, which significantly assisted the adsorption of fibrils onto graphene from the membrane. The π–π stacking interaction between Phe23 and graphene played a crucial role, providing the driving force for the adsorption at the graphene surface. This study renders new insight towards the importance of water during the interactions between amyloid peptides, the phospholipidic membrane, and graphene, which might shed some light on future developments of graphene-based nanomedicine for preventing/curing PCDs like type II diabetes mellitus

  16. Dewetting transition assisted clearance of (NFGAILS) amyloid fibrils from cell membranes by graphene

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jiajia; Yang, Zaixing; Gu, Zonglin [Institute of Quantitative Biology and Medicine, SRMP and RAD-X, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, and Jiangsu Provincial Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou 215123 (China); Li, Haotian [Bio-X Lab, Department of Physics, Zhejiang University, Hangzhou 310027 (China); Garate, Jose Antonio [IBM Thomas J. Watson Research Center, Yorktown Heights, New York 10598 (United States); Zhou, Ruhong, E-mail: ruhongz@us.ibm.com [Institute of Quantitative Biology and Medicine, SRMP and RAD-X, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, and Jiangsu Provincial Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou 215123 (China); IBM Thomas J. Watson Research Center, Yorktown Heights, New York 10598 (United States); Department of Chemistry, Columbia University, New York, New York 10027 (United States)

    2014-12-14

    Clearance of partially ordered oligomers and monomers deposited on cell membrane surfaces is believed to be an effective route to alleviate many potential protein conformational diseases (PCDs). With large-scale all-atom molecular dynamics simulations, here we show that graphene nanosheets can easily and quickly win a competitive adsorption of human islet amyloid polypeptides (hIAPP{sub 22-28}) NFGAILS and associated fibrils against cell membrane, due to graphene's unique two-dimensional, highly hydrophobic surface with its all-sp{sup 2} hybrid structure. A nanoscale dewetting transition was observed at the interfacial region between the fibril (originally deposited on the membrane) and the graphene nanosheet, which significantly assisted the adsorption of fibrils onto graphene from the membrane. The π–π stacking interaction between Phe23 and graphene played a crucial role, providing the driving force for the adsorption at the graphene surface. This study renders new insight towards the importance of water during the interactions between amyloid peptides, the phospholipidic membrane, and graphene, which might shed some light on future developments of graphene-based nanomedicine for preventing/curing PCDs like type II diabetes mellitus.

  17. Inhibition of human amylin fibril formation by insulin-mimetic vanadium complexes.

    Science.gov (United States)

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

    2014-05-01

    The toxicity of amyloid-forming proteins can be linked to many degenerative and systemic diseases. Human islet amyloid polypeptide (hIAPP, amylin) has been associated with type II diabetes. Methods for efficient inhibition of amyloid fibril formation are highly clinically important. This study demonstrated the significant inhibitory effects of six vanadium complexes on hIAPP aggregation. Vanadium complexes, such as bis(maltolato)-oxovanadium (BMOV), have been used as insulin-mimetic agents for the treatment of diabetes for many years. Different biophysical methods were applied to investigate the interaction between V complexes and hIAPP. The results indicated that the selected compounds affected the peptide aggregation by different action modes and protected the cells from the cytotoxicity induced by hIAPP. Both the high binding affinity and the ligand spatial effect on inhibiting hIAPP aggregation are significant. Although some of these compounds undergo biotransformation under the conditions of the experiments, and the active species are not identified, it is understood that the effect results from a particular compound and its conversion products. Importantly, our work provided information on the effects of the selected V complexes on hIAPP and demonstrated multiple levels of effects of V complexes against amyloid-related diseases.

  18. Epigallocatechin-3-gallate rapidly remodels PAP85-120, SEM1(45-107, and SEM2(49-107 seminal amyloid fibrils

    Directory of Open Access Journals (Sweden)

    Laura M. Castellano

    2015-09-01

    Full Text Available Semen harbors amyloid fibrils formed by proteolytic fragments of prostatic acid phosphatase (PAP248-286 and PAP85-120 and semenogelins (SEM1 and SEM2 that potently enhance HIV infectivity. Amyloid but not soluble forms of these peptides enhance HIV infection. Thus, agents that remodel these amyloid fibrils could prevent HIV transmission. Here, we confirm that the green tea polyphenol, epigallocatechin-3-gallate (EGCG, slowly remodels fibrils formed by PAP248-286 termed SEVI (semen derived enhancer of viral infection and also exerts a direct anti-viral effect. We elucidate for the first time that EGCG remodels PAP85-120, SEM1(45-107, and SEM2(49-107 fibrils more rapidly than SEVI fibrils. We establish EGCG as the first small molecule that can remodel all four classes of seminal amyloid. The combined anti-amyloid and anti-viral properties of EGCG could have utility in preventing HIV transmission.

  19. Raman study of lysozyme amyloid fibrils suspended on super-hydrophobic surfaces by shear flow

    KAUST Repository

    Moretti, Manola

    2017-05-19

    The shear flow generated at the rim of a drop evaporating on a micro-fabricated super-hydrophobic surface has been used to suspend and orient single/few lysozyme amyloid fibrils between two pillars for substrate-free characterization. Micro Raman spectroscopy performed on extended fibers evidenced a shift of the Amide I band main peak to the value attributed to β-sheet secondary structure, characteristic of the amyloid fibers. In addition, given the orientation sensitivity of the anisotropic molecule, the Raman signal of the main secondary structure was nicely enhanced for a fiber alignment parallel to the polarization direction of the laser. The substrate-free sample generated by this suspending technique is suitable for other structural analysis methods, where fiber crystals are investigated. It could be further employed for generation of arrays and patterns in a controllable fashion, where bio-compatible material is needed.

  20. Direct Correlation Between Ligand-Induced α-Synuclein Oligomers and Amyloid-like Fibril Growth

    DEFF Research Database (Denmark)

    Pedersen, Martin Nors; Foderà, Vito; Horvath, Istvan

    2015-01-01

    link to disease related degenerative activity. Fibrils formed in the presence and absence of FN075 are indistinguishable on microscopic and macroscopic levels. Using small angle X-ray scattering, we reveal that FN075 induced oligomers are similar, but not identical, to oligomers previously observed......Aggregation of proteins into amyloid deposits is the hallmark of several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. The suggestion that intermediate oligomeric species may be cytotoxic has led to intensified investigations of pre-fibrillar oligomers, which...

  1. Sequence-Dependent Self-Assembly and Structural Diversity of Islet Amyloid Polypeptide-Derived β-Sheet Fibrils

    International Nuclear Information System (INIS)

    Wang, Shih-Ting; Lin, Yiyang; Spencer, Ryan K.; Thomas, Michael R.; Nguyen, Andy I.

    2017-01-01

    Determining the structural origins of amyloid fibrillation is essential for understanding both the pathology of amyloidosis and the rational design of inhibitors to prevent or reverse amyloid formation. In this work, the decisive roles of peptide structures on amyloid self-assembly and morphological diversity were investigated by the design of eight amyloidogenic peptides derived from islet amyloid polypeptide. Among the segments, two distinct morphologies were highlighted in the form of twisted and planar (untwisted) ribbons with varied diameters, thicknesses, and lengths. In particular, transformation of amyloid fibrils from twisted ribbons into untwisted structures was triggered by substitution of the C-terminal serine with threonine, where the side chain methyl group was responsible for the distinct morphological change. This effect was confirmed following serine substitution with alanine and valine and was ascribed to the restriction of intersheet torsional strain through the increased hydrophobic interactions and hydrogen bonding. We also studied the variation of fibril morphology (i.e., association and helicity) and peptide aggregation propensity by increasing the hydrophobicity of the peptide side group, capping the N-terminus, and extending sequence length. Lastly, we anticipate that our insights into sequence-dependent fibrillation and morphological diversity will shed light on the structural interpretation of amyloidogenesis and development of structure-specific imaging agents and aggregation inhibitors.

  2. Degeneration of amyloidfibrils caused by exposure to low-temperature atmospheric-pressure plasma in aqueous solution

    OpenAIRE

    Takai, Eisuke; Ohashi, Gai; Yoshida, Tomonori; Sörgjerd, Karin Margareta; Zako, Tamotsu; Maeda, Mizuo; Kitano, Katsuhisa; Shiraki, Kentaro

    2014-01-01

    Low-temperature atmospheric-pressure plasma was applied to degenerate amyloid-ß (Aß) fibrils, which are a major component of neuritic plaque associated with Alzheimer's disease (AD). We showed that an Aß fibril exposed to a low-frequency (LF) plasma jet in aqueous solution retained its morphology, molecular weight, and cytotoxicity, but, intriguingly, decreased in protease resistance and ß-sheet content. These results suggested that an LF plasma jet could be utilized for the treatment of AD t...

  3. The architecture of amyloid-like peptide fibrils revealed by X-ray scattering, diffraction and electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Langkilde, Annette E., E-mail: annette.langkilde@sund.ku.dk [University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen (Denmark); Morris, Kyle L.; Serpell, Louise C. [University of Sussex, Falmer, Brighton (United Kingdom); Svergun, Dmitri I. [European Molecular Biology Laboratory, Hamburg Outstation, 22607 Hamburg (Germany); Vestergaard, Bente [University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen (Denmark)

    2015-04-01

    The aggregation process and the fibril state of an amyloidogenic peptide suggest monomer addition to be the prevailing mechanism of elongation and a model of the peptide packing in the fibrils has been obtained. Structural analysis of protein fibrillation is inherently challenging. Given the crucial role of fibrils in amyloid diseases, method advancement is urgently needed. A hybrid modelling approach is presented enabling detailed analysis of a highly ordered and hierarchically organized fibril of the GNNQQNY peptide fragment of a yeast prion protein. Data from small-angle X-ray solution scattering, fibre diffraction and electron microscopy are combined with existing high-resolution X-ray crystallographic structures to investigate the fibrillation process and the hierarchical fibril structure of the peptide fragment. The elongation of these fibrils proceeds without the accumulation of any detectable amount of intermediate oligomeric species, as is otherwise reported for, for example, glucagon, insulin and α-synuclein. Ribbons constituted of linearly arranged protofilaments are formed. An additional hierarchical layer is generated via the pairing of ribbons during fibril maturation. Based on the complementary data, a quasi-atomic resolution model of the protofilament peptide arrangement is suggested. The peptide structure appears in a β-sheet arrangement reminiscent of the β-zipper structures evident from high-resolution crystal structures, with specific differences in the relative peptide orientation. The complexity of protein fibrillation and structure emphasizes the need to use multiple complementary methods.

  4. Viologen-Phosphorus Dendrimers Inhibit α-Synuclein Fibrillation.

    Science.gov (United States)

    Milowska, Katarzyna; Grochowina, Justyna; Katir, Nadia; El Kadib, Abdelkrim; Majoral, Jean-Pierre; Bryszewska, Maria; Gabryelak, Teresa

    2013-03-04

    Inhibition of α-synuclein (ASN) fibril formation is a potential therapeutic strategy in Parkinson's disease and other synucleinopathies. The aim of this study was to examine the role of viologen-phosphorus dendrimers in the α-synuclein fibrillation process and to assess the structural changes in α-synuclein under the influence of dendrimers. ASN interactions with phosphonate and pegylated surface-reactive viologen-phosphorus dendrimers were examined by measuring the zeta potential, which allowed determining the number of dendrimer molecules that bind to the ASN molecule. The fibrillation kinetics and the structural changes were examined using ThT fluorescence and CD spectroscopy. Depending on the concentration of the used dendrimer and the nature of the reactive groups located on the surface, ASN fibrillation kinetics can be significantly reduced, and even, in the specific case of phosphonate dendrimers, the fibrillation can be totally inhibited at low concentrations. The presented results indicate that viologen-phosphorus dendrimers are able to inhibit ASN fibril formation and may be used as fibrillar regulating agents in neurodegenerative disorders.

  5. Engineering Metal Ion Coordination to Regulate Amyloid Fibril Assembly And Toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Dong, J.; Canfield, J.M.; Mehta, A.K.; Shokes, J.E.; Tian, B.; Childers, W.S.; Simmons, J.A.; Mao, Z.; Scott, R.A.; Warncke, K.; Lynn, D.G.

    2009-06-02

    Protein and peptide assembly into amyloid has been implicated in functions that range from beneficial epigenetic controls to pathological etiologies. However, the exact structures of the assemblies that regulate biological activity remain poorly defined. We have previously used Zn{sup 2+} to modulate the assembly kinetics and morphology of congeners of the amyloid {beta} peptide (A{beta}) associated with Alzheimer's disease. We now reveal a correlation among A{beta}-Cu{sup 2+} coordination, peptide self-assembly, and neuronal viability. By using the central segment of A{beta}, HHQKLVFFA or A{beta}(13-21), which contains residues H13 and H14 implicated in A{beta}-metal ion binding, we show that Cu{sup 2+} forms complexes with A{beta}(13-21) and its K16A mutant and that the complexes, which do not self-assemble into fibrils, have structures similar to those found for the human prion protein, PrP. N-terminal acetylation and H14A substitution, Ac-A{beta}(13-21)H14A, alters metal coordination, allowing Cu{sup 2+} to accelerate assembly into neurotoxic fibrils. These results establish that the N-terminal region of A{beta} can access different metal-ion-coordination environments and that different complexes can lead to profound changes in A{beta} self-assembly kinetics, morphology, and toxicity. Related metal-ion coordination may be critical to the etiology of other neurodegenerative diseases.

  6. Vibrational circular dichroism as a probe of fibrillogenesis: the origin of the anomalous intensity enhancement of amyloid-like fibrils.

    Science.gov (United States)

    Measey, Thomas J; Schweitzer-Stenner, Reinhard

    2011-02-02

    Amyloid fibrils are affiliated with various human pathologies. Knowledge of their molecular architecture is necessary for a detailed understanding of the mechanism of fibril formation. Vibrational circular dichroism (VCD) spectroscopy has recently shown sensitivity to amyloid fibrils [Ma et al. J. Am. Chem. Soc. 2007, 129, 12364 and Measey et al. J. Am. Chem. Soc. 2009, 131, 18218]. In particular, amyloid fibrils give rise to an intensity enhanced signal in the amide I band region of the corresponding VCD spectrum, offering promise of utilizing such a method for probing fibrillogenesis and the chiral structure of fibrils. Herein, we further investigate this phenomenon and demonstrate the use of VCD to probe the fibril formation kinetics of a short alanine-rich peptide. To elucidate the origin of the anomalous VCD intensity enhancement, we use an excitonic coupling model to simulate the VCD spectrum of stacked β-sheets containing one (Ising-like model) and two amide I oscillators per strand, as models for the underlying amyloid-fibril secondary structure. With this simple model, we show that the VCD intensity enhancement of amyloid-like fibrils results from intrasheet and, to a more limited extent, also from intersheet vibrational coupling between stacked β-sheets. The enhancement requires helically twisted sheets and is most pronounced for arrangements with parallel-oriented strands. Both the intersheet distance and the orientation of the amide I transition dipole moments of neighboring sheets are found to modulate the intensity enhancement of the amide I VCD signal. Moreover, our simulations suggest that, depending on the three-dimensional arrangement of the β-strands, the sign of the VCD signal of amyloid-like fibrils can be used to distinguish between right- and left-handed helical twists of parallel-oriented β-sheets. We compare the results of our simulation to experimental spectra of two short peptides, GNNQQNY, the N-terminal peptide fragment of the yeast

  7. Stabilization of a β-hairpin in monomeric Alzheimer's amyloid-β peptide inhibits amyloid formation

    Science.gov (United States)

    Hoyer, Wolfgang; Grönwall, Caroline; Jonsson, Andreas; Ståhl, Stefan; Härd, Torleif

    2008-01-01

    According to the amyloid hypothesis, the pathogenesis of Alzheimer's disease is triggered by the oligomerization and aggregation of the amyloid-β (Aβ) peptide into protein plaques. Formation of the potentially toxic oligomeric and fibrillar Aβ assemblies is accompanied by a conformational change toward a high content of β-structure. Here, we report the solution structure of Aβ(1–40) in complex with the phage-display selected affibody protein ZAβ3, a binding protein of nanomolar affinity. Bound Aβ(1–40) features a β-hairpin comprising residues 17–36, providing the first high-resolution structure of Aβ in β conformation. The positions of the secondary structure elements strongly resemble those observed for fibrillar Aβ. ZAβ3 stabilizes the β-sheet by extending it intermolecularly and by burying both of the mostly nonpolar faces of the Aβ hairpin within a large hydrophobic tunnel-like cavity. Consequently, ZAβ3 acts as a stoichiometric inhibitor of Aβ fibrillation. The selected Aβ conformation allows us to suggest a structural mechanism for amyloid formation based on soluble oligomeric hairpin intermediates. PMID:18375754

  8. Modeling of the Inhibitory Effect of Nanoparticles on Amyloid β Fibrillation.

    Science.gov (United States)

    Ramesh, Nirmal Kumar; Sudhakar, Swathi; Mani, Ethayaraja

    2018-03-22

    Experiments have shown that charged nanoparticles (NP) inhibit, partially or completely, the aggregation of Aβ protein monomers into fibrils. The equilibrium fibril content is found to be inversely proportional to the concentration of NP. In this work, we report a kinetic model for the fibrillation of Aβ protein in the presence of NP. In the model, apart from nucleation, elongation and fragmentation processes, the effect of NP is considered to cause a conformational change to the protein monomer, making the latter incompatible for aggregation. The simulated results explain the growth kinetics of pure Aβ (1-40) protein, and the kinetics in the presence of NP. The NP-monomer interaction considered in the model captures the significant effect of NP on the fibrillation process at a very molar ratio (NP to Aβ monomer) as low as 10 -4 . The model predictions are compared with two different NP systems, namely, gold and silica NP. The model can be applied to explain the inhibitory effect of other additives such as small molecules, NP, lipids, and surfactants that show a similar inhibition trend for fibril formation of Aβ and other proteins.

  9. Structure and intermolecular dynamics of aggregates populated during amyloid fibril formation studied by hydrogen/deuterium exchange.

    Science.gov (United States)

    Carulla, Natàlia; Zhou, Min; Giralt, Ernest; Robinson, Carol V; Dobson, Christopher M

    2010-08-17

    The aggregation of proteins into amyloid fibrils is a complex and fascinating process associated with debilitating clinical disorders such as Alzheimer's and Parkinson's diseases. The process of aggregation involves a series of steps during which many intermediate aggregation states are populated. Recent evidence points to these intermediate states as the toxic moieties primarily responsible for cell damage or cell death, which are critical steps in the origin and progression of these disorders. To understand the molecular basis of these diseases, it is crucial to investigate and define the details of the aggregation process, and to achieve this objective, researchers need the tools to characterize the structure and kinetics of interconversion of the various species present during amyloid fibril formation. Hydrogen-deuterium (HD) exchange experiments are based on solvent accessibilities and provide one means by which this kind of information may be acquired. In this Account, we describe research based on HD exchange processes that is directed toward better understanding the dynamics and structural reorganizations involved in the formation of amyloid fibrils. Amide hydrogens that normally undergo rapid exchange with solvent hydrogens experience much slower exchange when involved in H-bonded structures or when sterically inaccessible to the solvent. The rates of exchange can be monitored by replacing some hydrogens with deuterons. When peptide and protein molecules assemble into amyloid fibrils, the fibrils contain a core region based on repetitive arrays of beta-sheets oriented parallel to the fibril axis. HD experiments have been applied extensively to map such structures in different amyloid fibril systems. By an extension of this approach, we have observed that HD exchange can be governed by a mechanism through which molecules making up the fibrils are continuously dissolving and reforming, revealing that amyloid fibrils are not static but dynamic structures

  10. A Peptide Derived from the HIV-1 gp120 Coreceptor-Binding Region Promotes Formation of PAP248-286 Amyloid Fibrils to Enhance HIV-1 Infection.

    Directory of Open Access Journals (Sweden)

    Jinquan Chen

    Full Text Available Semen is a major vehicle for HIV transmission. Prostatic acid phosphatase (PAP fragments, such as PAP248-286, in human semen can form amyloid fibrils to enhance HIV infection. Other endogenous or exogenous factors present during sexual intercourse have also been reported to promote the formation of seminal amyloid fibrils.Here, we demonstrated that a synthetic 15-residue peptide derived from the HIV-1 gp120 coreceptor-binding region, designated enhancing peptide 2 (EP2, can rapidly self-assemble into nanofibers. These EP2-derivated nanofibers promptly accelerated the formation of semen amyloid fibrils by PAP248-286, as shown by Thioflavin T (ThT and Congo red assays. The amyloid fibrils presented similar morphology, assessed via transmission electron microscopy (TEM, in the presence or absence of EP2. Circular dichroism (CD spectroscopy revealed that EP2 accelerates PAP248-286 amyloid fibril formation by promoting the structural transition of PAP248-286 from a random coil into a cross-β-sheet. Newly formed semen amyloid fibrils effectively enhanced HIV-1 infection in TZM-bl cells and U87 cells by promoting the binding of HIV-1 virions to target cells.Nanofibers composed of EP2 promote the formation of PAP248-286 amyloid fibrils and enhance HIV-1 infection.

  11. Benzothiazole Aniline Tetra(ethylene glycol) and 3-Amino-1,2,4-triazole Inhibit Neuroprotection against Amyloid Peptides by Catalase Overexpression in Vitro

    Science.gov (United States)

    2013-01-01

    Alzheimer’s disease, Familial British dementia, Familial Danish dementia, Type 2 diabetes mellitus, plus Creutzfeldt-Jakob disease are associated with amyloid fibril deposition and oxidative stress. The antioxidant enzyme catalase is a neuroprotective amyloid binding protein. Herein the effects of catalase overexpression in SH-SY5Y neuronal cells on the toxicity of amyloid-β (Aβ), amyloid-Bri (ABri), amyloid-Dan (ADan), amylin (IAPP), and prion protein (PrP) peptides were determined. Results showed catalase overexpression was neuroprotective against Aβ, ABri, ADan, IAPP, and PrP peptides. The catalase inhibitor 3-amino-1,2,4-triazole (3-AT) and catalase-amyloid interaction inhibitor benzothiazole aniline tetra(ethylene glycol) (BTA-EG4) significantly enhanced neurotoxicity of amyloid peptides in catalase overexpressing neuronal cells. This suggests catalase neuroprotection involves breakdown of hydrogen peroxide (H2O2) plus a direct binding interaction between catalase and the Aβ, ABri, ADan, IAPP, and PrP peptides. Kisspeptin 45–50 had additive neuroprotective actions against the Aβ peptide in catalase overexpressing cells. The effects of 3-AT had an intracellular site of action, while catalase-amyloid interactions had an extracellular component. These results suggest that the 3-AT and BTA-EG4 compounds may be able to inhibit endogenous catalase mediated neuroprotection. Use of BTA-EG4, or compounds that inhibit catalase binding to amyloid peptides, as potential therapeutics for Neurodegenerative diseases may therefore result in unwanted effects. PMID:23968537

  12. Benzothiazole aniline tetra(ethylene glycol) and 3-amino-1,2,4-triazole inhibit neuroprotection against amyloid peptides by catalase overexpression in vitro.

    Science.gov (United States)

    Chilumuri, Amrutha; Odell, Mark; Milton, Nathaniel G N

    2013-11-20

    Alzheimer's disease, Familial British dementia, Familial Danish dementia, Type 2 diabetes mellitus, plus Creutzfeldt-Jakob disease are associated with amyloid fibril deposition and oxidative stress. The antioxidant enzyme catalase is a neuroprotective amyloid binding protein. Herein the effects of catalase overexpression in SH-SY5Y neuronal cells on the toxicity of amyloid-β (Aβ), amyloid-Bri (ABri), amyloid-Dan (ADan), amylin (IAPP), and prion protein (PrP) peptides were determined. Results showed catalase overexpression was neuroprotective against Aβ, ABri, ADan, IAPP, and PrP peptides. The catalase inhibitor 3-amino-1,2,4-triazole (3-AT) and catalase-amyloid interaction inhibitor benzothiazole aniline tetra(ethylene glycol) (BTA-EG4) significantly enhanced neurotoxicity of amyloid peptides in catalase overexpressing neuronal cells. This suggests catalase neuroprotection involves breakdown of hydrogen peroxide (H2O2) plus a direct binding interaction between catalase and the Aβ, ABri, ADan, IAPP, and PrP peptides. Kisspeptin 45-50 had additive neuroprotective actions against the Aβ peptide in catalase overexpressing cells. The effects of 3-AT had an intracellular site of action, while catalase-amyloid interactions had an extracellular component. These results suggest that the 3-AT and BTA-EG4 compounds may be able to inhibit endogenous catalase mediated neuroprotection. Use of BTA-EG4, or compounds that inhibit catalase binding to amyloid peptides, as potential therapeutics for Neurodegenerative diseases may therefore result in unwanted effects.

  13. Effect of heating on the stability of amyloid A (AA) fibrils and the intra- and cross-species transmission of AA amyloidosis.

    Science.gov (United States)

    Ogawa, Saki; Murakami, Tomoaki; Inoshima, Yasuo; Ishiguro, Naotaka

    2015-01-01

    Amyloid A (AA) amyloidosis is a protein misfolding disease characterized by extracellular deposition of AA fibrils. AA fibrils are found in several tissues from food animals with AA amyloidosis. For hygienic purposes, heating is widely used to inactivate microbes in food, but it is uncertain whether heating is sufficient to inactivate AA fibrils and prevent intra- or cross-species transmission. We examined the effect of heating (at 60 °C or 100 °C) and autoclaving (at 121 °C or 135 °C) on murine and bovine AA fibrils using Western blot analysis, transmission electron microscopy (TEM), and mouse model transmission experiments. TEM revealed that a mixture of AA fibrils and amorphous aggregates appeared after heating at 100 °C, whereas autoclaving at 135 °C produced large amorphous aggregates. AA fibrils retained antigen specificity in Western blot analysis when heated at 100 °C or autoclaved at 121 °C, but not when autoclaved at 135 °C. Transmissible pathogenicity of murine and bovine AA fibrils subjected to heating (at 60 °C or 100 °C) was significantly stimulated and resulted in amyloid deposition in mice. Autoclaving of murine AA fibrils at 121 °C or 135 °C significantly decreased amyloid deposition. Moreover, amyloid deposition in mice injected with murine AA fibrils was more severe than that in mice injected with bovine AA fibrils. Bovine AA fibrils autoclaved at 121 °C or 135 °C did not induce amyloid deposition in mice. These results suggest that AA fibrils are relatively heat stable and that similar to prions, autoclaving at 135 °C is required to destroy the pathogenicity of AA fibrils. These findings may contribute to the prevention of AA fibril transmission through food materials to different animals and especially to humans.

  14. Insights on the binding of thioflavin derivative markers to amyloid fibril models and Aβ{sub 1-40} fibrils from computational approaches

    Energy Technology Data Exchange (ETDEWEB)

    Alí-Torres, Jorge; Rimola, Albert; Sodupe, Mariona [Departament de Química, Universitat Autònoma de Barcelona, Bellaterra 08193 (Spain); Rodriguez-Rodríguez, Cristina [Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, V6T 1Z1 (Canada)

    2014-10-06

    The present contribution analyzes the binding of ThT and neutral ThT derivatives to a β-sheet model by means of quantum chemical calculations. In addition, we study the properties of four molecules: (2-(2-hydroxyphenyl)benzoxazole (HBX), 2-(2-hydroxyphenyl)benzothiazole (HBT) and their respective iodinated compounds, HBXI and HBTI, in binding to amyloid fibril models and Aβ{sub 1-40}fibrils by using a combination of docking, molecular dynamics and quantum mechanics calculations.

  15. Interaction with prefibrillar species and amyloid-like fibrils changes the stiffness of lipid bilayers

    DEFF Research Database (Denmark)

    Borro, Bruno C; Parolini, Lucia; Cicuta, Pietro

    2017-01-01

    and amyloid-like fibrils of α-lactalbumin. These effects range from substantially lowering the bending rigidity of the membranes to irreversible structural changes and complete disruption of the lipid bilayers. Our findings clearly indicate how the wide heterogeneity in structures occurring during protein......Evaluating the toxicity of self-assembled protein states is a key step towards developing effective strategies against amyloidogenic pathologies such as Alzheimer's and Parkinson's diseases. Such analysis is directly connected to quantitatively probing the stability of the cellular membrane upon...... interaction with different protein states. Using a combination of spectroscopic techniques, morphological observations, and spectral analysis of membrane fluctuations, we identify different destabilisation routes for giant unilamellar vesicles interacting with native-like states, prefibrillar species...

  16. Conformational stability of mammalian prion protein amyloid fibrils is dictated by a packing polymorphism within the core region.

    Science.gov (United States)

    Cobb, Nathan J; Apostol, Marcin I; Chen, Shugui; Smirnovas, Vytautas; Surewicz, Witold K

    2014-01-31

    Mammalian prion strains are believed to arise from the propagation of distinct conformations of the misfolded prion protein PrP(Sc). One key operational parameter used to define differences between strains has been conformational stability of PrP(Sc) as defined by resistance to thermal and/or chemical denaturation. However, the structural basis of these stability differences is unknown. To bridge this gap, we have generated two strains of recombinant human prion protein amyloid fibrils that show dramatic differences in conformational stability and have characterized them by a number of biophysical methods. Backbone amide hydrogen/deuterium exchange experiments revealed that, in sharp contrast to previously studied strains of infectious amyloid formed from the yeast prion protein Sup35, differences in β-sheet core size do not underlie differences in conformational stability between strains of mammalian prion protein amyloid. Instead, these stability differences appear to be dictated by distinct packing arrangements (i.e. steric zipper interfaces) within the amyloid core, as indicated by distinct x-ray fiber diffraction patterns and large strain-dependent differences in hydrogen/deuterium exchange kinetics for histidine side chains within the core region. Although this study was limited to synthetic prion protein amyloid fibrils, a similar structural basis for strain-dependent conformational stability may apply to brain-derived PrP(Sc), especially because large strain-specific differences in PrP(Sc) stability are often observed despite a similar size of the PrP(Sc) core region.

  17. Genesis of mammalian prions: from non-infectious amyloid fibrils to a transmissible prion disease.

    Directory of Open Access Journals (Sweden)

    Natallia Makarava

    2011-12-01

    Full Text Available The transmissible agent of prion disease consists of a prion protein in its abnormal, β-sheet rich state (PrP(Sc, which is capable of replicating itself according to the template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a PrP(Sc template. Here we report that authentic PrP(Sc and transmissible prion disease can be generated de novo in wild type animals by recombinant PrP (rPrP amyloid fibrils, which are structurally different from PrP(Sc and lack any detectable PrP(Sc particles. When induced by rPrP fibrils, a long silent stage that involved two serial passages preceded development of the clinical disease. Once emerged, the prion disease was characterized by unique clinical, neuropathological, and biochemical features. The long silent stage to the disease was accompanied by significant transformation in neuropathological properties and biochemical features of the proteinase K-resistant PrP material (PrPres before authentic PrP(Sc evolved. The current work illustrates that transmissible prion diseases can be induced by PrP structures different from that of authentic PrP(Sc and suggests that a new mechanism different from the classical templating exists. This new mechanism designated as "deformed templating" postulates that a change in the PrP folding pattern from the one present in rPrP fibrils to an alternative specific for PrP(Sc can occur. The current work provides important new insight into the mechanisms underlying genesis of the transmissible protein states and has numerous implications for understanding the etiology of neurodegenerative diseases.

  18. Contact between the β1 and β2 Segments of α-Synuclein that Inhibits Amyloid Formation.

    Science.gov (United States)

    Shaykhalishahi, Hamed; Gauhar, Aziz; Wördehoff, Michael M; Grüning, Clara S R; Klein, Antonia N; Bannach, Oliver; Stoldt, Matthias; Willbold, Dieter; Härd, Torleif; Hoyer, Wolfgang

    2015-07-20

    Conversion of the intrinsically disordered protein α-synuclein (α-syn) into amyloid aggregates is a key process in Parkinson's disease. The sequence region 35-59 contains β-strand segments β1 and β2 of α-syn amyloid fibril models and most disease-related mutations. β1 and β2 frequently engage in transient interactions in monomeric α-syn. The consequences of β1-β2 contacts are evaluated by disulfide engineering, biophysical techniques, and cell viability assays. The double-cysteine mutant α-synCC, with a disulfide linking β1 and β2, is aggregation-incompetent and inhibits aggregation and toxicity of wild-type α-syn. We show that α-syn delays the aggregation of amyloid-β peptide and islet amyloid polypeptide involved in Alzheimer's disease and type 2 diabetes, an effect enhanced in the α-synCC mutant. Tertiary interactions in the β1-β2 region of α-syn interfere with the nucleation of amyloid formation, suggesting promotion of such interactions as a potential therapeutic approach. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Ellagic acid promotes Aβ42 fibrillization and inhibits Aβ42-induced neurotoxicity

    International Nuclear Information System (INIS)

    Feng, Ying; Yang, Shi-gao; Du, Xue-ting; Zhang, Xi; Sun, Xiao-xia; Zhao, Min; Sun, Gui-yuan; Liu, Rui-tian

    2009-01-01

    Smaller, soluble oligomers of β-amyloid (Aβ) play a critical role in the pathogenesis of Alzheimer's disease (AD). Selective inhibition of Aβ oligomer formation provides an optimum target for AD therapy. Some polyphenols have potent anti-amyloidogenic activities and protect against Aβ neurotoxicity. Here, we tested the effects of ellagic acid (EA), a polyphenolic compound, on Aβ42 aggregation and neurotoxicity in vitro. EA promoted Aβ fibril formation and significant oligomer loss, contrary to previous results that polyphenols inhibited Aβ aggregation. The results of transmission electron microscopy (TEM) and Western blot displayed more fibrils in Aβ42 samples co-incubated with EA in earlier phases of aggregation. Consistent with the hypothesis that plaque formation may represent a protective mechanism in which the body sequesters toxic Aβ aggregates to render them harmless, our MTT results showed that EA could significantly reduce Aβ42-induced neurotoxicity toward SH-SY5Y cells. Taken together, our results suggest that EA, an active ingredient in many fruits and nuts, may have therapeutic potential in AD.

  20. Ellagic acid promotes A{beta}42 fibrillization and inhibits A{beta}42-induced neurotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Ying [Department of Histology and Embryology, College of Basic Medical Science, China Medical University, Shenyang 110001 (China); Tsinghua University School of Medicine, Haidian District, Beijing 100084 (China); Yang, Shi-gao; Du, Xue-ting; Zhang, Xi; Sun, Xiao-xia; Zhao, Min [Tsinghua University School of Medicine, Haidian District, Beijing 100084 (China); Sun, Gui-yuan, E-mail: sungy2004@sohu.com [Department of Histology and Embryology, College of Basic Medical Science, China Medical University, Shenyang 110001 (China); Liu, Rui-tian, E-mail: rtliu@tsinghua.edu.cn [Tsinghua University School of Medicine, Haidian District, Beijing 100084 (China)

    2009-12-25

    Smaller, soluble oligomers of {beta}-amyloid (A{beta}) play a critical role in the pathogenesis of Alzheimer's disease (AD). Selective inhibition of A{beta} oligomer formation provides an optimum target for AD therapy. Some polyphenols have potent anti-amyloidogenic activities and protect against A{beta} neurotoxicity. Here, we tested the effects of ellagic acid (EA), a polyphenolic compound, on A{beta}42 aggregation and neurotoxicity in vitro. EA promoted A{beta} fibril formation and significant oligomer loss, contrary to previous results that polyphenols inhibited A{beta} aggregation. The results of transmission electron microscopy (TEM) and Western blot displayed more fibrils in A{beta}42 samples co-incubated with EA in earlier phases of aggregation. Consistent with the hypothesis that plaque formation may represent a protective mechanism in which the body sequesters toxic A{beta} aggregates to render them harmless, our MTT results showed that EA could significantly reduce A{beta}42-induced neurotoxicity toward SH-SY5Y cells. Taken together, our results suggest that EA, an active ingredient in many fruits and nuts, may have therapeutic potential in AD.

  1. Solution NMR structure and inhibitory effect against amyloidfibrillation of Humanin containing a d-isomerized serine residue.

    Science.gov (United States)

    Alsanousi, Nesreen; Sugiki, Toshihiko; Furuita, Kyoko; So, Masatomo; Lee, Young-Ho; Fujiwara, Toshimichi; Kojima, Chojiro

    2016-09-02

    Humanin comprising 24 amino acid residues is a bioactive peptide that has been isolated from the brain tissue of patients with Alzheimer's disease. Humanin reportedly suppressed aging-related death of various cells due to amyloid fibrils and oxidative stress. There are reports that the cytoprotective activity of Humanin was remarkably enhanced by optical isomerization of the Ser14 residue from l to d form, but details of the molecular mechanism remained unclear. Here we demonstrated that Humanin d-Ser14 exhibited potent inhibitory activity against fibrillation of amyloid-β and remarkably higher binding affinity for amyloid-β than that of the Humanin wild-type and S14G mutant. In addition, we determined the solution structure of Humanin d-Ser14 by nuclear magnetic resonance (NMR) and showed that d-isomerization of the Ser14 residue enables drastic conformational rearrangement of Humanin. Furthermore, we identified an amyloid-β-binding site on Humanin d-Ser14 at atomic resolution by NMR. These biophysical and high-resolution structural analyses clearly revealed structure-function relationships of Humanin and explained the driving force of the drastic conformational change and molecular basis of the potent anti-amyloidfibrillation activity of Humanin caused by d-isomerization of the Ser14 residue. This is the first study to show correlations between the functional activity, tertiary structure, and partner recognition mode of Humanin and may lead to elucidation of the molecular mechanisms of the cytoprotective activity of Humanin. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Negatively charged food additive dye "Allura Red" rapidly induces SDS-soluble amyloid fibril in beta-lactoglobulin protein.

    Science.gov (United States)

    Al-Shabib, Nasser Abdulatif; Khan, Javed Masood; Malik, Ajamaluddin; Alsenaidy, Abdulrahman M; Alsenaidy, Mohammad A; Husain, Fohad Mabood; Shamsi, Monis Bilal; Hidayathulla, Syed; Khan, Rizwan Hasan

    2018-02-01

    Recent studies have led to an increased interest to categorize small molecular inhibitors of protein fibrillation. In this study, we used spectroscopy, microscopy and gel electrophoresis techniques that provides an elaborated description of the Allura Red-induced amyloid fibrillation in the β-LG protein at two pHs (7.4 and 3.5). The spectroscopy results show that β-LG protein form aggregates in the presence of Allura Red (0.04-15.0mM) at pH 3.5 due to electrostatic and hydrophobic interactions. However, at pH 7.4, the β-LG does not interact electrostatically with Allura Red and therefore no aggregation occurred. The Allura Red-induced aggregates have an amyloid-like structure that was confirmed by far-UV CD, Congo Red and transmission electron microscopy (TEM). The CD spectrum of β-LG contains single minima at ∼218nm, which shifts towards higher wavelength minima at ∼225nm in the presence of Allura Red, characteristics of the cross β-sheet structure. The TEM results suggest that β-LG form long straight fibril when exposed to Allura Red at pH 3.5. The Allura Red-induced amyloid fibril is SDS-soluble confirmed by SDS-PAGE techniques. A far UV CD result shows the conversion of Allura Red induced cross β-sheet structure into alpha-helical structure in the presence of increasing concentration of SDS. The results of this study suggest that the electrostatic, as well as hydrophobic interactions play an important role during Allura Red-induced β-LG fibrillation. Copyright © 2017. Published by Elsevier B.V.

  3. Antibody-conjugated, dual-modal, near-infrared fluorescent iron oxide nanoparticles for antiamyloidgenic activity and specific detection of amyloidfibrils

    Directory of Open Access Journals (Sweden)

    Skaat H

    2013-10-01

    Full Text Available Hadas Skaat,1 Enav Corem-Slakmon,1 Igor Grinberg,1 David Last,2 David Goez,2 Yael Mardor,2,3 Shlomo Margel1 1Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Ramat-Gan, Israel; 2Advanced Technology Center, Sheba Medical Center, Tel-Hashomer, Ramat-Gan, Israel; 3Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel Abstract: Amyloid-β (Aβ peptide is the main fibrillar component of plaque deposits found in brains affected by Alzheimer's disease (AD and is related to the pathogenesis of AD. Passive anti-Aβ immunotherapy has emerged as a promising approach for the therapy of AD, based on the administration of specific anti-Aβ monoclonal antibodies (aAβmAbs to delay Aβ aggregation in the brain. However, the main disadvantage of this approach is the required readministration of the aAβmAbs at frequent intervals. There are only a few reports describing in vitro study for the immobilization of aAβmAbs to nanoparticles as potential targeting agents of Aβ aggregates. In this article, we report the immobilization of the aAβmAb clone BAM10 to near-infrared fluorescent maghemite nanoparticles for the inhibition of Aβ40 fibrillation kinetics and the specific detection of Aβ40 fibrils. The BAM10-conjugated iron oxide nanoparticles were well-characterized, including their immunogold labeling and cytotoxic effect on PC-12 (pheochromocytoma cell line. Indeed, these antibody-conjugated nanoparticles significantly inhibit the Aβ40 fibrillation kinetics compared with the same concentration, or even five times higher, of the free BAM10. This inhibitory effect was confirmed by different assays such as the photo-induced crosslinking of unmodified proteins combined with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. A cell viability assay also confirmed that these antibody-conjugated nanoparticles significantly reduced the Aβ40-induced cytotoxicity to PC-12 cells. Furthermore, the selective

  4. Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli.

    Science.gov (United States)

    Li, Qiaojing; Richard, Charles-Adrien; Moudjou, Mohammed; Vidic, Jasmina

    2015-12-19

    The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni(2+)-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

  5. β-Amyloid-derived pentapeptide RIIGLa inhibits Aβ1-42 aggregation and toxicity

    International Nuclear Information System (INIS)

    Fueloep, Livia; Zarandi, Marta; Datki, Zsolt; Soos, Katalin; Penke, Botond

    2004-01-01

    Pr-IIGL a , a derivative of the tetrapeptide β-amyloid 31-34 (Aβ 31-34 ), exerts controversial effects: it is toxic in a neuroblastoma culture, but it protects glial cells from the cytotoxic action of Aβ 1-42 . For an understanding of this phenomenon, a new pentapeptide, RIIGL a was synthetized, and both compounds were studied by different physicochemical and biological methods. Transmission electron microscopic (TEM) studies revealed that Pr-IIGL a forms fibrillar aggregates, whereas RIIGL a does not form fibrils. Congo red binding studies furnished the same results. Aggregated Pr-IIGL a acts as a cytotoxic agent in neuroblastoma cultures, but RIIGL a does not display inherent toxicity. RIIGL a co-incubated with Aβ 1-42 inhibits the formation of mature amyloid fibres (TEM studies) and reduces the cytotoxic effect of fibrillar Aβ 1-42 . These results indicate that RIIGL a is an effective inhibitor of both the aggregation and the toxic effects of Aβ 1-42 and can serve as a lead compound for the design of novel neuroprotective peptidomimetics

  6. Polymorphism of amyloid fibrils formed by a peptide from the yeast prion protein Sup35: AFM and Tip-Enhanced Raman Scattering studies

    Energy Technology Data Exchange (ETDEWEB)

    Krasnoslobodtsev, Alexey V., E-mail: akrasnos@unomaha.edu [Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198 (United States); Department of Physics, University of Nebraska Omaha, Omaha, NE 68182 (United States); Deckert-Gaudig, Tanja [IPHT-Leibniz Institute of Photonic Technology, Albert-Einstein-Str. 9, D-07745 Jena (Germany); Zhang, Yuliang [Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198 (United States); Deckert, Volker [IPHT-Leibniz Institute of Photonic Technology, Albert-Einstein-Str. 9, D-07745 Jena (Germany); Institute for Physical Chemistry and Abbe Center of Photonics, University of Jena, Helmholtzweg 4, D-07743 Jena (Germany); Lyubchenko, Yuri L., E-mail: ylyubchenko@unmc.edu [Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198 (United States)

    2016-06-15

    Aggregation of prion proteins is the cause of various prion related diseases. The infectious form of prions, amyloid aggregates, exist as multiple strains. The strains are thought to represent structurally different prion protein molecules packed into amyloid aggregates, but the knowledge on the structure of different types of aggregates is limited. Here we report on the use of AFM (Atomic Force Microscopy) and TERS (Tip-Enhanced Raman Scattering) to study morphological heterogeneity and access underlying conformational features of individual amyloid aggregates. Using AFM we identified the morphology of amyloid fibrils formed by the peptide (CGNNQQNY) from the yeast prion protein Sup35 that is critically involved in the aggregation of the full protein. TERS results demonstrate that morphologically different amyloid fibrils are composed of a distinct set of conformations. Fibrils formed at pH 5.6 are composed of a mixture of peptide conformations (β-sheets, random coil and α-helix) while fibrils formed in pH~2 solution primarily have β-sheets. Additionally, peak positions in the amide III region of the TERS spectra suggested that peptides have parallel arrangement of β-sheets for pH~2 fibrils and antiparallel arrangement for fibrils formed at pH 5.6. We also developed a methodology for detailed analysis of the peptide secondary structure by correlating intensity changes of Raman bands in different regions of TERS spectra. Such correlation established that structural composition of peptides is highly localized with large contribution of unordered secondary structures on a fibrillar surface. - Highlights: • Amyloid polymorphs were characterized by AFM and TERS. • A mixture of peptide secondary structures in fibrils were identified using TERS. • TERS recognizes packing arrangement (parallel versus antiparallel) of peptides. • TERS is a powerful tool for high resolution structural analysis of fibrils.

  7. Oil Palm Phenolics Inhibit the In Vitro Aggregation of β-Amyloid Peptide into Oligomeric Complexes

    Directory of Open Access Journals (Sweden)

    Robert P. Weinberg

    2018-01-01

    Full Text Available Alzheimer’s disease is a severe neurodegenerative disease characterized by the aggregation of amyloid-β peptide (Aβ into toxic oligomers which activate microglia and astrocytes causing acute neuroinflammation. Multiple studies show that the soluble oligomers of Aβ42 are neurotoxic and proinflammatory, whereas the monomers and insoluble fibrils are relatively nontoxic. We show that Aβ42 aggregation is inhibited in vitro by oil palm phenolics (OPP, an aqueous extract from the oil palm tree (Elaeis guineensis. The data shows that OPP inhibits stacking of β-pleated sheets, which is essential for oligomerization. We demonstrate the inhibition of Aβ42 aggregation by (1 mass spectrometry; (2 Congo Red dye binding; (3 2D-IR spectroscopy; (4 dynamic light scattering; (5 transmission electron microscopy; and (6 transgenic yeast rescue assay. In the yeast rescue assay, OPP significantly reduces the cytotoxicity of aggregating neuropeptides in yeast genetically engineered to overexpress these peptides. The data shows that OPP inhibits (1 the aggregation of Aβ into oligomers; (2 stacking of β-pleated sheets; and (3 fibrillar growth and coalescence. These inhibitory effects prevent the formation of neurotoxic oligomers and hold potential as a means to reduce neuroinflammation and neuronal death and thereby may play some role in the prevention or treatment of Alzheimer’s disease.

  8. Dispersible amyloid β-protein oligomers, protofibrils, and fibrils represent diffusible but not soluble aggregates: their role in neurodegeneration in amyloid precursor protein (APP) transgenic mice.

    Science.gov (United States)

    Rijal Upadhaya, Ajeet; Capetillo-Zarate, Estibaliz; Kosterin, Irina; Abramowski, Dorothee; Kumar, Sathish; Yamaguchi, Haruyasu; Walter, Jochen; Fändrich, Marcus; Staufenbiel, Matthias; Thal, Dietmar Rudolf

    2012-11-01

    Soluble amyloid β-protein (Aβ) aggregates have been identified in the Alzheimer's disease (AD) brain. Dispersed Aβ aggregates in the brain parenchyma are different from soluble, membrane-associated and plaque-associated solid aggregates. They are in mixture with the extra- or intracellular fluid but can be separated from soluble proteins by ultracentrifugation. To clarify the role of dispersible Aβ aggregates for neurodegeneration we analyzed 2 different amyloid precursor protein (APP)-transgenic mouse models. APP23 mice overexpress human mutant APP with the Swedish mutation. APP51/16 mice express high levels of human wild type APP. Both mice develop Aβ-plaques. Dendritic degeneration, neuron loss, and loss of asymmetric synapses were seen in APP23 but not in APP51/16 mice. The soluble and dispersible fractions not separated from one another were received as supernatant after centrifugation of native forebrain homogenates at 14,000 × g. Subsequent ultracentrifugation separated the soluble, i.e., the supernatant, from the dispersible fraction, i.e., the resuspended pellet. The major biochemical difference between APP23 and APP51/16 mice was that APP23 mice exhibited higher levels of dispersible Aβ oligomers, protofibrils and fibrils precipitated with oligomer (A11) and protofibril/fibril (B10AP) specific antibodies than APP51/16 mice. These differences, rather than soluble Aβ and Aβ plaque pathology were associated with dendritic degeneration, neuron, and synapse loss in APP23 mice in comparison with APP51/16 mice. Immunoprecipitation of dispersible Aβ oligomers, protofibrils, and fibrils revealed that they were associated with APP C-terminal fragments (APP-CTFs). These results indicate that dispersible Aβ oligomers, protofibrils, and fibrils represent an important pool of Aβ aggregates in the brain that critically interact with membrane-associated APP C-terminal fragments. The concentration of dispersible Aβ aggregates, thereby, presumably determines

  9. The molecular mass of dextran used to modify magnetite nanoparticles affects insulin amyloid aggregation

    Energy Technology Data Exchange (ETDEWEB)

    Siposova, Katarina [Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Kosice (Slovakia); Pospiskova, Kristyna [Regional Centre of Advanced Technologies and Materials, Palacky University, Olomouc (Czech Republic); Bednarikova, Zuzana [Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Kosice (Slovakia); Department of Biochemistry, Faculty of Science, Safarik University, Kosice (Slovakia); Safarik, Ivo [Regional Centre of Advanced Technologies and Materials, Palacky University, Olomouc (Czech Republic); Department of Nanobiotechnology, Biology Centre, ISB, CAS, Ceske Budejovice (Czech Republic); Safarikova, Mirka [Department of Nanobiotechnology, Biology Centre, ISB, CAS, Ceske Budejovice (Czech Republic); Kubovcikova, Martina; Kopcansky, Peter [Department of Magnetism, Institute of Experimental Physics, Slovak Academy of Sciences, Kosice (Slovakia); Gazova, Zuzana, E-mail: gazova@saske.sk [Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Kosice (Slovakia)

    2017-04-01

    Protein transformation from its soluble state into amyloid aggregates is associated with amyloid-related diseases. Amyloid deposits of insulin fibrils have been found in the sites of subcutaneous insulin application in patients with prolonged diabetes. Using atomic force microscopy and ThT fluorescence assay we have investigated the interference of insulin amyloid aggregation with superparamagnetic Fe{sub 3}O{sub 4}-based nanoparticles (SPIONs) coated with dextran (DEX); molecular mass of dextran was equal to 15–20, 40 or 70 kDa. The obtained data indicate that all three types of dextran coated nanoparticles (NP-FeDEXs) are able to inhibit insulin fibrillization and to destroy amyloid fibrils. The extent of anti-amyloid activities depends on the properties of NP-FeDEXs, mainly on the size of nanoparticles which is determined by molecular mass of dextran molecules. The most effective inhibiting activity was observed for the smallest nanoparticles coated with 15–20 kDa dextran. Contrary, the highest destroying activity was observed for the largest NP-FeDEX (70 kDa dextran). - Highlights: • Interference of dextran- magnetite nanoparticles with insulin amyloid aggregation. • Nanoparticles inhibited insulin fibrillization and depolymerized insulin amyloid fibrils. • Size of nanoparticles significantly influences their anti-amyloid activities. • The most effective inhibition of insulin amyloid fibrillization was detected for the smallest nanoparticles. • Contrary, DC{sub 50} values decreased with increasing size of nanoparticles.

  10. Nonequilibrium all-atom molecular dynamics simulation of the bubble cavitation and application to dissociate amyloid fibrils

    Science.gov (United States)

    Hoang Viet, Man; Derreumaux, Philippe; Nguyen, Phuong H.

    2016-11-01

    The cavitation of gas bubbles in liquids has been applied to different disciplines in life and natural sciences, and in technologies. To obtain an appropriate theoretical description of effects induced by the bubble cavitation, we develop an all-atom nonequilibrium molecular-dynamics simulation method to simulate bubbles undergoing harmonic oscillation in size. This allows us to understand the mechanism of the bubble cavitation-induced liquid shear stress on surrounding objects. The method is then employed to simulate an Aβ fibril model in the presence of bubbles, and the results show that the bubble expansion and contraction exert water pressure on the fibril. This yields to the deceleration and acceleration of the fibril kinetic energy, facilitating the conformational transition between local free energy minima, and leading to the dissociation of the fibril. Our work, which is a proof-of-concept, may open a new, efficient way to dissociate amyloid fibrils using the bubble cavitation technique, and new venues to investigate the complex phenomena associated with amyloidogenesis.

  11. Keampferol-3-O-rhamnoside abrogates amyloid beta toxicity by modulating monomers and remodeling oligomers and fibrils to non-toxic aggregates

    Directory of Open Access Journals (Sweden)

    Sharoar Md

    2012-12-01

    Full Text Available Abstract Background Aggregation of soluble, monomeric β- amyloid (Aβ to oligomeric and then insoluble fibrillar Aβ is a key pathogenic feature in development of Alzheimer’s disease (AD. Increasing evidence suggests that toxicity is linked to diffusible Aβ oligomers, rather than to insoluble fibrils. The use of naturally occurring small molecules for inhibition of Aβ aggregation has recently attracted significant interest for development of effective therapeutic strategies against the disease. A natural polyphenolic flavone, Kaempferol-3-O-rhamnoside (K-3-rh, was utilized to investigate its effects on aggregation and cytotoxic effects of Aβ42 peptide. Several biochemical techniques were used to determine the conformational changes and cytotoxic effect of the peptide in the presence and absence of K-3-rh. Results K-3-rh showed a dose-dependent effect against Aβ42 mediated cytotoxicity. Anti-amyloidogenic properties of K-3-rh were found to be efficient in inhibiting fibrilogenesis and secondary structural transformation of the peptide. The consequence of these inhibitions was the accumulation of oligomeric structural species. The accumulated aggregates were smaller, soluble, non-β-sheet and non-toxic aggregates, compared to preformed toxic Aβ oligomers. K-3-rh was also found to have the remodeling properties of preformed soluble oligomers and fibrils. Both of these conformers were found to remodel into non-toxic aggregates. The results showed that K-3-rh interacts with different Aβ conformers, which affects fibril formation, oligomeric maturation and fibrillar stabilization. Conclusion K-3-rh is an efficient molecule to hinder the self assembly and to abrogate the cytotoxic effects of Aβ42 peptide. Hence, K-3-rh and small molecules with similar structure might be considered for therapeutic development against AD.

  12. Solution NMR structure and inhibitory effect against amyloidfibrillation of Humanin containing a D-isomerized serine residue

    Energy Technology Data Exchange (ETDEWEB)

    Alsanousi, Nesreen [Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Sugiki, Toshihiko, E-mail: sugiki@protein.osaka-u.ac.jp [Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Furuita, Kyoko; So, Masatomo; Lee, Young-Ho; Fujiwara, Toshimichi [Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kojima, Chojiro, E-mail: kojima-chojiro-xk@ynu.ac.jp [Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Graduate School of Engineering, Yokohama National University, Tokiwadai 79-5, Hodogaya-ku, Yokohama 240-8501 (Japan)

    2016-09-02

    Humanin comprising 24 amino acid residues is a bioactive peptide that has been isolated from the brain tissue of patients with Alzheimer's disease. Humanin reportedly suppressed aging-related death of various cells due to amyloid fibrils and oxidative stress. There are reports that the cytoprotective activity of Humanin was remarkably enhanced by optical isomerization of the Ser14 residue from L to D form, but details of the molecular mechanism remained unclear. Here we demonstrated that Humanin D-Ser14 exhibited potent inhibitory activity against fibrillation of amyloid-β and remarkably higher binding affinity for amyloid-β than that of the Humanin wild-type and S14G mutant. In addition, we determined the solution structure of Humanin D-Ser14 by nuclear magnetic resonance (NMR) and showed that D-isomerization of the Ser14 residue enables drastic conformational rearrangement of Humanin. Furthermore, we identified an amyloid-β-binding site on Humanin D-Ser14 at atomic resolution by NMR. These biophysical and high-resolution structural analyses clearly revealed structure–function relationships of Humanin and explained the driving force of the drastic conformational change and molecular basis of the potent anti-amyloidfibrillation activity of Humanin caused by D-isomerization of the Ser14 residue. This is the first study to show correlations between the functional activity, tertiary structure, and partner recognition mode of Humanin and may lead to elucidation of the molecular mechanisms of the cytoprotective activity of Humanin. - Highlights: • Humanin D-Ser14 showed the strongest inhibitory activity against Aβ40 fibrillation. • NMR structure of Humanin D-Ser14 was determined in alcohol/water mixture solution. • Humanin D-Ser14 directly bound Aβ40 stronger than Humanin wild-type and Humanin S14G. • Aβ40 and zinc ion binding sites of Humanin D-Ser14 were identified. • Structure around Ser14 of Humanin is critical for Aβ40 binding and

  13. A Synchrotron-Based Hydroxyl Radical Footprinting Analysis of Amyloid Fibrils and Prefibrillar Intermediates with Residue-Specific Resolution

    Energy Technology Data Exchange (ETDEWEB)

    Klinger, Alexandra L. [Univ. of Pennsylvania, Philadelphia, PA (United States); Kiselar, Janna [Case Western Reserve Univ., Cleveland, OH (United States); Ilchenko, Serguei [Case Western Reserve Univ., Cleveland, OH (United States); Komatsu, Hiroaki [Univ. of Pennsylvania, Philadelphia, PA (United States); Chance, Mark R. [Case Western Reserve Univ., Cleveland, OH (United States); Axelsen, Paul H. [Univ. of Pennsylvania, Philadelphia, PA (United States)

    2014-11-09

    The structural models of the fibrils formed by the 40-residue amyloid-β (Aβ40) peptide in Alzheimer’s disease typically consist of linear polypeptide segments, oriented approximately perpendicular to the long axis of the fibril, and joined together as parallel in-register β-sheets to form filaments. However, various models differ in the number of filaments that run the length of a fibril, and in the topological arrangement of these filaments. In addition to questions about the structure of Aβ40 monomers in fibrils, there are important unanswered questions about their structure in prefibrillar intermediates, which are of interest because they may represent the most neurotoxic form of Aβ40. To assess different models of fibril structure and to gain insight into the structure of prefibrillar intermediates, the relative solvent accessibility of amino acid residue side chains in fibrillar and prefibrillar Aβ40 preparations was characterized in solution by hydroxyl radical footprinting and structural mass spectrometry. A key to the application of this technology was the development of hydroxyl radical reactivity measures for individual side chains of Aβ40. When we combined mass-per-length measurements performed by dark-field electron microscopy, we determined that the results of our study were consistent with the core filament structure represented by two- and three-filament solid state nuclear magnetic resonance-based models of the Aβ40 fibril (such as 2LMN, 2LMO, 2LMP, and 2LMQ), with minor refinements, but they are inconsistent with the more recently proposed 2M4J model. Our results also demonstrate that individual Aβ40 fibrils exhibit structural heterogeneity or polymorphism, where regions of two-filament structure alternate with regions of three-filament structure. The footprinting approach utilized in this study will be valuable for characterizing various fibrillar and nonfibrillar forms of the Aβ peptide.

  14. Albumin-induced circular dichroism in Congo red: Applications for studies of amyloid-like fibril aggregates and binding sites.

    Science.gov (United States)

    de Vasconcelos, Débora Naliati; Ximenes, Valdecir Farias

    2015-01-01

    Congo red (CR), one of the most commonly used dyes for the identification of amyloid fibril aggregates, is also a ligand of native bovine serum albumin (BSA). Induced circular dichroism (ICD) is a phenomenon observed when a chiral compound induces chirality in an achiral one. Here, we study the spectral properties and analytical applications of ICD in Congo red provoked by its interaction with BSA. The complex BSA:CR displays a strong ICD spectrum with a positive band at 412 nm and two negative bands at 356 and 490 nm. The use of site I and site II albumin ligands as warfarin and ibuprofen, respectively, provoked different alterations in the Congo red ICD spectrum. The BSA binding sites were modified by oxidation and the ICD signal was sensitive to this alteration. The thermal treatment of the BSA:CR complex (30-90 °C) was monitored by ICD at 490 nm and showed a sigmoidal pattern typical of phase transition in proteins. The altered ICD spectrum is consistent with the formation of amyloid-like fibril aggregates in BSA, which was confirmed by thioflavin T and Rayleigh scattering assays. In conclusion, the ICD provoked by the binding of Congo red to albumin may represent a new spectroscopic technique for studying alterations in the structure of albumin regarding its binding sites and the formation of amyloid aggregates. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Dynamic domains of amyloid fibrils can be site-specifically assigned with proton detected 3D NMR spectroscopy

    International Nuclear Information System (INIS)

    Falk, Alexander S.; Siemer, Ansgar B.

    2016-01-01

    Several amyloid fibrils have cores framed by highly dynamic, intrinsically disordered, domains that can play important roles for function and toxicity. To study these domains in detail using solid-state NMR spectroscopy, site-specific resonance assignments are required. Although the rapid dynamics of these domains lead to considerable averaging of orientation-dependent NMR interactions and thereby line-narrowing, the proton linewidths observed in these samples is far larger than what is regularly observed in solution. Here, we show that it is nevertheless possible to record 3D HNCO, HNCA, and HNcoCA spectra on these intrinsically disordered domains and to obtain site-specific assignments.

  16. Dynamic domains of amyloid fibrils can be site-specifically assigned with proton detected 3D NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Falk, Alexander S.; Siemer, Ansgar B., E-mail: asiemer@usc.edu [Keck School of Medicine of USC, Department of Biochemistry and Molecular Medicine, Zilkha Neurogenetic Institute (United States)

    2016-11-15

    Several amyloid fibrils have cores framed by highly dynamic, intrinsically disordered, domains that can play important roles for function and toxicity. To study these domains in detail using solid-state NMR spectroscopy, site-specific resonance assignments are required. Although the rapid dynamics of these domains lead to considerable averaging of orientation-dependent NMR interactions and thereby line-narrowing, the proton linewidths observed in these samples is far larger than what is regularly observed in solution. Here, we show that it is nevertheless possible to record 3D HNCO, HNCA, and HNcoCA spectra on these intrinsically disordered domains and to obtain site-specific assignments.

  17. Hydrogen/deuterium exchange mass spectrometry identifies two highly protected regions in recombinant full-length prion protein amyloid fibrils.

    Science.gov (United States)

    Nazabal, Alexis; Hornemann, Simone; Aguzzi, Adriano; Zenobi, Renato

    2009-06-01

    Understanding the structural basis that distinguishes the amyloid form of the prion protein from its monomeric homologue is of crucial importance to elucidate the mechanism of the lethal diseases related to this protein. Recently, an in vitro conversion system was established which reproduces the transition of recombinant prion protein PrP(23-230) from its native alpha-helical rich form into an aggregated amyloid beta-sheet rich form with physicochemical properties reminiscent to those of the disease-related isoform of the prion protein, PrPSc. To study the tertiary and quaternary structural organization within recombinant amyloid fibrils from mouse, mPrP(23-231)betaf; bovine, bPrP(23-230)betaf; and elk, ePrP(23-230)betaf; we utilized hydrogen/deuterium (H/D) exchange analyzed by matrix-assisted laser desorption/ionization (MALDI) and nano-electrospray (nano-ESI) mass spectrometry. No significant differences were found by measuring the deuterium exchange kinetics of the aggregated fibrillar forms for mPrP(23-231)betaf, bPrP(23-230)betaf and ePrP(23-230)betaf, indicating a similar overall structural organization of the fibrils from all three species. Next, we characterized the solvent accessibility for the soluble and fibrillar forms of the mouse prion protein by hydrogen exchange, pepsin proteolysis and nano-ESI ion trap mass spectrometry analysis. In its amyloid form, two highly protected regions of mPrP(23-231) comprising residues [24-98] and [182-212] were identified. The residues between the two highly protected stretches were found to be more solvent exposed, but less than in the soluble protein, and might therefore rather form part of a fibrillar interface. Copyright 2009 John Wiley & Sons, Ltd.

  18. Investigation of amyloid formation inhibition of chemically and biogenically from Citrus aurantium L. blossoms and Rose damascena oils of gold nanoparticles: Toxicity evaluation in rat pheochromocytoma PC12 cells.

    Science.gov (United States)

    Sattarahmady, N; Firoozabadi, V; Nazari-Vanani, R; Azarpira, N

    2018-02-06

    Fibrillation inhibition effects of chemically and biogenically gold nanoparticles (GNPs) were investigated in vitro using human insulin as a model for fibrillation of protein. This inspection was followed using the Congo red assay, thioflavin T fluorescence measurements, transmission electron microscopy, and evaluation of cytotoxicity effects on rat pheochromocytoma PC12 cells. Biogenic GNPs were synthesized using oil extracts of Citrus aurantium L. blossoms and Rose damascena blossoms as reducing and concomitant agents. Congo red assay showed development of fibril formation of insulin at acidic media at 60°C over a period of 48h. In these circumstances, transmission electron micrographs confirmed the progress of fibril state from globular chains to amyloid. However, the results of ThT fluorescence measurements indicated a concentration-dependent inhibiting effect of chemically synthesized GNPs on insulin fibrillation in vitro, simultaneously by conversion of the formed fibrils into amorphous aggregates. Furthermore, biogenic GNPs were found to more effectively inhibit the fibril formation, compared to chemically synthesized GNPs. Accordingly, just 0.05nmolL -1 of the biogenic GNPs showed similar inhibition property of chemically synthesized GNPs with a concentration of 10nmolL -1 . Both types of GNPs diminished toxicity of insulin fibrils in rat pheochromocytoma PC12 cells viability. Copyright © 2017. Published by Elsevier B.V.

  19. Basic Equations in Statics and Kinetics of Protein Polymerization and the Mechanism of the Formation and Dissociation of Amyloid Fibrils Revealed by Pressure Perturbation.

    Science.gov (United States)

    Tachibana, Hideki

    2015-01-01

    Studies of the pressure-dissociation of several amyloid or amyloid-like fibrils have shown that the fibril state is considerably voluminous. Quantitative characterization of the protein fibrillation reaction with respect to volumetric parameters is necessary to elucidate mechanisms of amyloid fibrillation in molecular terms such as protein cavity and hydration. Here we discuss, firstly, basic equations in statics and kinetics of protein polymerization as employed to obtain thermodynamic, volumetric, and kinetic parameters. Equilibrium treatment of the reactions with the scheme such as one-step polymerization, linear-association polymerization, or nucleation-dependent polymerization, and kinetic treatment of seeded linear-polymerization or spontaneous nucleation-elongation polymerization are described. In particular we will detail kinetics of the dissociation of fibrils which have been produced under the linear-association mechanism and therefore the length-distribution of which conforms to a geometric sequence in the degree of polymerization with a common ratio r, which is less than, and usually very close to, unity. In this case, an observed macroscopic rate of dissociation is shown to be a product of the microscopic elementary dissociation rate constant and a factor (1-r), extremely reduced compared with the intrinsic elementary rate. Secondly, we discuss protein conformational states in fibrillogenesis with molecular and volumetric observations reported, such as the unfolded state responsible for the association with seeds and the extension of amyloid fibrils, the transition state in which protein cavity formation and dehydration occur to intermediate levels, and the fibril state in which they occur to final respective levels which, in some cases, depend on the maturity of the fibril.

  20. The Associative Memory, Water Mediated, Structure and Energy Model (AWSEM)-Amylometer: Predicting Amyloid Propensity and Fibril Topology Using an Optimized Folding Landscape Model.

    Science.gov (United States)

    Chen, Mingchen; Schafer, Nicholas P; Zheng, Weihua; Wolynes, Peter G

    2018-01-10

    Amyloids are fibrillar protein aggregates with simple repeated structural motifs in their cores, usually β-strands but sometimes α-helices. Identifying the amyloid-prone regions within protein sequences is important both for understanding the mechanisms of amyloid-associated diseases and for understanding functional amyloids. Based on the crystal structures of seven cross-β amyloidogenic peptides with different topologies and one recently solved cross-α fiber structure, we have developed a computational approach for identifying amyloidogenic segments in protein sequences using the Associative memory, Water mediated, Structure and Energy Model (AWSEM). The AWSEM-Amylometer performs favorably in comparison with other predictors in predicting aggregation-prone sequences in multiple data sets. The method also predicts well the specific topologies (the relative arrangement of β-strands in the core) of the amyloid fibrils. An important advantage of the AWSEM-Amylometer over other existing methods is its direct connection with an efficient, optimized protein folding simulation model, AWSEM. This connection allows one to combine efficient and accurate search of protein sequences for amyloidogenic segments with the detailed study of the thermodynamic and kinetic roles that these segments play in folding and aggregation in the context of the entire protein sequence. We present new simulation results that highlight the free energy landscapes of peptides that can take on multiple fibril topologies. We also demonstrate how the Amylometer methodology can be straightforwardly extended to the study of functional amyloids that have the recently discovered cross-α fibril architecture.

  1. Identification of Plant Extracts that Inhibit the Formation of Diabetes-Linked IAPP Amyloid.

    Science.gov (United States)

    Fuentes, Ana Lucia; Hennessy, Kathleen; Pascual, Jacob; Pepe, Nicole; Wang, In; Santiago, Alexander; Chaggan, Cynthia; Martinez, Jessica; Rivera, Evelyn; Cota, Paola; Cunha, Christina; Nogaj, Luiza A; Moffet, David A

    2016-03-01

    The extracts of 27 vegetables, spices and herbs were screened for their functional ability to inhibit the aggregation of islet amyloid polypeptide (IAPP, amylin) into toxic amyloid aggregates. The aggregation of IAPP has been directly linked to the death of pancreatic β-islet cells in type 2 diabetes. Inhibiting the aggregation of IAPP is believed to have the potential to slow, if not prevent entirely, the progression of this disease. As vegetables, spices and herbs are known to possess many different positive health effects, the extracts of 27 plants (abundant within the United States and spanning several plant families) were screened for their ability to inhibit the formation of toxic IAPP aggregates. Their anti-amyloid activities were assessed through (1) thioflavin T binding assays, (2) visualization of amyloid fibers using atomic force microscopy and (3) cell rescue studies. From this research, mint, peppermint, red bell pepper and thyme emerged as possessing the greatest anti-amyloid activity.

  2. 9,10-Anthraquinone hinders β-aggregation: How does a small molecule interfere with Aβ-peptide amyloid fibrillation?

    Science.gov (United States)

    Convertino, Marino; Pellarin, Riccardo; Catto, Marco; Carotti, Angelo; Caflisch, Amedeo

    2009-01-01

    Amyloid aggregation is linked to a number of neurodegenerative syndromes, the most prevalent one being Alzheimer's disease. In this pathology, the β-amyloid peptides (Aβ) aggregate into oligomers, protofibrils, and fibrils and eventually into plaques, which constitute the characteristic hallmark of Alzheimer's disease. Several low-molecular-weight compounds able to impair the Aβ aggregation process have been recently discovered; yet, a detailed description of their interactions with oligomers and fibrils is hitherto missing. Here, molecular dynamics simulations are used to investigate the influence of two relatively similar tricyclic, planar compounds, that is, 9, 10-anthraquinone (AQ) and anthracene (AC), on the early phase of the aggregation of the Aβ heptapeptide segment H14QKLVFF20, the hydrophobic stretch that promotes the Aβ self-assembly. The simulations show that AQ interferes with β-sheet formation more than AC. In particular, AQ intercalates into the β-sheet because polar interactions between the compound and the peptide backbone destabilize the interstrand hydrogen bonds, thereby favoring disorder. The thioflavin T-binding assay indicates that AQ, but not AC, sensibly reduces the amount of aggregated Aβ1–40 peptide. Taken together, the in silico and in vitro results provide evidence that structural perturbations by AQ can remarkably affect ordered oligomerization. Moreover, the simulations shed light at the atomic level on the interactions between AQ and Aβ oligomers, providing useful insights for the design of small-molecule inhibitors of aggregation with therapeutic potential in Alzheimer's disease. PMID:19309732

  3. Differential recruitment efficacy of patient-derived amyloidogenic and myeloma light chain proteins by synthetic fibrils-A metric for predicting amyloid propensity.

    Directory of Open Access Journals (Sweden)

    Emily B Martin

    Full Text Available Monoclonal free light chain (LC proteins are present in the circulation of patients with immunoproliferative disorders such as light chain (AL amyloidosis and multiple myeloma (MM. Light chain-associated amyloid is a complex pathology composed of proteinaceous fibrils and extracellular matrix proteins found in all patients with AL and in ~10-30% of patients who presented with MM. Amyloid deposits systemically in multiple organs and tissues leading to dysfunction and ultimately death. The overall survival of patients with amyloidosis is worse than for those with early stage MM.We have developed a sensitive binding assay quantifying the recruitment of full length, patient-derived LC proteins by synthetic amyloid fibrils, as a method for studying their amyloidogenic potential. In a survey of eight urinary LC, both AL and MM-associated proteins were recruited by synthetic amyloid fibrils; however, AL-associated LC bound significantly more efficiently (p < 0.05 than did MM LCs. The LC proteins used in this study were isolated from urine and presumed to represent a surrogate of serum free light chains.The binding of LC to synthetic fibrils in this assay accurately differentiated LC with amyloidogenic propensity from MM LC that were not associated with clinical amyloid disease. Notably, the LC from a MM patient who subsequently developed amyloid behaved as an AL-associated protein in the assay, indicating the possibility for identifying MM patients at risk for developing amyloidosis based on the light chain recruitment efficacy. With this information, at risk patients can be monitored more closely for the development of amyloidosis, allowing timely administration of novel, amyloid-directed immunotherapies-this approach may improve the prognosis for these patients.

  4. S-sulfonation of transthyretin is an important trigger step in the formation of transthyretin-related amyloid fibril.

    Science.gov (United States)

    Nakanishi, Toyofumi; Yoshioka, Masanori; Moriuchi, Kazuyoshi; Yamamoto, Daisuke; Tsuji, Motomu; Takubo, Takayuki

    2010-07-01

    Senile systemic amyloidosis and familial amyloid polyneuropathy are caused by oxidative deposition of conformationally altered transthyretin (TTR). We identified oxidative modification of the 10th cysteine of TTR through S-sulfonation in vitro. Based on mass spectrometric analysis, we determined the spectrophotometric, western blotting, and fluorescent microscopic properties of TTR incubated with and without cysteine-S-sulfonate in acidic (pH 4) and alkaline (pH 8) conditions at 37 degrees. The absorption of the aggregated TTR molecules increased more with incubation time and the concentration of cysteine-S-sulfonate at pH 4 than at pH 8. The Congo red binding to the S-sulfonated TTR at pH 4 was saturated with an apparent Bmax of 2.01 mol per mole of the S-sulfonated TTR and apparent KD of 7.75x10(-6) M. On the other hand, the Bmax of cysteinyl TTR was 1.38, and its KD was 3.52x10(-6) M while the Bmax of reduced TTR was 0.86, and its KD was 2.86x10(-6) M. Moreover, we detected positive amyloid fibril staining using Thioflavin T and Congo red with the S-sulfonated TTR but not with untreated or reduced TTR by microscopic fluorescent analysis. After modification of TTR in vitro, oligomers resisted reduction and denaturation was irreversibly induced, and which contributed differences in the Western blotting patterns obtained with four anti-TTR antibodies. In conclusion, this study showed that the formation of S-sulfonation of TTR through oxidative modifications of the thiol residue on the 10th cysteine of TTR is an important trigger step in the formation of transthyretin-related amyloid fibril. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  5. The conformation of the Congo-red ligand bound to amyloid fibrils HET-s(218-289): a solid-state NMR study.

    Science.gov (United States)

    Gowda, Chandrakala; Zandomeneghi, Giorgia; Zimmermann, Herbert; Schütz, Anne K; Böckmann, Anja; Ernst, Matthias; Meier, Beat H

    2017-12-01

    We have previously shown that Congo red (CR) binds site specifically to amyloid fibrils formed by HET-s(218-289) with the long axis of the CR molecule almost parallel to the fibril axis. HADDOCK docking studies indicated that CR adopts a roughly planar conformation with the torsion angle ϕ characterizing the relative orientation of the two phenyl rings being a few degrees. In this study, we experimentally determine the torsion angle ϕ at the center of the CR molecule when bound to HET-s(218-289) amyloid fibrils using solid-state NMR tensor-correlation experiments. The method described here relies on the site-specific 13 C labeling of CR and on the analysis of the two-dimensional magic-angle spinning tensor-correlation spectrum of 13 C 2 -CR. We determined the torsion angle ϕ to be 19°.

  6. Stabilization of a β-hairpin in monomeric Alzheimer's amyloid-β peptide inhibits amyloid formation

    OpenAIRE

    Hoyer, Wolfgang; Grönwall, Caroline; Jonsson, Andreas; Ståhl, Stefan; Härd, Torleif

    2008-01-01

    According to the amyloid hypothesis, the pathogenesis of Alzheimer's disease is triggered by the oligomerization and aggregation of the amyloid-β (Aβ) peptide into protein plaques. Formation of the potentially toxic oligomeric and fibrillar Aβ assemblies is accompanied by a conformational change toward a high content of β-structure. Here, we report the solution structure of Aβ(1–40) in complex with the phage-display selected affibody protein ZAβ3, a binding protein of nanomolar affinity. Boun...

  7. Congo red, an amyloid-inhibiting compound, alleviates various types of cellular dysfunction triggered by mutant protein kinase cγ that causes spinocerebellar ataxia type 14 (SCA14) by inhibiting oligomerization and aggregation.

    Science.gov (United States)

    Seki, Takahiro; Takahashi, Hideyuki; Yamamoto, Kazuhiro; Ogawa, Kota; Onji, Tomoya; Adachi, Naoko; Tanaka, Shigeru; Hide, Izumi; Saito, Naoaki; Sakai, Norio

    2010-01-01

    Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is susceptible to aggregation that induces apoptotic cell death. Congo red is widely used as a histological dye for amyloid detection. Recent evidence has revealed that Congo red has the property to inhibit amyloid oligomers and fibril formation of misfolded proteins. In the present study, we examine whether Congo red inhibits aggregate formation and cytotoxicity of mutant γPKC. Congo red likely inhibits aggregate formation of mutant γPKC – green fluorescent protein (GFP) without affecting its expression level in SH-SY5Y cells. Congo red counteracts the insolubilization of recombinant mutant γPKC, suggesting that the dye inhibits aggregation of mutant γPKC by a direct mechanism. Congo red also inhibits aggregation and oligomerization of mutant γPKC-GFP in primary cultured cerebellar Purkinje cells. Moreover, the dye reverses the improper development of dendrites and inhibits apoptotic cell death in Purkinje cells that express mutant γPKC-GFP. These results indicate that amyloid-inhibiting compounds like Congo red may be novel therapeutics for SCA14.

  8. Synergistic Inhibition of Protein Fibrillation by Proline and Sorbitol: Biophysical Investigations.

    Directory of Open Access Journals (Sweden)

    Sinjan Choudhary

    Full Text Available We report here interesting synergistic effects of proline and sorbitol, two well-known chemical chaperones, in the inhibition of fibrillation of two proteins, insulin and lysozyme. A combination of many biophysical techniques has been used to understand the structural morphology and modes of interaction of the chaperones with the proteins during fibrillation. Both the chaperones establish stronger polar interactions in the elongation and saturation stages of fibrillation compared to that in the native stage. However, when presented as a mixture, we also see contribution of hydrophobic interactions. Thus, a co-operative adjustment of polar and hydrophobic interactions between the chaperones and the protein surface seems to drive the synergistic effects in the fibrillation process. In insulin, this synergy is quantitatively similar in all the stages of the fibrillation process. These observations would have significant implications for understanding protein folding concepts, in general, and for designing combination therapies against protein fibrillation, in particular.

  9. Kinetic modeling and determination of reaction constants of Alzheimer's beta-amyloid fibril extension and dissociation using surface plasmon resonance.

    Science.gov (United States)

    Hasegawa, Kazuhiro; Ono, Kenjiro; Yamada, Masahito; Naiki, Hironobu

    2002-11-19

    To establish the kinetic model of the extension and dissociation of beta-amyloid fibrils (f(A)beta) in vitro, we analyzed these reactions using a surface plasmon resonance (SPR) biosensor. Sonicated f(A)beta were immobilized on the surface of the SPR sensor chip as seeds. The SPR signal increased linearly as a function of time after amyloid beta-peptides (Abeta) were injected into the f(A)beta-immobilized chips. The extension of f(A)beta was confirmed by atomic force microscopy. When flow cells were washed with running buffer, the SPR signal decreased with time after the extension reaction. The curve fitting resolved the dissociation reaction into the fast exponential and slow linear decay phases. Kinetic analysis of the effect of Abeta/f(A)beta concentrations on the reaction rate indicated that both the extension reaction and the slow linear phase of the dissociation were consistent with a first-order kinetic model; i.e., the extension/dissociation reactions proceed via consecutive association/dissociation of Abeta onto/from the end of existing fibrils. On the basis of this model, the critical monomer concentration ([M](e)) and the equilibrium association constant (K) were calculated, for the first time, to be 20 nM and 5 x 10(7) M(-1), respectively. Alternatively, [M](e) was directly measured as 200 nM, which may represent the equilibrium between the extension reaction and the fast phase of the dissociation. The SPR biosensor is a useful quantitative tool for the kinetic and thermodynamic study of the molecular mechanisms of f9A)beta formation in vitro.

  10. The Investigation on Resorcinarenes towards either Inhibiting or Promoting Insulin Fibrillation.

    Science.gov (United States)

    Han, Xu; Tian, Chuan; Gandra, Ingrid; Eslava, Valeria; Galindres, Diana; Vargas, Edgar; Leblanc, Roger

    2017-12-19

    Different tail-engineered resorcinarenes have been examined for insulin fibrillation by experimental and computational studies. The resorcinarene showed a promising effect on the inhibition of insulin fibrillation, studied using a ThT assay, circular dichroism spectroscopy, and atomic force microscopy. Both the ThT assay and computational results indicate the tail from the resorcinarene has an impact on insulin fibrillation by either inhibition or promotion because of the resident position on insulin. These observations have significant biological implications in the design of drug molecules as well as the development of potential therapeutic strategies. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Fibril polymorphism affects immobilized non-amyloid flanking domains of huntingtin exon1 rather than its polyglutamine core

    Science.gov (United States)

    Lin, Hsiang-Kai; Boatz, Jennifer C.; Krabbendam, Inge E.; Kodali, Ravindra; Hou, Zhipeng; Wetzel, Ronald; Dolga, Amalia M.; Poirier, Michelle A.; van der Wel, Patrick C. A.

    2017-05-01

    Polyglutamine expansion in the huntingtin protein is the primary genetic cause of Huntington's disease (HD). Fragments coinciding with mutant huntingtin exon1 aggregate in vivo and induce HD-like pathology in mouse models. The resulting aggregates can have different structures that affect their biochemical behaviour and cytotoxic activity. Here we report our studies of the structure and functional characteristics of multiple mutant htt exon1 fibrils by complementary techniques, including infrared and solid-state NMR spectroscopies. Magic-angle-spinning NMR reveals that fibrillar exon1 has a partly mobile α-helix in its aggregation-accelerating N terminus, and semi-rigid polyproline II helices in the proline-rich flanking domain (PRD). The polyglutamine-proximal portions of these domains are immobilized and clustered, limiting access to aggregation-modulating antibodies. The polymorphic fibrils differ in their flanking domains rather than the polyglutamine amyloid structure. They are effective at seeding polyglutamine aggregation and exhibit cytotoxic effects when applied to neuronal cells.

  12. Inhibition of amyloid fiber assembly by both BiP and its target peptide.

    Energy Technology Data Exchange (ETDEWEB)

    Davis, D. P.; Raffen, R.; Vogen, S.; Williamson, E.; Stevens, F. J.; Argon, Y.; Biosciences Division; Univ. of Chicago

    2000-10-01

    Immunoglobulin light chain (LC) normally is a soluble, secreted protein, but some LC assemble into ordered fibrils whose deposition in tissues results in amyloidosis and organ failure. Here we reconstitute fibril formation in vitro and show that preformed fibrils can nucleate polymerization of soluble LC. This prion-like behavior has important physiological implications, since somatic mutations generate multiple related LC sequences. Furthermore, we demonstrate that fibril formation in vitro and aggregation of whole LC within cells are inhibited by BiP and by a synthetic peptide that is identical to a major LC binding site for BiP. We propose that LC form fibrils via an interprotein loop swap and that the underlying conformational change should be amenable to drug therapy.

  13. Structural Characterization of Fibrils from Recombinant Human Islet Amyloid Polypeptide by Solid-State NMR: The Central FGAILS Segment Is Part of the β-Sheet Core.

    Directory of Open Access Journals (Sweden)

    Franziska Weirich

    Full Text Available Amyloid deposits formed from islet amyloid polypeptide (IAPP are a hallmark of type 2 diabetes mellitus and are known to be cytotoxic to pancreatic β-cells. The molecular structure of the fibrillar form of IAPP is subject of intense research, and to date, different models exist. We present results of solid-state NMR experiments on fibrils of recombinantly expressed and uniformly 13C, 15N-labeled human IAPP in the non-amidated, free acid form. Complete sequential resonance assignments and resulting constraints on secondary structure are shown. A single set of chemical shifts is found for most residues, which is indicative of a high degree of homogeneity. The core region comprises three to four β-sheets. We find that the central 23-FGAILS-28 segment, which is of critical importance for amyloid formation, is part of the core region and forms a β-strand in our sample preparation. The eight N-terminal amino acid residues of IAPP, forming a ring-like structure due to a disulfide bridge between residues C2 and C7, appear to be well defined but with an increased degree of flexibility. This study supports the elucidation of the structural basis of IAPP amyloid formation and highlights the extent of amyloid fibril polymorphism.

  14. Structural Characterization of Fibrils from Recombinant Human Islet Amyloid Polypeptide by Solid-State NMR: The Central FGAILS Segment Is Part of the β-Sheet Core

    Science.gov (United States)

    Weirich, Franziska; Gremer, Lothar; Mirecka, Ewa A.; Schiefer, Stephanie; Hoyer, Wolfgang; Heise, Henrike

    2016-01-01

    Amyloid deposits formed from islet amyloid polypeptide (IAPP) are a hallmark of type 2 diabetes mellitus and are known to be cytotoxic to pancreatic β-cells. The molecular structure of the fibrillar form of IAPP is subject of intense research, and to date, different models exist. We present results of solid-state NMR experiments on fibrils of recombinantly expressed and uniformly 13C, 15N-labeled human IAPP in the non-amidated, free acid form. Complete sequential resonance assignments and resulting constraints on secondary structure are shown. A single set of chemical shifts is found for most residues, which is indicative of a high degree of homogeneity. The core region comprises three to four β-sheets. We find that the central 23-FGAILS-28 segment, which is of critical importance for amyloid formation, is part of the core region and forms a β-strand in our sample preparation. The eight N-terminal amino acid residues of IAPP, forming a ring-like structure due to a disulfide bridge between residues C2 and C7, appear to be well defined but with an increased degree of flexibility. This study supports the elucidation of the structural basis of IAPP amyloid formation and highlights the extent of amyloid fibril polymorphism. PMID:27607147

  15. Adsorption and Fibrillization of Islet Amyloid Polypeptide at Self-Assembled Monolayers Studied by QCM-D, AFM, and PM-IRRAS.

    Science.gov (United States)

    Hajiraissi, Roozbeh; Hanke, Marcel; Yang, Yu; Duderija, Belma; Gonzalez Orive, Alejandro; Grundmeier, Guido; Keller, Adrian

    2018-03-20

    Aggregation and fibrillization of human islet amyloid polypeptide (hIAPP) plays an important role in the development of type 2 diabetes mellitus. Understanding the interaction of hIAPP with interfaces such as cell membranes at a molecular level therefore represents an important step toward new therapies. Here, we investigate the fibrillization of hIAPP at different self-assembled alkanethiol monolayers (SAMs) by quartz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM), and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). We find that hydrophobic interactions with the CH 3 -terminated SAM tend to retard hIAPP fibrillization compared to the carboxylic acid-terminated SAM where attractive electrostatic interactions lead to the formation of a three-dimensional network of interwoven fibrils. At the hydroxyl- and amino-terminated SAMs, fibrillization appears to be governed by hydrogen bonding between the peptide and the terminating groups which may even overcome electrostatic repulsion. These results thus provide fundamental insights into the molecular mechanisms governing amyloid assembly at interfaces.

  16. Spatially resolved spectroscopic differentiation of hydrophilic and hydrophobic domains on individual insulin amyloid fibrils

    DEFF Research Database (Denmark)

    Deckert-Gaudig, Tanja; Kurouski, Dmitry; Hedegaard, Martin A B

    2016-01-01

    on the identification of the secondary structure via the amide III band and the differentiation of hydrophobic and hydrophilic domains on the surface. In addition multivariate data analysis, specifically the N-FINDR procedure, was employed to generate structure-specific maps. The ability of TERS to localize specific...... structural domains on fibril surfaces shows promise to the development of new fibril dissection strategies and can be generally applied to any (bio)chemical surface when structural variations at the nanometer level are of interest....

  17. Amyloidinhibits PDGFβ receptor activation and prevents PDGF-BB-induced neuroprotection.

    Science.gov (United States)

    Liu, Hui; Saffi, Golam T; Vasefi, Maryam S; Choi, Youngjik; Kruk, Jeff S; Ahmed, Nawaz; Gondora, Nyasha; Mielke, John; Leonenko, Zoya; Beazely, Michael A

    2018-01-09

    PDGFβ receptors and their ligand, PDGF-BB, are upregulated in vivo after neuronal insults such as ischemia. When applied exogenously, PDGF-BB is neuroprotective against excitotoxicity and HIV proteins. Given this growth factor's neuroprotective ability, we sought to determine if PDGF-BB would be neuroprotective against amyloid-β (1-42), one of the pathological agents associated with Alzheimer's disease (AD). In both primary hippocampal neurons and the human-derived neuroblastoma cell line, SH-SY5Y, amyloid- treatment for 24 h decreased surviving cell number in a concentration-dependent manner. Pretreatment with PDGF-BB failed to provide any neuroprotection against amyloid-β in primary neurons and only very limited protective effects in SH-SY5Y cells. In addition to its neuroprotective action, PDGF promotes cell growth and division in several systems, and the application of PDGF-BB alone to serum-starved SH-SY5Y cells resulted in an increase in cell number. Amyloid-β attenuated the mitogenic effects of PDGF-BB, inhibited PDGF-BB-induced PDGFβ receptor phosphorylation, and attenuated the ability of PDGF-BB to protect neurons against NMDA-induced excitotoxicity. Despite the ability of amyloid-β to inhibit PDGF receptor activation, immunoprecipitation experiments failed to detect a physical interaction between amyloid-β and PDGF-BB or the PDGFβ receptor. However, G protein-coupled receptor transactivation of the PDGFβ receptor (an exclusively intracellular signaling pathway) remained unaffected by the presence of amyloid-β. As the PDGF system is upregulated upon neuronal damage, the ability of amyloid-β to inhibit this endogenous neuroprotective system should be further investigated in the context of AD pathophysiology. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. Synthesis and structural investigation of 2-aminomethyl-3-(4-methoxy-phenyl)-propionic acid containing a peptide analogue of the amyloidogenic AS(6-7) sequence: inhibition of fibril formation.

    Science.gov (United States)

    Paikar, Arpita; Debnath, Mintu; Podder, Debasish; Sasmal, Supriya; Haldar, Debasish

    2017-05-16

    The incorporation of a single β-amino acid moiety in a highly amyloidogenic peptide sequence resulted in the complete inhibition of amyloid fibril formation. The Boc-l-Phe-l-Leu-OMe sequence 1, which has sequence identity with the N-terminal AS(6-7) of the non-immunoglobulin amyloid fibril protein AS, which is responsible for rheumatoid arthritis, self-associates to produce fibrils. The d-Phe analogue peptide 2 shows an elongated ribbon-like morphology. However, the 2-aminomethyl-3-(4-methoxy-phenyl)-propionic acid containing analogue peptide 3 exhibits a polydisperse microsphere morphology. Moreover, fibrils from peptides 1 and 2 exhibit typical green-gold birefringence upon Congo red (CR) staining and show an amyloid-like morphological resemblance. However, the 2-aminomethyl-3-(4-methoxy-phenyl)-propionic acid modified peptide 3 does not respond to the Congo red assay. From X-ray crystallography, peptide 1 with the l-Phe residue adopts an extended structure, whereas the d-Phe analogue 2 adopts a kink-like structure. Both peptides 1 and 2 show twisted anti-parallel sheet-like structures at higher order assembly. However, peptide 3 adopts a nine-membered hydrogen bonded δ-turn-like structure in the solid state and self-associates to form a loop-like supramolecular structure through multiple intermolecular hydrogen bonds. The structural analysis presented herein may foster new studies for de novo design and therapeutics.

  19. Tritium-labeled (E,E)-2,5-Bis(4’-hydroxy-3’-carboxystyryl)benzene as a Probe for β-Amyloid Fibrils

    Science.gov (United States)

    Matveev, Sergey V.; Kwiatkowski, Stefan; Sviripa, Vitaliy M.; Fazio, Robert C.; Watt, David S.; LeVine, Harry

    2014-01-01

    Accumulation of Aβ in the brains of Alzheimer disease (AD) patients reflects an imbalance between Aβ production and clearance from their brains. Alternative cleavage of amyloid precursor protein (APP) by processing proteases generates soluble APP fragments including the neurotoxic amyloid Aβ40 and Aβ42 peptides that assemble into fibrils and form plaques. Plaque-buildup occurs over an extended time-frame, and the early detection and modulation of plaque formation are areas of active research. Radiolabeled probes for the detection of amyloid plaques and fibrils in living subjects are important for noninvasive evaluation of AD diagnosis, progression, and differentiation of AD from other neurodegenerative diseases and age-related cognitive decline. Tritium-labeled (E,E)-1-[3H]-2,5-bis(4’-hydroxy-3’-carbomethoxystyryl)benzene possesses an improved level of chemical stability relative to a previously reported radioiodinated analog for radiometric quantification of Aβ plaque and tau pathology in brain tissue and in vitro studies with synthetic Aβ and tau fibrils. PMID:25452000

  20. Tritium-labeled (E,E)-2,5-bis(4'-hydroxy-3'-carboxystyryl)benzene as a probe for β-amyloid fibrils.

    Science.gov (United States)

    Matveev, Sergey V; Kwiatkowski, Stefan; Sviripa, Vitaliy M; Fazio, Robert C; Watt, David S; LeVine, Harry

    2014-12-01

    Accumulation of Aβ in the brains of Alzheimer disease (AD) patients reflects an imbalance between Aβ production and clearance from their brains. Alternative cleavage of amyloid precursor protein (APP) by processing proteases generates soluble APP fragments including the neurotoxic amyloid Aβ40 and Aβ42 peptides that assemble into fibrils and form plaques. Plaque-buildup occurs over an extended time-frame, and the early detection and modulation of plaque formation are areas of active research. Radiolabeled probes for the detection of amyloid plaques and fibrils in living subjects are important for noninvasive evaluation of AD diagnosis, progression, and differentiation of AD from other neurodegenerative diseases and age-related cognitive decline. Tritium-labeled (E,E)-1-[(3)H]-2,5-bis(4'-hydroxy-3'-carbomethoxystyryl)benzene possesses an improved level of chemical stability relative to a previously reported radioiodinated analog for radiometric quantification of Aβ plaque and tau pathology in brain tissue and in vitro studies with synthetic Aβ and tau fibrils. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Amyloid-β secretion, generation, and lysosomal sequestration in response to proteasome inhibition

    DEFF Research Database (Denmark)

    Agholme, Lotta; Hallbeck, Martin; Benedikz, Eirikur

    2012-01-01

    , as the autophagosome has been suggested as a site of amyloid-β (Aβ) generation. In this study, we investigated the effect of proteasome inhibition on Aβ accumulation and secretion, as well as the processing of amyloid-β protein precursor (AβPP) in AβPP(Swe) transfected SH-SY5Y neuroblastoma cells. We show...... makes C99 available for γ-secretase cleavage, leading to Aβ generation. Inhibition of autophagy after proteasome inhibition led to reduced levels of intracellular, but not secreted Aβ, and tended to further increase the C99 to AβPP ratio, supporting involvement of the autophagosome in Aβ generation....... Furthermore, proteasome inhibition caused a reduction in cellular viability, which was reverted by inhibition of autophagy. Dysfunction of the proteasome could cause lysosomal accumulation of Aβ, as well as increased generation and secretion of Aβ, which is partly facilitated by autophagy. As a decrease...

  2. Calpain inhibition prevents amyloid-beta-induced neurodegeneration and associated behavioral dysfunction in rats

    NARCIS (Netherlands)

    Granic, Ivica; Nyakas, Csaba; Luiten, Paul G. M.; Eisel, Ulrich L. M.; Halmy, Laszlo G.; Gross, Gerhard; Schoemaker, Hans; Moeller, Achim; Nimmrich, Volker

    2010-01-01

    Amyloid-beta (A beta) is toxic to neurons and such toxicity is - at least in part - mediated via the NMDA receptor. Calpain, a calcium dependent cystein protease, is part of the NMDA receptor-induced neurodegeneration pathway, and we previously reported that inhibition of calpain prevents

  3. Ameliorating AmyloidFibrils Triggered Inflammation via Curcumin-Loaded Polymeric Nanoconstructs

    Directory of Open Access Journals (Sweden)

    Andrea Ameruoso

    2017-10-01

    Full Text Available Inflammation is a common hallmark in several diseases, including atherosclerosis, cancer, obesity, and neurodegeneration. In Alzheimer’s disease (AD, growing evidence directly correlates neuronal damage with inflammation of myeloid brain cells, such as microglia. Here, polymeric nanoparticles were engineered and characterized for the delivery of anti-inflammatory molecules to macrophages stimulated via direct incubation with amyloid-β fibers. 200 nm spherical polymeric nanoconstructs (SPNs and 1,000 nm discoidal polymeric nanoconstructs (DPNs were synthesized using poly(lactic-co-glycolic acid (PLGA, polyethylene glycol (PEG, and lipid chains as building blocks. First, the internalization propensity in macrophages of both nanoparticles was assessed via cytofluorimetric and confocal microscopy analyses, demonstrating that SPNs are by far more rapidly taken up as compared to DPNs (99.6 ± 0.11 vs 14.4 ± 0.06%, within 24 h. Then, Curcumin-loaded SPNs (Curc-SPNs were realized by encapsulating Curcumin, a natural anti-inflammatory molecule, within the PLGA core of SPNs. Finally, Curc-SPNs were shown to diminish up to 6.5-fold the production of pro-inflammatory cytokines—IL-1β; IL-6, and TNF-α—in macrophages stimulated via amyloid-β fibers. Although more sophisticated in vitro models and systematic analyses on the blood–brain barrier permeability are critically needed, these findings hold potential in the development of nanoparticles for modulating inflammation in AD.

  4. Amyloid-beta binds catalase with high affinity and inhibits hydrogen peroxide breakdown.

    OpenAIRE

    Milton, N G

    1999-01-01

    Amyloid-beta (Abeta) specifically bound purified catalase with high affinity and inhibited catalase breakdown of H(2)O(2). The Abeta-induced catalase inhibition involved formation of the inactive catalase Compound II and was reversible. CatalaseAbeta interactions provide rapid functional assays for the cytotoxic domain of Abeta and suggest a mechanism for some of the observed actions of Abeta plus catalase in vitro.

  5. Graphene-supporting films and low-voltage STEM in SEM toward imaging nanobio materials without staining: Observation of insulin amyloid fibrils.

    Science.gov (United States)

    Ogawa, Takashi; Gang, Geun Won; Thieu, Minh Thu; Kwon, Hyuksang; Ahn, Sang Jung; Ha, Tai Hwan; Cho, Boklae

    2017-05-01

    Utilization of graphene-supporting films and low-voltage scanning transmission electron microscopy (LV-STEM) in scanning electron microscopy (SEM) is shown to be an effective means of observing unstained nanobio materials. Insulin amyloid fibrils, which are implicated as a cause of type II diabetes, are formed in vitro and observed without staining at room temperature. An in-lens cold field-emission SEM, equipped with an additional homemade STEM detector, provides dark field (DF)-STEM images in the low energy range of 5-30keV, together with secondary electron (SE) images. Analysis based on Lenz's theory is used to interpret the experimental results. Graphene films, where the fibrils are deposited, reduce the background level of the STEM images compared with instances when conventional amorphous carbon films are used. Using 30keV, which is lower than that for conventional TEM (100-300keV), together with low detection angles (15-55mrad) enhances the signals from the fibrils. These factors improve image quality, which enables observation of thin fibrils with widths of 7-8nm. STEM imaging clearly reveals a twisted-ribbon structure of a fibril, and SE imaging shows an emphasized striped pattern of the fibril. The LV-STEM in SEM enables acquisition of two types of images of an identical fibril in a single instrument, which is useful for understanding the structure. This study expands the application of SEM to other systems of interest, which is beneficial to a large number of users. The method in this study can be applied to the observation of various nanobio materials and analysis of their native structures, thus contributing to research in materials and life sciences. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Molecular Insight into Human Lysozyme and Its Ability to Form Amyloid Fibrils in High Concentrations of Sodium Dodecyl Sulfate: A View from Molecular Dynamics Simulations.

    Directory of Open Access Journals (Sweden)

    Majid Jafari

    Full Text Available Changes in the tertiary structure of proteins and the resultant fibrillary aggregation could result in fatal heredity diseases, such as lysozyme systemic amyloidosis. Human lysozyme is a globular protein with antimicrobial properties with tendencies to fibrillate and hence is known as a fibril-forming protein. Therefore, its behavior under different ambient conditions is of great importance. In this study, we conducted two 500000 ps molecular dynamics (MD simulations of human lysozyme in sodium dodecyl sulfate (SDS at two ambient temperatures. To achieve comparative results, we also performed two 500000 ps human lysozyme MD simulations in pure water as controls. The aim of this study was to provide further molecular insight into all interactions in the lysozyme-SDS complexes and to provide a perspective on the ability of human lysozyme to form amyloid fibrils in the presence of SDS surfactant molecules. SDS, which is an anionic detergent, contains a hydrophobic tail with 12 carbon atoms and a negatively charged head group. The SDS surfactant is known to be a stabilizer for helical structures above the critical micelle concentration (CMC [1]. During the 500000 ps MD simulations, the helical structures were maintained by the SDS surfactant above its CMC at 300 K, while at 370 K, human lysozyme lost most of its helices and gained β-sheets. Therefore, we suggest that future studies investigate the β-amyloid formation of human lysozyme at SDS concentrations above the CMC and at high temperatures.

  7. Molecular Insight into Human Lysozyme and Its Ability to Form Amyloid Fibrils in High Concentrations of Sodium Dodecyl Sulfate: A View from Molecular Dynamics Simulations.

    Science.gov (United States)

    Jafari, Majid; Mehrnejad, Faramarz

    2016-01-01

    Changes in the tertiary structure of proteins and the resultant fibrillary aggregation could result in fatal heredity diseases, such as lysozyme systemic amyloidosis. Human lysozyme is a globular protein with antimicrobial properties with tendencies to fibrillate and hence is known as a fibril-forming protein. Therefore, its behavior under different ambient conditions is of great importance. In this study, we conducted two 500000 ps molecular dynamics (MD) simulations of human lysozyme in sodium dodecyl sulfate (SDS) at two ambient temperatures. To achieve comparative results, we also performed two 500000 ps human lysozyme MD simulations in pure water as controls. The aim of this study was to provide further molecular insight into all interactions in the lysozyme-SDS complexes and to provide a perspective on the ability of human lysozyme to form amyloid fibrils in the presence of SDS surfactant molecules. SDS, which is an anionic detergent, contains a hydrophobic tail with 12 carbon atoms and a negatively charged head group. The SDS surfactant is known to be a stabilizer for helical structures above the critical micelle concentration (CMC) [1]. During the 500000 ps MD simulations, the helical structures were maintained by the SDS surfactant above its CMC at 300 K, while at 370 K, human lysozyme lost most of its helices and gained β-sheets. Therefore, we suggest that future studies investigate the β-amyloid formation of human lysozyme at SDS concentrations above the CMC and at high temperatures.

  8. A bifunctional curcumin analogue for two-photon imaging and inhibiting crosslinking of amyloid beta in Alzheimer's disease.

    Science.gov (United States)

    Zhang, Xueli; Tian, Yanli; Yuan, Peng; Li, Yuyan; Yaseen, Mohammad A; Grutzendler, Jaime; Moore, Anna; Ran, Chongzhao

    2014-10-09

    In this report, we designed a highly bright bifunctional curcumin analogue CRANAD-28. In vivo two-photon imaging suggested that CRANAD-28 could penetrate the blood brain barrier (BBB) and label plaques and cerebral amyloid angiopathies (CAAs). We also demonstrated that this imaging probe could inhibit the crosslinking of amyloid beta induced either by copper or by natural conditions.

  9. Binding and Inhibitory Effect of the Dyes Amaranth and Tartrazine on Amyloid Fibrillation in Lysozyme.

    Science.gov (United States)

    Basu, Anirban; Suresh Kumar, Gopinatha

    2017-02-16

    Interaction of two food colorant dyes, amaranth and tartrazine, with lysozyme was studied employing multiple biophysical techniques. The dyes exhibited hypochromic changes in the presence of lysozyme. The intrinsic fluorescence of lysozyme was quenched by both dyes; amaranth was a more efficient quencher than tartrazine. The equilibrium constant of amaranth was higher than that of tartarzine. From FRET analysis, the binding distances for amaranth and tartrazine were calculated to be 4.51 and 3.93 nm, respectively. The binding was found to be dominated by non-polyelectrolytic forces. Both dyes induced alterations in the microenvironment surrounding the tryptophan and tyrosine residues of the protein, with the alterations being comparatively higher for the tryptophans than the tyrosines. The interaction caused significant loss in the helicity of lysozyme, the change being higher with amaranth. The binding of both dyes was exothermic. The binding of amaranth was enthalpy driven, while that of tartrazine was predominantly entropy driven. Amaranth delayed lysozyme fibrillation at 25 μM, while tartrazine had no effect even at 100 μM. Nevertheless, both dyes had a significant inhibitory effect on fibrillogenesis. The present study explores the potential antiamyloidogenic property of these azo dyes used as food colorants.

  10. Oxidized epigallocatechin gallate inhibited lysozyme fibrillation more strongly than the native form

    Directory of Open Access Journals (Sweden)

    Ting-Ting An

    2017-04-01

    Full Text Available Epigallocatechin gallate (EGCG, the most abundant flavanoid in green tea, is currently being evaluated in the clinic due to its benefits in the treatment of amyloid disorders. Its anti-amyloidogenic effect has been attributed to direct interaction of the intact molecule with misfolded polypeptides. In addition, antioxidant activity is also involved in the anti-amyloidogenic role. The detailed molecular mechanism is still unclear and requires further investigation. In the present study, the kinetics of EGCG oxidation and the anti-amyloidogenic effect of the resultant oxidation substances have been examined. The results indicate that EGCG degrades in a medium at pH 8.0 with a half-life less than 2 h. By utilizing lysozyme as an in vitro model, the oxidized EGCG demonstrates a more potent anti-amyloidogenic capacity than the intact molecule, as shown by ThT and ANS fluorescence, TEM determination, and hemolytic assay. The oxidized EGCG also has a stronger disruptive effect on preformed fibrils than the native form. Ascorbic acid eliminates the disruptive role of native EGCG on the fibrils, suggesting that oxidation is a prerequisite in fibril disruption. The results of this work demonstrate that oxidized EGCG plays a more important role than the intact molecule in anti-amyloidogenic activity. These insights into the action of EGCG may provide a novel route to understand the anti-amyloidogenic activity of natural polyphenols.

  11. Deconstructing glucagon fibrillation

    DEFF Research Database (Denmark)

    Ghodke, Shirin

    Aggregation of misfolded proteins into ordered amyloid fibrils has deep implications in disease pathology as well as in pharmaceutical production of therapeutic proteins. This thesis elucidates the fibrillation pathway of the therapeutic peptide hormone, glucagon and explores the biophysical basis...... of its inherent fibril polymorphism by varying physicochemical factors like temperature, pressure and co-solvents. This work demonstrates the important role of hydration in the fibrillation process as well as in fibril polymorphism....

  12. Donepezil loaded PLGA-b-PEG nanoparticles: their ability to induce destabilization of amyloid fibrils and to cross blood brain barrier in vitro.

    Science.gov (United States)

    Baysal, Ipek; Ucar, Gulberk; Gultekinoglu, Merve; Ulubayram, Kezban; Yabanoglu-Ciftci, Samiye

    2017-01-01

    Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disease. Cholinesterase inhibitors (ChEIs) are commonly used for symptomatic treatment of neural transmission improvement in AD. Donepezil is a reversible and non-competitive ChEI which is clinically used for palliative treatment of AD. The aim of the present study was to investigate the destabilizing effect of donepezil loaded poly(lactic-co-glycolic acid)-block-poly (ethylene glycol) [PLGA-b-PEG] nanoparticles on fibril formation in vitro and the ability of these nanoparticles to cross blood brain barrier (BBB) using in vitro BBB model and the neuroprotective effects of free donepezil and donepezil loaded PLGA-b-PEG nanoparticles. Donepezil loaded PLGA-b-PEG nanoparticles were prepared with double emulsion method. Destabilizing effect of these donepezil loaded particles on the amyloid-beta fibril (Aβ 1-40 and Aβ 1-42 ) formation was determined in vitro. Nanoparticles were found to have small particle size and have destabilizing effect on fibril formation. In vitro BBB model was successfully prepared. Nanoparticles showed the ability to cross the BBB and showed a controlled release profile in this system. IL-1β, IL-6, GM-CSF, TGF-β, MCP-1 and TNF-α levels were found to be increased in both gene and protein expression levels in astrocytes incubated with amyloid fibrils in in vitro BBB model suggesting an increased inflammation. Free donepezil and donepezil loaded nanoparticle administration caused a significant dose-dependent decrease in both gene and protein expression levels of IL-1β, IL-6, GM-CSF and TNF-α. No significant changes were observed for TGF-β and MCP-1.

  13. Selective inhibition of aggregation/fibrillation of bovine serum albumin by osmolytes: Mechanistic and energetics insights.

    Directory of Open Access Journals (Sweden)

    Moumita Dasgupta

    Full Text Available Bovine serum albumin (BSA is an important transport protein of the blood and its aggregation/fibrillation would adversely affect its transport ability leading to metabolic disorder. Therefore, understanding the mechanism of fibrillation/aggregation of BSA and design of suitable inhibitor molecules for stabilizing its native conformation, are of utmost importance. The qualitative and quantitative aspects of the effect of osmolytes (proline, hydroxyproline, glycine betaine, sarcosine and sorbitol on heat induced aggregation/fibrillation of BSA at physiological pH (pH 7.4 have been studied employing a combination of fluorescence spectroscopy, Rayleigh scattering, isothermal titration calorimetry (ITC, dynamic light scattering (DLS and transmission electron microscopy (TEM. Formation of fibrils by BSA under the given conditions was confirmed from increase in fluorescence emission intensities of Thioflavin T over a time period of 600 minutes and TEM images. Absence of change in fluorescence emission intensities of 8-Anilinonaphthalene-1-sulfonic acid (ANS in presence of native and aggregated BSA signify the absence of any amorphous aggregates. ITC results have provided important insights on the energetics of interaction of these osmolytes with different stages of the fibrillar aggregates of BSA, thereby suggesting the possible modes/mechanism of inhibition of BSA fibrillation by these osmolytes. The heats of interaction of the osmolytes with different stages of fibrillation of BSA do not follow a trend, suggesting that the interactions of stages of BSA aggregates are osmolyte specific. Among the osmolytes used here, we found glycine betaine to be supporting and promoting the aggregation process while hydroxyproline to be maximally efficient in suppressing the fibrillation process of BSA, followed by sorbitol, sarcosine and proline in the following order of their decreasing potency: Hydroxyproline> Sorbitol> Sarcosine> Proline> Glycine betaine.

  14. Ginkgolide B inhibits the neurotoxicity of prions or amyloid-β1-42

    Directory of Open Access Journals (Sweden)

    Williams Alun

    2004-05-01

    response to platelet activating factor, amyloid-β1-42 or sPrP106. These results are compatible with prior reports that ginkgolides inhibit platelet-activating factor, and that platelet-activating factor antagonists block the toxicity of amyloid-β1-42 or sPrP106. The results presented here suggest that platelet-activating factor antagonists such as the ginkgolides may be relevant treatments for prion or Alzheimer's diseases.

  15. Inhibition of amyloid oligomerization into different supramolecular architectures by small molecules: mechanistic insights and design rules.

    Science.gov (United States)

    Brahmachari, Sayanti; Paul, Ashim; Segal, Daniel; Gazit, Ehud

    2017-05-01

    Protein misfolding and aggregation have been associated with several human disorders, including Alzheimer's, Parkinson's and Huntington's diseases, as well as senile systemic amyloidosis and Type II diabetes. However, there is no current disease-modifying therapy available for the treatment of these disorders. In spite of extensive academic, pharmaceutical, medicinal and clinical research, a complete mechanistic model for this family of diseases is still lacking. In this review, we primarily discuss the different types of small molecular entities which have been used for the inhibition of the aggregation process of different amyloidogenic proteins under diseased conditions. These include small peptides, polyphenols, inositols, quinones and their derivatives, and metal chelator molecules. In recent years, these groups of molecules have been extensively studied using in vitro, in vivo and computational models to understand their mechanism of action and common structural features underlying the process of inhibition. A salient feature found to be instrumental in the process of inhibition is the balance between the aromatic unit that functions as the amyloid recognition unit and the hydrophilic amyloid breaker unit. The establishment of structure-function relationship for amyloid-modifying therapies by the various functional entities should serve as an important step toward the development of efficient therapeutics.

  16. Inhibition of Src kinase activity attenuates amyloid associated microgliosis in a murine model of Alzheimer’s disease

    Directory of Open Access Journals (Sweden)

    Dhawan Gunjan

    2012-07-01

    Full Text Available Abstract Background Microglial activation is an important histologic characteristic of the pathology of Alzheimer’s disease (AD. One hypothesis is that amyloid beta (Aβ peptide serves as a specific stimulus for tyrosine kinase-based microglial activation leading to pro-inflammatory changes that contribute to disease. Therefore, inhibiting Aβ stimulation of microglia may prove to be an important therapeutic strategy for AD. Methods Primary murine microglia cultures and the murine microglia cell line, BV2, were used for stimulation with fibrillar Aβ1-42. The non-receptor tyrosine kinase inhibitor, dasatinib, was used to treat the cells to determine whether Src family kinase activity was required for the Aβ stimulated signaling response and subsequent increase in TNFα secretion using Western blot analysis and enzyme-linked immunosorbent assay (ELISA, respectively. A histologic longitudinal analysis was performed using an AD transgenic mouse model, APP/PS1, to determine an age at which microglial protein tyrosine kinase levels increased in order to administer dasatinib via mini osmotic pump diffusion. Effects of dasatinib administration on microglial and astroglial activation, protein phosphotyrosine levels, active Src kinase levels, Aβ plaque deposition, and spatial working memory were assessed via immunohistochemistry, Western blot, and T maze analysis. Results Aβ fibrils stimulated primary murine microglia via a tyrosine kinase pathway involving Src kinase that was attenuated by dasatinib. Dasatinib administration to APP/PS1 mice decreased protein phosphotyrosine, active Src, reactive microglia, and TNFα levels in the hippocampus and temporal cortex. The drug had no effect on GFAP levels, Aβ plaque load, or the related tyrosine kinase, Lyn. These anti-inflammatory changes correlated with improved performance on the T maze test in dasatinib infused animals compared to control animals. Conclusions These data suggest that amyloid

  17. Effect of human serum on the electrical detection of amyloidfibrils in biological environments using azo-dye immobilized field effect transistor (FET biosensor

    Directory of Open Access Journals (Sweden)

    Sho Hideshima

    2018-02-01

    Full Text Available As amyloid-β peptide 1–42 (Aβ42 was found to be an emerging and important biomarker for Alzheimer's disease, the detection of this peptide in biological samples such as human serum (HS has become very important for evaluating the potential disease state and determining the appropriate treatment. In this study, we developed an electrical analysis strategy based on a field effect transistor (FET biosensor as a simple and reliable technique for confirming the presence of Aβ42 aggregates (fibrils in biological samples. By utilizing Congo red immobilized on the FET gate surface as a biorecognition element, we observed remarkable sensitivity and specificity for detecting Aβ42 fibrils. Furthermore, we optimized the procedure to minimize the interference of abundant human serum albumin for the detection system using HS samples. The optimized system of Congo red-immobilized FET enables measurement of Aβ42 fibrils in the 100-pM level in HS samples, which is lower than its clinical concentration. The FET device can be applied as a biosensing system for mass and routine screening of peptide biomarkers related to Alzheimer's disease.

  18. Protein disulfide isomerase interacts with tau protein and inhibits its fibrillization.

    Directory of Open Access Journals (Sweden)

    Li-Rong Xu

    Full Text Available BACKGROUND: Tau protein is implicated in the pathogenesis of neurodegenerative disorders such as tauopathies including Alzheimer disease, and Tau fibrillization is thought to be related to neuronal toxicity. Physiological inhibitors of Tau fibrillization hold promise for developing new strategies for treatment of Alzheimer disease. Because protein disulfide isomerase (PDI is both an enzyme and a chaperone, and implicated in neuroprotection against Alzheimer disease, we want to know whether PDI can prevent Tau fibrillization. In this study, we have investigated the interaction between PDI and Tau protein and the effect of PDI on Tau fibrillization. METHODOLOGY/PRINCIPAL FINDINGS: As evidenced by co-immunoprecipitation and confocal laser scanning microscopy, human PDI interacts and co-locates with some endogenous human Tau on the endoplasmic reticulum of undifferentiated SH-SY5Y neuroblastoma cells. The results from isothermal titration calorimetry show that one full-length human PDI binds to one full-length human Tau (or human Tau fragment Tau244-372 monomer with moderate, micromolar affinity at physiological pH and near physiological ionic strength. As revealed by thioflavin T binding assays, Sarkosyl-insoluble SDS-PAGE, and transmission electron microscopy, full-length human PDI remarkably inhibits both steps of nucleation and elongation of Tau244-372 fibrillization in a concentration-dependent manner. Furthermore, we find that two molecules of the a-domain of human PDI interact with one Tau244-372 molecule with sub-micromolar affinity, and inhibit both steps of nucleation and elongation of Tau244-372 fibrillization more strongly than full-length human PDI. CONCLUSIONS/SIGNIFICANCE: We demonstrate for the first time that human PDI binds to Tau protein mainly through its thioredoxin-like catalytic domain a, forming a 1∶1 complex and preventing Tau misfolding. Our findings suggest that PDI could act as a physiological inhibitor of Tau

  19. The effect of tachykinin neuropeptides on amyloid {beta} aggregation

    Energy Technology Data Exchange (ETDEWEB)

    Flashner, Efrat [The Institute of Chemistry, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 91904 (Israel); Raviv, Uri, E-mail: raviv@chem.ch.huji.ac.il [The Institute of Chemistry, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 91904 (Israel); Friedler, Assaf, E-mail: assaf@chem.ch.huji.ac.il [The Institute of Chemistry, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 91904 (Israel)

    2011-04-01

    Research highlights: {yields} Mechanistic explanation of how tachykinin neuropeptides reduce A{beta}-induced neurotoxicity. {yields} Biophysical studies suggest that tachykinins do not modulate the distribution of A{beta} oligomeric states, but rather may incorporate into the fibrils. {yields} A possible strategy to inhibit toxicity of amyloid fibrils. -- Abstract: A hallmark of Alzheimer's disease is production of amyloid {beta} peptides resulting from aberrant cleavage of the amyloid precursor protein. Amyloid {beta} assembles into fibrils under physiological conditions, through formation of neurotoxic intermediate oligomers. Tachykinin peptides are known to affect amyloid {beta} neurotoxicity in cells. To understand the mechanism of this effect, we studied how tachykinins affect A{beta}(1-40) aggregation in vitro. Fibrils grown in the presence of tachykinins exhibited reduced thioflavin T (ThT) fluorescence, while their morphology, observed in transmission electron microscopy (TEM), did not alter. Cross linking studies revealed that the distribution of low molecular weight species was not affected by tachykinins. Our results suggest that there may be a specific interaction between tachykinins and A{beta}(1-40) that allows them to co-assemble. This effect may explain the reduction of A{beta}(1-40) neurotoxicity in cells treated with tachykinins.

  20. Serum amyloid P component inhibits influenza A virus infections: in vitro and in vivo studies

    DEFF Research Database (Denmark)

    Horvath, A; Andersen, I; Junker, K

    2001-01-01

    . These studies were extended to comprise five mouse-adapted influenza A strains, two swine influenza A strains, a mink influenza A virus, a ferret influenza A reassortant virus, a influenza B virus and a parainfluenza 3 virus. The HA activity of all these viruses was inhibited by SAP. Western blotting showed......Serum amyloid P component (SAP) binds in vitro Ca(2+)-dependently to several ligands including oligosaccharides with terminal mannose and galactose. We have earlier reported that SAP binds to human influenza A virus strains, inhibiting hemagglutinin (HA) activity and virus infectivity in vitro...... that SAP bound to HA trimers, monomers and HA1 and HA2 subunits of influenza A virus. Binding studies indicated that galactose, mannose and fucose moieties contributed to the SAP reacting site(s). Intranasal administration of human SAP to mice induced no demonstrable toxic reactions, and circulating...

  1. Inhibition of potassium currents is involved in antiarrhythmic effect of moderate ethanol on atrial fibrillation.

    Science.gov (United States)

    Yang, Baode; Li, Chenxing; Sun, Junyi; Wang, Xinghui; Liu, Xinling; Yang, Chun; Chen, Lina; Zhou, Jun; Hu, Hao

    2017-05-01

    Excessive consumption of alcohol is a well-established risk factor of atrial fibrillation (AF). However, the effects of moderate alcohol drinking remain to be elucidated. This study was designed to determine the effects of moderate ethanol ingestion on atrial fibrillation and the electrophysiological mechanisms. In acetylcholine-induced canine and mouse AF models, the moderate ethanol prevented the generation and persistence of AF through prolonging the latent period of AF and shortening the duration of AF. The action potential duration (APD) was remarkably prolonged under the concentration range of 12.5-50.0mM ethanol in guinea pig atrial myocytes. Ultra-rapid delayed rectified potassium currents (I Kv1.5 ) were markedly inhibited by 12.5-50.0mM ethanol in a concentration-dependent manner. Ethanol with 50.0mM could inhibit rapid delayed rectifier potassium currents (I hERG ). Ethanol under 6.25-50.0mM did not affect on inward rectifier potassium currents (I Kir2.1 ). Collectively, the present study provided an evidence that moderate ethanol intake can prolong the APD of atrial myocytes by inhibition of I Kv1.5 and I hERG , which contributed to preventing the development and duration of AF. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Pressure effects on .alpha.-synuclein amyloid fibrils: an experimental investigation on their dissociation and reversible nature

    Czech Academy of Sciences Publication Activity Database

    Piccirilli, F.; Plotegher, N.; Spinozzi, F.; Bubacco, L.; Mariani, P.; Beltramini, M.; Tessari, I.; Militello, V.; Perucchi, A.; Amenitsch, H.; Baldassarri Jr., E.; Steinhart, Miloš; Lupi, S.; Ortore, M. G.

    2017-01-01

    Roč. 627, 1 August (2017), s. 46-55 ISSN 0003-9861 Institutional support: RVO:61389013 Keywords : amyloid * high-pressure * SAXS Subject RIV: CD - Macromolecular Chemistry OBOR OECD: Polymer science Impact factor: 3.165, year: 2016

  3. The architecture of amyloid-like peptide fibrils revealed by X-ray scattering, diffraction and electron microscopy

    DEFF Research Database (Denmark)

    Langkilde, Annette Eva; Morris, Kyle L; Serpell, Louise C

    2015-01-01

    hierarchical layer is generated via the pairing of ribbons during fibril maturation. Based on the complementary data, a quasi-atomic resolution model of the protofilament peptide arrangement is suggested. The peptide structure appears in a β-sheet arrangement reminiscent of the β-zipper structures evident from...

  4. Effects on atrial fibrillation in aged hypertensive rats by Ca(2+)-activated K(+) channel inhibition

    DEFF Research Database (Denmark)

    Diness, Jonas Goldin; Skibsbye, Lasse; Jespersen, Thomas

    2011-01-01

    hypertensive rats were more vulnerable to AF induction both by S2 stimulation and burst pacing. Vehicle affected neither the atrial effective refractory period nor AF duration. SK channel inhibition with NS8593 and UCL1684 significantly increased the atrial effective refractory period and decreased AF duration......We have shown previously that inhibition of small conductance Ca(2+)-activated K(+) (SK) channels is antiarrhythmic in models of acutely induced atrial fibrillation (AF). These models, however, do not take into account that AF derives from a wide range of predisposing factors, the most prevalent...... being hypertension. In this study we assessed the effects of two different SK channel inhibitors, NS8593 and UCL1684, in aging, spontaneously hypertensive rats to examine their antiarrhythmic properties in a setting of hypertension-induced atrial remodeling. Male spontaneously hypertensive rats...

  5. A new molecular model for Congo Red-β amyloid interaction: implications for diagnosis and inhibition of brain plaque formation in Alzheimer's disease

    Science.gov (United States)

    Zhang, Kristine A.; Li, Yat

    2015-08-01

    Alzheimer's disease (AD), an age-related neurodegenerative disorder, is the seventh leading cause of death in the United States. One strong pathological indicator of AD is senile plaques, which are aggregates of fibrils formed from amyloid β (Aβ) peptides. Thus, detection and inhibition of Aβ aggregation are critical for the prevention and treatment of AD. Congo red (CR) is one of the most widely used dye molecules for probing as well as inhabiting Aβ aggregation. However, the nature of interaction between CR and Aβ is not well understood. In this research, we systematically studied the interaction between CR and Aβ using a combination of optical techniques, including electronic absorption, fluorescence, Raman scattering, and circular dichroism, to provide detailed information with molecular specificity and high sensitivity. Compared to CR alone, interaction of the dye with Aβ results in a new absorption peak near 540 nm and significantly enhanced photoluminescence as well as Raman signal. Our results led us to propose a new model suggesting that CR exists primarily in a micellar form, resembling H-aggregates, in water and dissociates into monomers upon interaction with Aβ. This model has significant implications for the development of new strategies to detect and inhibit brain plaques for treatment of neurological diseases like AD.

  6. Semen amyloids participate in spermatozoa selection and clearance

    Science.gov (United States)

    Roan, Nadia R; Sandi-Monroy, Nathallie; Kohgadai, Nargis; Usmani, Shariq M; Hamil, Katherine G; Neidleman, Jason; Montano, Mauricio; Ständker, Ludger; Röcker, Annika; Cavrois, Marielle; Rosen, Jared; Marson, Kara; Smith, James F; Pilcher, Christopher D; Gagsteiger, Friedrich; Sakk, Olena; O’Rand, Michael; Lishko, Polina V; Kirchhoff, Frank

    2017-01-01

    Unlike other human biological fluids, semen contains multiple types of amyloid fibrils in the absence of disease. These fibrils enhance HIV infection by promoting viral fusion to cellular targets, but their natural function remained unknown. The similarities shared between HIV fusion to host cell and sperm fusion to oocyte led us to examine whether these fibrils promote fertilization. Surprisingly, the fibrils inhibited fertilization by immobilizing sperm. Interestingly, however, this immobilization facilitated uptake and clearance of sperm by macrophages, which are known to infiltrate the female reproductive tract (FRT) following semen exposure. In the presence of semen fibrils, damaged and apoptotic sperm were more rapidly phagocytosed than healthy ones, suggesting that deposition of semen fibrils in the lower FRT facilitates clearance of poor-quality sperm. Our findings suggest that amyloid fibrils in semen may play a role in reproduction by participating in sperm selection and facilitating the rapid removal of sperm antigens. DOI: http://dx.doi.org/10.7554/eLife.24888.001 PMID:28653619

  7. Interactions of laminin with the amyloid ß peptide: Implications for Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Morgan C.

    2001-01-01

    Full Text Available Extensive neuronal cell loss is observed in Alzheimer's disease. Laminin immunoreactivity colocalizes with senile plaques, the characteristic extracellular histopathological lesions of Alzheimer brain, which consist of the amyloid ß (Aß peptide polymerized into amyloid fibrils. These lesions have neurotoxic effects and have been proposed to be a main cause of neurodegeneration. In order to understand the pathological significance of the interaction between laminin and amyloid, we investigated the effect of laminin on amyloid structure and toxicity. We found that laminin interacts with the Aß1-40 peptide, blocking fibril formation and even inducing depolymerization of preformed fibrils. Protofilaments known to be intermediate species of Aß fibril formation were also detected as intermediate species of laminin-induced Aß fibril depolymerization. Moreover, laminin-amyloid interactions inhibited the toxic effects on rat primary hippocampal neurons. As a whole, our results indicate a putative anti-amyloidogenic role of laminin which may be of biological and therapeutic interest for controlling amyloidosis, such as those observed in cerebral angiopathy and Alzheimer's disease.

  8. Acute gamma-secretase inhibition of nonhuman primate CNS shifts amyloid precursor protein (APP) metabolism from amyloid-beta production to alternative APP fragments without amyloid-beta rebound.

    Science.gov (United States)

    Cook, Jacquelynn J; Wildsmith, Kristin R; Gilberto, David B; Holahan, Marie A; Kinney, Gene G; Mathers, Parker D; Michener, Maria S; Price, Eric A; Shearman, Mark S; Simon, Adam J; Wang, Jennifer X; Wu, Guoxin; Yarasheski, Kevin E; Bateman, Randall J

    2010-05-12

    The accumulation of amyloid beta (Abeta) in Alzheimer's disease is caused by an imbalance of production and clearance, which leads to increased soluble Abeta species and extracellular plaque formation in the brain. Multiple Abeta-lowering therapies are currently in development: an important goal is to characterize the molecular mechanisms of action and effects on physiological processing of Abeta, as well as other amyloid precursor protein (APP) metabolites, in models which approximate human Abeta physiology. To this end, we report the translation of the human in vivo stable-isotope-labeling kinetics (SILK) method to a rhesus monkey cisterna magna ported (CMP) nonhuman primate model, and use the model to test the mechanisms of action of a gamma-secretase inhibitor (GSI). A major concern of inhibiting the enzymes which produce Abeta (beta- and gamma-secretase) is that precursors of Abeta may accumulate and cause a rapid increase in Abeta production when enzyme inhibition discontinues. In this study, the GSI MK-0752 was administered to conscious CMP rhesus monkeys in conjunction with in vivo stable-isotope-labeling, and dose-dependently reduced newly generated CNS Abeta. In contrast to systemic Abeta metabolism, CNS Abeta production was not increased after the GSI was cleared. These results indicate that most of the CNS APP was metabolized to products other than Abeta, including C-terminal truncated forms of Abeta: 1-14, 1-15 and 1-16; this demonstrates an alternative degradation pathway for CNS amyloid precursor protein during gamma-secretase inhibition.

  9. Alzheimer's associated β-amyloid protein inhibits influenza A virus and modulates viral interactions with phagocytes.

    Directory of Open Access Journals (Sweden)

    Mitchell R White

    Full Text Available Accumulation of β-Amyloid (βA is a key pathogenetic factor in Alzheimer's disease; however, the normal function of βA is unknown. Recent studies have shown that βA can inhibit growth of bacteria and fungi. In this paper we show that βA also inhibits replication of seasonal and pandemic strains of H3N2 and H1N1 influenza A virus (IAV in vitro. The 42 amino acid fragment of βA (βA42 had greater activity than the 40 amino acid fragment. Direct incubation of the virus with βA42 was needed to achieve optimal inhibition. Using quantitative PCR assays βA42 was shown to reduce viral uptake by epithelial cells after 45 minutes and to reduce supernatant virus at 24 hours post infection. βA42 caused aggregation of IAV particles as detected by light transmission assays and electron and confocal microscopy. βA42 did not stimulate neutrophil H2O2 production or extracellular trap formation on its own, but it increased both responses stimulated by IAV. In addition, βA42 increased uptake of IAV by neutrophils. βA42 reduced viral protein synthesis in monocytes and reduced IAV-induced interleukin-6 production by these cells. Hence, we demonstrate for the first time that βA has antiviral activity and modulates viral interactions with phagocytes.

  10. Rigid Organization of Fluorescence-Active Ligands by Artificial Macrocyclic Receptor to Achieve the Thioflavin T-Amyloid Fibril Level Association.

    Science.gov (United States)

    Zhang, Ying-Ming; Zhang, Xu-Jie; Xu, Xiufang; Fu, Xiao-Ning; Hou, Hong-Biao; Liu, Yu

    2016-04-28

    The push-pull molecules with an intramolecular charge transfer from donor to acceptor sides upon excitation exhibit a wide variety of biological and electronic activities, as exemplified by the in vivo fluorescence imaging probes for amyloid fibrils in the diagnosis and treatment of amyloid diseases. Interestingly, the structurally much simpler bis(4,8-disulfonato-1,5-naphtho)-32-crown-8 (DNC), in keen contrast to the conventional macrocyclic receptors, was found to dramatically enhance the fluorescence of twisted intramolecular charge-transfer molecules possessing various benzothiazolium and stilbazolium fluorophores upon complexation. Spectroscopic and microcalorimetric titrations jointly demonstrated the complex structures and the interactions that promote the extremely strong complexation, revealing that the binding affinity in these artificial host-guest pairs could reach up to a nearly 10(7) M(-1) order of magnitude in water, and the sandwich-type complexation is driven by electrostatic, hydrophobic, π-stacking, and hydrogen-bonding interactions. Quantum chemical calculations on free molecules and their DNC-bound species in both the ground and excited states elucidated that the encapsulation by DNC could greatly deter the central single and double chemical bonds from free intramolecular rotation in the singlet excited state, thus leading to the unique and unprecedented fluorescence enhancement upon sandwich-type complexation. This complexation-induced structural reorganization mechanism may also apply to the binding of other small-molecule ligands by functional receptors and contribute to the molecular-level understanding of the receptor-ligand interactions in many biology-related systems.

  11. Structural insights into mechanisms for inhibiting amyloid β42 aggregation by non-catechol-type flavonoids.

    Science.gov (United States)

    Hanaki, Mizuho; Murakami, Kazuma; Akagi, Ken-ichi; Irie, Kazuhiro

    2016-01-15

    The prevention of 42-mer amyloid β-protein (Aβ42) aggregation is promising for the treatment of Alzheimer's disease. We previously described the site-specific inhibitory mechanism for Aβ42 aggregation by a catechol-type flavonoid, (+)-taxifolin, targeting Lys16,28 after its autoxidation. In contrast, non-catechol-type flavonoids (morin, datiscetin, and kaempferol) inhibited Aβ42 aggregation without targeting Lys16,28 with almost similar potencies to that of (+)-taxifolin. We herein provided structural insights into their mechanisms for inhibiting Aβ42 aggregation. Physicochemical analyses revealed that their inhibition did not require autoxidation. The (1)H-(15)N SOFAST-HMQC NMR of Aβ42 in the presence of morin and datiscetin revealed the significant perturbation of chemical shifts of His13,14 and Gln15, which were close to the intermolecular β-sheet region, Gln15-Ala21. His13,14 also played a role in radical formation at Tyr10, thereby inducing the oxidation of Met35, which has been implicated in Aβ42 aggregation. These results suggest the direct interaction of morin and datiscetin with the Aβ42 monomer. Although only kaempferol was oxidatively-degraded during incubation, its degradation products as well as kaempferol itself suppressed Aβ42 aggregation. However, neither kaempferol nor its decomposed products perturbed the chemical shifts of the Aβ42 monomer. Aggregation experiments using 1,1,1,3,3,3-hexafluoro-2-propanol-treated Aβ42 demonstrated that kaempferol and its degradation products inhibited the elongation rather than nucleation phase, implying that they interacted with small aggregates of Aβ42, but not with the monomer. In contrast, morin and datiscetin inhibited both phases. The position and number of hydroxyl groups on the B-ring of non-catechol-type flavonoids could be important for their inhibitory potencies and mechanisms against Aβ42 aggregation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Fibrillar dimer formation of islet amyloid polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Chiu, Chi-cheng [Univ. of Chicago, IL (United States); Argonne National Lab. (ANL), Argonne, IL (United States); de Pablo, Juan J. [Univ. of Chicago, IL (United States); Argonne National Lab. (ANL), Argonne, IL (United States)

    2015-05-08

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

  13. Fibrillar dimer formation of islet amyloid polypeptides

    Science.gov (United States)

    Chiu, Chi-cheng; de Pablo, Juan J.

    2015-09-01

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 - 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 - 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

  14. BACE1 Function and Inhibition: Implications of Intervention in the Amyloid Pathway of Alzheimer’s Disease Pathology

    Directory of Open Access Journals (Sweden)

    Gerald Koelsch

    2017-10-01

    Full Text Available Alzheimer’s disease (AD is a fatal progressive neurodegenerative disorder characterized by increasing loss in memory, cognition, and function of daily living. Among the many pathologic events observed in the progression of AD, changes in amyloid β peptide (Aβ metabolism proceed fastest, and precede clinical symptoms. BACE1 (β-secretase 1 catalyzes the initial cleavage of the amyloid precursor protein to generate Aβ. Therefore inhibition of BACE1 activity could block one of the earliest pathologic events in AD. However, therapeutic BACE1 inhibition to block Aβ production may need to be balanced with possible effects that might result from diminished physiologic functions BACE1, in particular processing of substrates involved in neuronal function of the brain and periphery. Potentials for beneficial or consequential effects resulting from pharmacologic inhibition of BACE1 are reviewed in context of ongoing clinical trials testing the effect of BACE1 candidate inhibitor drugs in AD populations.

  15. MMP-7 cleaves amyloid β fragment peptides and copper ion inhibits the degradation.

    Science.gov (United States)

    Taniguchi, Masanari; Matsuura, Kazuki; Nakamura, Rina; Kojima, Aya; Konishi, Motomi; Akizawa, Toshifumi

    2017-10-01

    The extracellular deposition of amyloid β (Aβ) is known to be the fundamental cause of Alzheimer's disease (AD). Aβ1-42, generated by β-secretases from the amyloid precursor protein (APP), is the main component of neuritic plaque, and the aggregation of this protein is shown to be dependent to an extent on metal ions such as copper and zinc. However, the mechanism by which Cu 2+ affects the physicochemical properties of Aβ1-42 or the central nervous system is still under debate. A recent series of studies have demonstrated that both the soluble-type matrix metalloproteinases (MMP-2 and MMP-9) and the membrane-type matrix metalloproteinase (MT1-MMP) are capable of degrading Aβ peptides. MMP-7, one of the soluble-type matrix metalloproteinases, is expressed in hippocampal tissue; however, less information is available concerning the pathophysiological roles of this enzyme in the process and/or progress of Alzheimer's disease. In this study, we examined the degradation activity of MMP-7 against various Aβ1-42's fragment peptides and the effect of Cu 2+ . Although Aβ22-40 was degraded by MMP-7 regardless of Cu 2+ , Cu 2+ inhibited the degradation of Aβ1-19, Aβ11-20, and Aβ11-29 by MMP-7. These results indicate that MMP-7 is capable of degrading Aβ1-42, and that Aβ1-42 acquired resistance against MMP-7 cleavage through Cu 2+ -binding and structure changes. Our results demonstrate that MMP-7 may play an important role in the defensive mechanism against the aggregation of Aβ1-42, which gives rise to the pathology of AD.

  16. The molecular mass of dextran used to modify magnetite nanoparticles affects insulin amyloid aggregation

    Science.gov (United States)

    Siposova, Katarina; Pospiskova, Kristyna; Bednarikova, Zuzana; Safarik, Ivo; Safarikova, Mirka; Kubovcikova, Martina; Kopcansky, Peter; Gazova, Zuzana

    2017-04-01

    Protein transformation from its soluble state into amyloid aggregates is associated with amyloid-related diseases. Amyloid deposits of insulin fibrils have been found in the sites of subcutaneous insulin application in patients with prolonged diabetes. Using atomic force microscopy and ThT fluorescence assay we have investigated the interference of insulin amyloid aggregation with superparamagnetic Fe3O4-based nanoparticles (SPIONs) coated with dextran (DEX); molecular mass of dextran was equal to 15-20, 40 or 70 kDa. The obtained data indicate that all three types of dextran coated nanoparticles (NP-FeDEXs) are able to inhibit insulin fibrillization and to destroy amyloid fibrils. The extent of anti-amyloid activities depends on the properties of NP-FeDEXs, mainly on the size of nanoparticles which is determined by molecular mass of dextran molecules. The most effective inhibiting activity was observed for the smallest nanoparticles coated with 15-20 kDa dextran. Contrary, the highest destroying activity was observed for the largest NP-FeDEX (70 kDa dextran).

  17. Amyloid cascade in Alzheimer's disease: Recent advances in medicinal chemistry.

    Science.gov (United States)

    Mohamed, Tarek; Shakeri, Arash; Rao, Praveen P N

    2016-05-04

    Alzheimer's disease is of major concern all over the world due to a number of factors including (i) an aging population (ii) increasing life span and (iii) lack of effective pharmacotherapy options. The past decade has seen intense research in discovering disease-modifying multitargeting small molecules as therapeutic options. The pathophysiology of Alzheimer's disease is attributed to a number of factors such as the cholinergic dysfunction, amyloid/tau toxicity and oxidative stress/mitochondrial dysfunction. In recent years, targeting the amyloid cascade has emerged as an attractive strategy to discover novel neurotherapeutics. Formation of beta-amyloid species, with different degrees of solubility and neurotoxicity is associated with the gradual decline in cognition leading to dementia. The two commonly used approaches to prevent beta-amyloid accumulation in the brain include (i) development of beta-secretase inhibitors and (ii) designing direct inhibitors of beta-amyloid (self-induced) aggregation. This review highlights the amyloid cascade hypothesis and the key chemical features required to design small molecules that inhibit lower and higher order beta-amyloid aggregates. Several recent examples of small synthetic molecules with disease-modifying properties were considered and their molecular docking studies were conducted using either a dimer or steric-zipper assembly of beta-amyloid. These investigations provide a mechanistic understanding on the structural requirements needed to design novel small molecules with anti-amyloid aggregation properties. Significantly, this work also demonstrates that the structural requirements to prevent aggregation of various amyloid species differs considerably, which explains the fact that many small molecules do not exhibit similar inhibition profile toward diverse amyloid species such as dimers, trimers, tetramers, oligomers, protofibrils and fibrils. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  18. Protein Polymers and Amyloids

    DEFF Research Database (Denmark)

    Risør, Michael Wulff

    2014-01-01

    Several human disorders are caused by a common general disease mechanism arising from abnormal folding and aggregation of the underlying protein. These include the prevalent dementias like Alzheimer’s and Parkinson’s, where accumulation of protein fibrillar structures, known as amyloid fibrils...... that inhibits its target protease through a large conformational change but mutations compromise this function and cause premature structural collapse into hyperstable polymers. Understanding the conformational disorders at a molecular level is not only important for our general knowledge on protein folding......, underlining the importance of understanding this relationship. The monomeric C-36 peptide was investigated by liquid-state NMR spectroscopy and found to be intrinsically disordered with minor propensities towards β-sheet structure. The plasticity of such a peptide makes it suitable for a whole range...

  19. Inhibition of lysozyme amyloidogenesis by phospholipids. Focus on long-chain dimyristoylphosphocholine.

    Science.gov (United States)

    Ponikova, Slavomira; Kubackova, Jana; Bednarikova, Zuzana; Marek, Jozef; Demjen, Erna; Antosova, Andrea; Musatov, Andrey; Gazova, Zuzana

    2017-11-01

    Protein amyloid aggregation is an important pathological feature of a group of different degenerative human diseases called amyloidosis. We tested effect of two phospholipids, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) on amyloid aggregation of hen egg white (HEW) lysozyme in vitro. Effect of phospholipids was investigated using spectroscopic techniques (fluorescence and CD spectroscopy), atomic force microscopy and image analysis. Phospholipids DMPC and DHPC are able dose-dependently inhibit lysozyme fibril formation. The length of the phospholipid tails and different structural arrangement of the phospholipid molecules affect inhibitory activity; long-chain DMPC inhibits fibrillization more efficiently. Interestingly, interference of DMPC with lysozyme amyloid fibrils has no effect on their morphology or amount. Phospholipid molecules have significant effect on lysozyme amyloid fibrillization. We suggest that inhibitory activity is due to the interference of phospholipids with lysozyme leading to the blocking of the intermolecular protein interactions important for formation of the cross-β structure within the core of the fibrils. The higher inhibitory activity of DMPC is probably due to adsorption of protein molecules on the liposome surfaces which caused decrease of species needed for fibrillization. Interaction of the phospholipids with formed fibrils is not sufficient enough to interrupt the bonds in β-sheets which are required for destroying of amyloid fibrils. The obtained results contribute to a better understanding of the effect of phospholipids on amyloid fibrillization of the lysozyme. The data suggest that DMPC and DHPC phospholipids represent agents able to modulate lysozyme amyloid aggregation. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Why are Functional Amyloids Non-Toxic in Humans?

    Directory of Open Access Journals (Sweden)

    Matthew P. Jackson

    2017-09-01

    Full Text Available Amyloids were first identified in association with amyloidoses, human diseases in which proteins and peptides misfold into amyloid fibrils. Subsequent studies have identified an array of functional amyloid fibrils that perform physiological roles in humans. Given the potential for the production of toxic species in amyloid assembly reactions, it is remarkable that cells can produce these functional amyloids without suffering any obvious ill effect. Although the precise mechanisms are unclear, there are a number of ways in which amyloid toxicity may be prevented. These include regulating the level of the amyloidogenic peptides and proteins, minimising the production of prefibrillar oligomers in amyloid assembly reactions, sequestrating amyloids within membrane bound organelles, controlling amyloid assembly by other molecules, and disassembling the fibrils under physiological conditions. Crucially, a better understanding of how toxicity is avoided in the production of functional amyloids may provide insights into the prevention of amyloid toxicity in amyloidoses.

  1. PROTEIN AGREGATION MODELS OF PARKINSONS DISEASE USING VIRAL VECTORS, PROTEASOME INHIBITION AND INOCULATION OF PREFORMED FIBRILS IN THE GOTTINGEN MINIPIG CNS

    DEFF Research Database (Denmark)

    Glud, Andreas Nørgaard; Lillethorup, Thea Pinholt; Landeck, Natalie

    SYN) fibrils, overexpressing aSYN using Lentivirus (LV) and Adeno Assosiated Virus (AAV) vectors or proteasome inhibition in the nigrostriatal system, we hope to create a new porcine models for PD. Methods: Using conventional human-intended stereotaxic neurosurgery methods, we apply aSYN or preformed fibrils...

  2. Alginate-Derived Oligosaccharide Inhibits Neuroinflammation and Promotes Microglial Phagocytosis of β-Amyloid

    Directory of Open Access Journals (Sweden)

    Rui Zhou

    2015-09-01

    Full Text Available Alginate from marine brown algae has been widely applied in biotechnology. In this work, the effects of alginate-derived oligosaccharide (AdO on lipopolysaccharide (LPS/β-amyloid (Aβ-induced neuroinflammation and microglial phagocytosis of Aβ were studied. We found that pretreatment of BV2 microglia with AdO prior to LPS/Aβ stimulation led to a significant inhibition of production of nitric oxide (NO and prostaglandin E2 (PGE2, expression of inducible nitric oxide synthase (iNOS and cyclooxygenase-2 (COX-2 and secretion of proinflammatory cytokines. We further demonstrated that AdO remarkably attenuated the LPS-activated overexpression of toll-like receptor 4 (TLR4 and nuclear factor (NF-κB in BV2 cells. In addition to the impressive inhibitory effect on neuroinflammation, we also found that AdO promoted the phagocytosis of Aβ through its interaction with TLR4 in microglia. Our results suggested that AdO exerted the inhibitory effect on neuroinflammation and the promotion effect on microglial phagocytosis, indicating its potential as a nutraceutical or therapeutic agent for neurodegenerative diseases, particularly Alzheimer’s disease (AD.

  3. Phenylpropanoids from cinnamon bark reduced β-amyloid production by the inhibition of β-secretase in Chinese hamster ovarian cells stably expressing amyloid precursor protein.

    Science.gov (United States)

    Kang, Yu Jeong; Seo, Dae-Gun; Park, So-Young

    2016-11-01

    β-Amyloid (Aβ) is a substance of Alzheimer disease (AD), which is generated via the amyloidogenic pathway from amyloid precursor protein (APP) by β-secretase and γ-secretase. Inhibition of Aβ production is a potential therapeutic approach to AD. Thus, we tested the hypothesis that cinnamon bark (Cinnamomi Cortex Spissus), the dried bark of Cinnamomum cassia Blume (Lauraceae), and its constituents are beneficial to AD. The methanol extract of cinnamon bark efficiently reduced Aβ40 production in Chinese hamster ovarian (CHO) cells stably expressing APP as determined by enzyme-linked immunosorbent assay. Bioassay-guided isolation of cinnamon bark extract was carried out using open column chromatography and high-performance liquid chromatography, and the following 6 phenylpropanoids were isolated: syringaresinol (1); medioresinol (2); coumarin (3); 2-hydroxycinnamaldehyde (4); cryptamygin A (5); and 3',5,7-trimethoxy epicatechin (6). Among these, 4 μg/mL medioresinol and cryptamygin A reduced Aβ40 production by 50% and 60%, respectively, compared with dimethyl sulfoxide-treated control cells. The IC 50 values of medioresinol and cryptamygin A for the inhibition of Aβ40 production were 10.8 and 8.2 μg/mL, respectively. Furthermore, treatment of APP-CHO cells with either compound decreased the amount of β-secretase and sAPPβ (the proteolytic fragment of APP catalyzed by β-secretase). These results suggest that the antiamyloidogenic activity of cinnamon bark extract was exerted by medioresinol and cryptamygin A via a reduction in the amount of β-secretase. The extract of cinnamon bark contains potentially valuable antiamyloidogenic agents for the prevention and treatment of AD. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Human Islet Amyloid Polypeptide

    DEFF Research Database (Denmark)

    Kosicka, Iga

    2014-01-01

    Diabetes mellitus type II is a metabolic disease affecting millions of people worldwide. The disease is associated with occurence of insoluble, fibrillar, protein aggregates in islets of Langerhans in the pancreas - islet amyloid. The main constituent of these protein fibers is the human islet...... of diabetes type II, while revealing the structure(s) of islet amyloid fibrils is necessary for potential design of therapeutic agents....

  5. Donepezil inhibits the amyloid-beta oligomer-induced microglial activation in vitro and in vivo.

    Science.gov (United States)

    Kim, Hyo Geun; Moon, Minho; Choi, Jin Gyu; Park, Gunhyuk; Kim, Ae-Jung; Hur, Jinyoung; Lee, Kyung-Tae; Oh, Myung Sook

    2014-01-01

    Recent studies on Alzheimer's disease (AD) have focused on soluble oligomeric forms of amyloid-beta (Aβ oligomer, AβO) that are directly associated with AD-related pathologies, such as cognitive decline, neurodegeneration, and neuroinflammation. Donepezil is a well-known anti-dementia agent that increases acetylcholine levels through inhibition of acetylcholinesterase. However, a growing body of experimental and clinical studies indicates that donepezil may also provide neuroprotective and disease-modifying effects in AD. Additionally, donepezil has recently been demonstrated to have anti-inflammatory effects against lipopolysaccharides and tau pathology. However, it remains unknown whether donepezil has anti-inflammatory effects against AβO in cultured microglial cells and the brain in animals. Further, the effects of donepezil against AβO-mediated neuronal death, astrogliosis, and memory impairment have also not yet been investigated. Thus, in the present study, we examined the anti-inflammatory effect of donepezil against AβO and its neuroinflammatory mechanisms. Donepezil significantly attenuated the release of inflammatory mediators (prostaglandin E2, interleukin-1 beta, tumor necrosis factor-α, and nitric oxide) from microglia. Donepezil also decreased AβO-induced up-regulation of inducible nitric oxide synthase and cyclooxygenase-2 protein and phosphorylation of p38 mitogen-activated protein kinase as well as translocation of nuclear factor-kappa B. We next showed that donepezil suppresses activated microglia-mediated toxicity in primary hippocampal cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In intrahippocampal AβO-injected mice, donepezil significantly inhibited microgliosis and astrogliosis. Furthermore, behavioral tests revealed that donepezil (2 mg/kg/day, 5 days, p.o.) significantly ameliorated AβO-induced memory impairment. These results suggest that donepezil directly inhibits microglial activation

  6. Vitamin B12 Inhibits Tau Fibrillization via Binding to Cysteine Residues of Tau.

    Science.gov (United States)

    Rafiee, Saharnaz; Asadollahi, Kazem; Riazi, Gholamhossein; Ahmadian, Shahin; Saboury, Ali Akbar

    2017-12-20

    Two mechanisms underlie the inhibitory/acceleratory action of chemical compounds on tau aggregation including the regulation of cellular kinases and phosphatases activity and direct binding to tau protein. Vitamin B12 is one of the tau polymerization inhibitors, and its deficiency is linked to inactivation of protein phosphatase 2A and subsequently hyperphosphorylation and aggregation of tau protein. Regarding the structure and function of vitamin B12 and tau protein, we assumed that vitamin B12 is also able to directly bind to tau protein. Hence, we investigated the interaction of vitamin B12 with tau protein in vitro using fluorometry and circular dichrosim. Interaction studies was followed by investigation into the effect of vitamin B12 on tau aggregation using ThT fluorescence, circular dichroism, transmission electron microscopy, and SDS-PAGE. The results indicated that vitamin B12 interacts with tau protein and prevents fibrillization of tau protein. Blocking the cysteine residues of tau confirmed the cysteine-mediated binding of vitamin B12 to tau and showed that binding to cysteine is essential for inhibitory effect of vitamin B12 on tau aggregation. SDS-PAGE analysis indicated that vitamin B12 inhibits tau aggregation and that tau oligomers formed in the presence of vitamin B12 are mostly SDS-soluble. We propose that direct binding of vitamin B12 is another mechanism underlying the inhibitory role of vitamin B12 on tau aggregation and neurodegeneration.

  7. Sortilin inhibits amyloid pathology by regulating non-specific degradation of APP.

    Science.gov (United States)

    Ruan, Chun-Sheng; Liu, Jia; Yang, Miao; Saadipour, Khalil; Zeng, Yue-Qin; Liao, Hong; Wang, Yan-Jiang; Bobrovskaya, Larisa; Zhou, Xin-Fu

    2018-01-01

    Amyloid plaque is one of the hallmarks of Alzheimer's disease (AD). The key component beta-amyloid (Aβ) is generated via proteolytic processing of amyloid precursor protein (APP). Sortilin (encoded by the gene Sort1) is a vacuolar protein sorting 10 protein domain-containing receptor, which is up-regulated in the brain of AD, colocalizes with amyloid plaques and interacts with APP. However, its role in amyloidogenesis remains unclear. In this study, we first found that the protein level of sortilin was up-regulated in the neocortex of aged (7 and 9months old) but not young (2 and 5months old) AD mice (APP/PS1). 9months old APP/PS1 transgenic mice with Sort1 gene knockout showed increased amyloid pathology in the brain; and this phenotype was rescued by intrahippocampal injection of AAV-hSORT1. Moreover, the 9months old APP/PS1 mice without Sort1 also displayed a decreased number of neurons and increased astrocyte activation in the hippocampus. In addition, the present study showed that the intracellular domain of sortilin was involved in the regulation of the non-specific degradation of APP. Together, our findings indicate that sortilin is a beneficial protein for the reduction of amyloid pathology in APP/PS1 mice by promoting APP degradation. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Continuation of exercise is necessary to inhibit high fat diet-induced β-amyloid deposition and memory deficit in amyloid precursor protein transgenic mice.

    Directory of Open Access Journals (Sweden)

    Masato Maesako

    Full Text Available High fat diet (HFD is prevalent in many modern societies and HFD-induced metabolic condition is a growing concern worldwide. It has been previously reported that HFD clearly worsens cognitive function in amyloid precursor protein (APP transgenic mice. On the other hand, we have demonstrated that voluntary exercise in an enriched environment is an effective intervention to rescue HFD-induced β-amyloid (Aβ deposition and memory deficit. However, it had been unclear whether consumption of HFD after exercising abolished the beneficial effect of exercise on the inhibition of Alzheimer's disease (AD pathology. To examine this question, we exposed wild type (WT and APP mice fed with HFD to exercise conditions at different time periods. In our previous experiment, we gave HFD to mice for 20 weeks and subjected them to exercise during weeks 10-20. In the present study, mice were subjected to exercise conditions during weeks 0-10 or weeks 5-15 while being on HFD. Interestingly, we found that the effect of exercise during weeks 0-10 or weeks 5-15 on memory function was not abolished in WT mice even if they kept having HFD after finishing exercise. However, in APP transgenic mice, HFD clearly disrupted the effect of exercise during weeks 0-10 or weeks 5-15 on memory function. Importantly, we observed that the level of Aβ oligomer was significantly elevated in the APP mice that exercised during weeks 0-10: this might have been caused by the up-regulation of Aβ production. These results provide solid evidence that continuation of exercise is necessary to rescue HFD-induced aggravation of cognitive decline in the pathological setting of AD.

  9. Inhibition of amyloid-beta-induced cell death in human brain pericytes in vitro.

    NARCIS (Netherlands)

    Rensink, A.A.M.; Verbeek, M.M.; Otte-Holler, I.; Donkelaar, H.J. ten; Waal, R.M.W. de; Kremer, H.P.H.

    2002-01-01

    Amyloid-beta protein (A beta) deposition in the cerebral vascular walls is one of the key features of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). A beta(1-40) carrying the 'Dutch' mutation (HCHWA-D A beta(1-40)) induces pronounced degeneration of

  10. Insulin inhibits amyloid beta-induced cell death in cultured human brain pericytes

    NARCIS (Netherlands)

    Rensink, Annemieke A M; Otte-Höller, Irene; de Boer, Roelie; Bosch, Remko R; ten Donkelaar, Hans J; de Waal, Robert M W; Verbeek, Marcel M; Kremer, Berry

    Amyloid-beta (Abeta) deposition in the cerebral arterial and capillary walls is one of the characteristics of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type. In vitro, Abeta1-40, carrying the "Dutch" mutation (DAbeta1-40), induced reproducible degeneration of

  11. GM1 Ganglioside Inhibits β-Amyloid Oligomerization Induced by Sphingomyelin

    Czech Academy of Sciences Publication Activity Database

    Amaro, Mariana; Šachl, Radek; Aydogan, Gokcan; Mikhalyov, I.; Vácha, R.; Hof, Martin

    2016-01-01

    Roč. 55, č. 32 (2016), s. 9411-9415 ISSN 1433-7851 R&D Projects: GA ČR(CZ) GBP208/12/G016 Grant - others:GA MŠk(CZ) LM2010005 Institutional support: RVO:61388955 Keywords : amyloid beta-peptides * Alzheimer's disease * diffusion coefficients Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 11.994, year: 2016

  12. GM1 Ganglioside Inhibits β-Amyloid Oligomerization Induced by Sphingomyelin

    Czech Academy of Sciences Publication Activity Database

    Amaro, Mariana; Šachl, Radek; Aydogan, Gokcan; Mikhalyov, I.; Vácha, R.; Hof, Martin

    2016-01-01

    Roč. 55, č. 32 (2016), s. 9411-9415 ISSN 1433-7851 R&D Projects: GA ČR(CZ) GBP208/12/G016 Grant - others:GA MŠk(CZ) LM2010005 Institutional support: RVO:61388955 Keywords : amyloid beta-peptides * Alzheimer's disease * diffusion coefficients Subject RIV: CF - Physical ; The oretical Chemistry Impact factor: 11.994, year: 2016

  13. Inhibition of small-conductance Ca2+-activated K+ channels terminates and protects against atrial fibrillation

    DEFF Research Database (Denmark)

    Diness, Jonas Goldin; Sørensen, Ulrik S; Nissen, Jakob Dahl

    2010-01-01

    Recently, evidence has emerged that small-conductance Ca(2+)-activated K(+) (SK) channels are predominantly expressed in the atria in a number of species including human. In rat, guinea pig, and rabbit ex vivo and in vivo models of atrial fibrillation (AF), we used 3 different SK channel inhibitors...

  14. The prototypic tissue pentraxin PTX3, in contrast to the short pentraxin serum amyloid P, inhibits phagocytosis of late apoptotic neutrophils by macrophages

    NARCIS (Netherlands)

    van Rossum, AP; Fazzini, F; Limburg, PC; Manfredi, AA; Rovere-Querini, P; Mantovani, A; Kallenberg, CGM

    Objective. Phagocytosis of apoptotic cells can be facilitated by complement components and short pentraxins, such as serum amyloid P (SAP). In contrast, the long pentraxin PTX3 was shown to inhibit phagocytosis of apoptotic Jurkat cells by dendritic cells and to bind late apoptotic polymorphonuclear

  15. β-Amyloid promotes accumulation of lipid peroxides by inhibiting CD36-mediated clearance of oxidized lipoproteins

    Directory of Open Access Journals (Sweden)

    Khan Tayeba

    2004-11-01

    Full Text Available Abstract Background Recent studies suggest that hypercholesterolemia, an established risk factor for atherosclerosis, is also a risk factor for Alzheimer's disease. The myeloid scavenger receptor CD36 binds oxidized lipoproteins that accumulate with hypercholesterolemia and mediates their clearance from the circulation and peripheral tissues. Recently, we demonstrated that CD36 also binds fibrillar β-amyloid and initiates a signaling cascade that regulates microglial recruitment and activation. As increased lipoprotein oxidation and accumulation of lipid peroxidation products have been reported in Alzheimer's disease, we investigated whether β-amyloid altered oxidized lipoprotein clearance via CD36. Methods The availability of mice genetically deficient in class A (SRAI & II and class B (CD36 scavenger receptors has facilitated studies to discriminate their individual actions. Using primary microglia and macrophages, we assessed the impact of Aβ on: (a cholesterol ester accumulation by GC-MS and neutral lipid staining, (b binding, uptake and degradation of 125I-labeled oxidized lipoproteins via CD36, SR-A and CD36/SR-A-independent pathways, (c expression of SR-A and CD36. In addition, using mice with targeted deletions in essential kinases in the CD36-signaling cascade, we investigated whether Aβ-CD36 signaling altered metabolism of oxidized lipoproteins. Results In primary microglia and macrophages, Aβ inhibited binding, uptake and degradation of oxidized low density lipoprotein (oxLDL in a dose-dependent manner. While untreated cells accumulated abundant cholesterol ester in the presence of oxLDL, cells treated with Aβ were devoid of cholesterol ester. Pretreatment of cells with Aβ did not affect subsequent degradation of oxidized lipoproteins, indicating that lysosomal accumulation of Aβ did not disrupt this degradation pathway. Using mice with targeted deletions of the scavenger receptors, we demonstrated that Aβ inhibited oxidized

  16. Antibodies to human serum amyloid P component eliminate visceral amyloid deposits.

    Science.gov (United States)

    Bodin, Karl; Ellmerich, Stephan; Kahan, Melvyn C; Tennent, Glenys A; Loesch, Andrzej; Gilbertson, Janet A; Hutchinson, Winston L; Mangione, Palma P; Gallimore, J Ruth; Millar, David J; Minogue, Shane; Dhillon, Amar P; Taylor, Graham W; Bradwell, Arthur R; Petrie, Aviva; Gillmore, Julian D; Bellotti, Vittorio; Botto, Marina; Hawkins, Philip N; Pepys, Mark B

    2010-11-04

    Accumulation of amyloid fibrils in the viscera and connective tissues causes systemic amyloidosis, which is responsible for about one in a thousand deaths in developed countries. Localized amyloid can also have serious consequences; for example, cerebral amyloid angiopathy is an important cause of haemorrhagic stroke. The clinical presentations of amyloidosis are extremely diverse and the diagnosis is rarely made before significant organ damage is present. There is therefore a major unmet need for therapy that safely promotes the clearance of established amyloid deposits. Over 20 different amyloid fibril proteins are responsible for different forms of clinically significant amyloidosis and treatments that substantially reduce the abundance of the respective amyloid fibril precursor proteins can arrest amyloid accumulation. Unfortunately, control of fibril-protein production is not possible in some forms of amyloidosis and in others it is often slow and hazardous. There is no therapy that directly targets amyloid deposits for enhanced clearance. However, all amyloid deposits contain the normal, non-fibrillar plasma glycoprotein, serum amyloid P component (SAP). Here we show that administration of anti-human-SAP antibodies to mice with amyloid deposits containing human SAP triggers a potent, complement-dependent, macrophage-derived giant cell reaction that swiftly removes massive visceral amyloid deposits without adverse effects. Anti-SAP-antibody treatment is clinically feasible because circulating human SAP can be depleted in patients by the bis-d-proline compound CPHPC, thereby enabling injected anti-SAP antibodies to reach residual SAP in the amyloid deposits. The unprecedented capacity of this novel combined therapy to eliminate amyloid deposits should be applicable to all forms of systemic and local amyloidosis.

  17. Two distinct β-sheet structures in Italian-mutant amyloid-beta fibrils : a potential link to different clinical phenotypes

    NARCIS (Netherlands)

    Hubin, Ellen; Deroo, Stéphanie; Schierle, Gabriele Kaminksi; Kaminski, Clemens; Serpell, Louise; Subramaniam, Vinod; van Nuland, Nico; Broersen, Kerensa; Raussens, Vincent; Sarroukh, Rabia

    2015-01-01

    Most Alzheimer's disease (AD) cases are late-onset and characterized by the aggregation and deposition of the amyloid-beta (Aβ) peptide in extracellular plaques in the brain. However, a few rare and hereditary Aβ mutations, such as the Italian Glu22-to-Lys (E22K) mutation, guarantee the development

  18. Amyloid Beta Peptides Block New Synapse Assembly by Nogo Receptor-Mediated Inhibition of T-Type Calcium Channels.

    Science.gov (United States)

    Zhao, Yanjun; Sivaji, Sivaprakash; Chiang, Michael C; Ali, Haadi; Zukowski, Monica; Ali, Sareen; Kennedy, Bryan; Sklyar, Alex; Cheng, Alice; Guo, Zihan; Reed, Alexander K; Kodali, Ravindra; Borowski, Jennifer; Frost, Georgia; Beukema, Patrick; Wills, Zachary P

    2017-10-11

    Compelling evidence links amyloid beta (Aβ) peptide accumulation in the brains of Alzheimer's disease (AD) patients with the emergence of learning and memory deficits, yet a clear understanding of the events that drive this synaptic pathology are lacking. We present evidence that neurons exposed to Aβ are unable to form new synapses, resulting in learning deficits in vivo. We demonstrate the Nogo receptor family (NgR1-3) acts as Aβ receptors mediating an inhibition of synapse assembly, plasticity, and learning. Live imaging studies reveal Aβ activates NgRs on the dendritic shaft of neurons, triggering an inhibition of calcium signaling. We define T-type calcium channels as a target of Aβ-NgR signaling, mediating Aβ's inhibitory effects on calcium, synapse assembly, plasticity, and learning. These studies highlight deficits in new synapse assembly as a potential initiator of cognitive pathology in AD, and pinpoint calcium dysregulation mediated by NgRs and T-type channels as key components. VIDEO ABSTRACT. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Back to the oligomeric state pH-induced dissolution of concanavalin A amyloid-like fibrils into non-native oligomers

    DEFF Research Database (Denmark)

    Santangelo, M. G.; Foderà, V.; Militello, V.

    2016-01-01

    ) or they are promptly dissolved leaving in solution non-native oligomers (pH > 10). The latter result can be ascribed to the change of the charge state for the ConA amino acid side chain with high pKa values. Our results support the idea of fibrils as a reservoir of oligomeric species that can be released if changes...

  20. Interactions driving the collapse of islet amyloid polypeptide: Implications for amyloid aggregation

    Science.gov (United States)

    Cope, Stephanie M.

    forms amyloid fibrils in vitro, whereas the rat variant (rIAPP), differing by six amino acids, does not. In addition to being highly soluble, rIAPP is an effective inhibitor of hIAPP fibril formation . Both of these properties have been attributed to rIAPP's three proline residues: A25P, S28P and S29P. Single proline mutants of hIAPP have also been shown to kinetically inhibit hIAPP fibril formation. Because of their intrinsic dihedral angle preferences, prolines are expected to affect conformational ensembles of intrinsically disordered proteins. The specific effect of proline substitutions on IAPP structure and dynamics has not yet been explored, as the detection of such properties is experimentally challenging due to the low molecular weight, fast reconfiguration times, and very low solubility of IAPP peptides. High-resolution techniques able to measure tertiary contact formations are needed to address this issue. We employ a nanosecond laser spectroscopy technique to measure end-to-end contact formation rates in IAPP mutants. We explore the proline substitutions in IAPP and quantify their effects in terms of intrinsic chain stiffness. We find that the three proline mutations found in rIAPP increase chain stiffness. Interestingly, we also find that residue R18 plays an important role in rIAPP's unique chain stiffness and, together with the proline residues, is a determinant for its non-amyloidogenic properties. We discuss the implications of our findings on the role of prolines in IDPs.

  1. Triptolide Inhibited Cytotoxicity of Differentiated PC12 Cells Induced by Amyloid-Beta25–35 via the Autophagy Pathway

    Science.gov (United States)

    Xu, Pengjuan; Li, Zhigui; Wang, Hui; Zhang, Xiaochen; Yang, Zhuo

    2015-01-01

    Evidence shows that an abnormal deposition of amyloid beta-peptide25–35 (Aβ25–35) was the primary cause of the pathogenesis of Alzheimer’s disease (AD). And the elimination of Aβ25–35 is considered an important target for the treatment of AD. Triptolide (TP), isolated from Tripterygium wilfordii Hook.f. (TWHF), has been shown to possess a broad spectrum of biological profiles, including neurotrophic and neuroprotective effects. In our study investigating the effect and potential mechanism of triptolide on cytotoxicity of differentiated rat pheochromocytoma cell line (the PC12 cell line is often used as a neuronal developmental model) induced by Amyloid-Beta25–35 (Aβ25–35), we used 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) assay, flow cytometry, Western blot, and acridine orange staining to detect whether triptolide could inhibit Aβ25–35–induced cell apoptosis. We focused on the potential role of the autophagy pathway in Aβ25–35-treated differentiated PC12 cells. Our experiments show that cell viability is significantly decreased, and the apoptosis increased in Aβ25–35-treated differentiated PC12 cells. Meanwhile, Aβ25–35 treatment increased the expression of microtubule-associated protein light chain 3 II (LC3 II), which indicates an activation of autophagy. However, triptolide could protect differentiated PC12 cells against Aβ25–35-induced cytotoxicity and attenuate Aβ25–35-induced differentiated PC12 cell apoptosis. Triptolide could also suppress the level of autophagy. In order to assess the effect of autophagy on the protective effects of triptolide in differentiated PC12 cells treated with Aβ25–35, we used 3-Methyladenine (3-MA, an autophagy inhibitor) and rapamycin (an autophagy activator). MTT assay showed that 3-MA elevated cell viability compared with the Aβ25–35-treated group and rapamycin inhibits the protection of triptolide. These results suggest that triptolide will repair the

  2. Hybrid Amyloid Membranes for Continuous Flow Catalysis.

    Science.gov (United States)

    Bolisetty, Sreenath; Arcari, Mario; Adamcik, Jozef; Mezzenga, Raffaele

    2015-12-29

    Amyloid fibrils are promising nanomaterials for technological applications such as biosensors, tissue engineering, drug delivery, and optoelectronics. Here we show that amyloid-metal nanoparticle hybrids can be used both as efficient active materials for wet catalysis and as membranes for continuous flow catalysis applications. Initially, amyloid fibrils generated in vitro from the nontoxic β-lactoglobulin protein act as templates for the synthesis of gold and palladium metal nanoparticles from salt precursors. The resulting hybrids possess catalytic features as demonstrated by evaluating their activity in a model catalytic reaction in water, e.g., the reduction of 4-nitrophenol into 4-aminophenol, with the rate constant of the reduction increasing with the concentration of amyloid-nanoparticle hybrids. Importantly, the same nanoparticles adsorbed onto fibrils surface show improved catalytic efficiency compared to the same unattached particles, pointing at the important role played by the amyloid fibril templates. Then, filter membranes are prepared from the metal nanoparticle-decorated amyloid fibrils by vacuum filtration. The resulting membranes serve as efficient flow catalysis active materials, with a complete catalytic conversion achieved within a single flow passage of a feeding solution through the membrane.

  3. Amyloid formation via supramolecular peptide assemblies.

    Science.gov (United States)

    Moore, Roger A; Hayes, Stanley F; Fischer, Elizabeth R; Priola, Suzette A

    2007-06-19

    Amyloid fibrils have been classically defined as linear, nonbranched polymeric proteins with a cross beta-sheet structure and the ability to alter the optical properties of the amyloid-specific dye Congo Red. Mounting evidence suggests that soluble oligomeric peptide assemblies approximately 2-20 nm in diameter are critical intermediates in amyloid formation. Using a pathogenic prion protein peptide comprised of residues 23-144, we demonstrate that, under quiescent but not agitated conditions, much larger globular assemblies up to 1 mum in diameter are made. These globules precede fibril formation and directly interact with growing fibril bundles. Fibrils made via these large spherical peptide assemblies displayed a remarkable diversity of ultrastructural features. Fibrillization of the Abeta1-40 peptide under similar conditions yielded similar results, suggesting a mechanism of general amyloid formation that can proceed through intermediates much larger than those previously described. Our data suggest that simply changing the physical microenvironment can profoundly influence the mechanism of amyloid formation and yield fibrils with novel ultrastructural properties.

  4. Computational Modelling of the Human Islet Amyloid Polypeptide

    DEFF Research Database (Denmark)

    Skeby, Katrine Kirkeby

    2014-01-01

    of a specific protein into amyloid fibrils. During this process, a cytotoxic event occurs which can be a serious actor in the evolvement of the disease. This thesis is concerned with elucidating the biological processes concerning amyloid proteins, more specifically, the peptide hormone human islet amyloid...... setup. We have exploited these strengths to study the interactions between an amyloid fibril and amyloid imaging agents. Imaging agents are promising tools for the detection of amyloid deposits in the brain of AD patients. This could aid in the early diagnosis as well as evaluation of new treatments......When proteins do not fold correctly, it can lead to very serious diseases. One such group of diseases is the amyloid diseases, of which Alzheimer’s disease (AD), Parkinson’s disease, and type 2 diabetes mellitus (T2DM) are members. The amyloid diseases are characterized by the aggregation...

  5. Amyloid beta inhibits olfactory bulb activity and the ability to smell.

    Directory of Open Access Journals (Sweden)

    Reynaldo Alvarado-Martínez

    Full Text Available Early olfactory dysfunction has been consistently reported in both Alzheimer's disease (AD and in transgenic mice that reproduce some features of this disease. In AD transgenic mice, alteration in olfaction has been associated with increased levels of soluble amyloid beta protein (Aβ as well as with alterations in the oscillatory network activity recorded in the olfactory bulb (OB and in the piriform cortex. However, since AD is a multifactorial disease and transgenic mice suffer a variety of adaptive changes, it is still unknown if soluble Aβ, by itself, is responsible for OB dysfunction both at electrophysiological and behavioral levels. Thus, here we tested whether or not Aβ directly affects OB network activity in vitro in slices obtained from mice and rats and if it affects olfactory ability in these rodents. Our results show that Aβ decreases, in a concentration- and time-dependent manner, the network activity of OB slices at clinically relevant concentrations (low nM and in a reversible manner. Moreover, we found that intrabulbar injection of Aβ decreases the olfactory ability of rodents two weeks after application, an effect that is not related to alterations in motor performance or motivation to seek food and that correlates with the presence of Aβ deposits. Our results indicate that Aβ disrupts, at clinically relevant concentrations, the network activity of the OB in vitro and can trigger a disruption in olfaction. These findings open the possibility of exploring the cellular mechanisms involved in early pathological AD as an approach to reduce or halt its progress.

  6. Differential Effects of Structural Modifications on the Competition of Chalcones for the PIB Amyloid Imaging Ligand-Binding Site in Alzheimer's Disease Brain and Synthetic Aβ Fibrils.

    Science.gov (United States)

    Fosso, Marina Y; McCarty, Katie; Head, Elizabeth; Garneau-Tsodikova, Sylvie; LeVine, Harry

    2016-02-17

    Alzheimer's disease (AD) is a complex brain disorder that still remains ill defined. In order to understand the significance of binding of different clinical in vivo imaging ligands to the polymorphic pathological features of AD brain, the molecular characteristics of the ligand interacting with its specific binding site need to be defined. Herein, we observed that tritiated Pittsburgh Compound B ((3)H-PIB) can be displaced from synthetic Aβ(1-40) and Aβ(1-42) fibrils and from the PIB binding complex purified from human AD brain (ADPBC) by molecules containing a chalcone structural scaffold. We evaluated how substitution on the chalcone scaffold alters its ability to displace (3)H-PIB from the synthetic fibrils and ADPBC. By comparing unsubstituted core chalcone scaffolds along with the effects of bromine and methyl substitution at various positions, we found that attaching a hydroxyl group on the ring adjacent to the carbonyl group (ring I) of the parent member of the chalcone family generally improved the binding affinity of chalcones toward ADPBC and synthetic fibrils F40 and F42. Furthermore, any substitution on ring I at the ortho-position of the carbonyl group greatly decreases the binding affinity of the chalcones, potentially as a result of steric hindrance. Together with the finding that neither our chalcones nor PIB interact with the Congo Red/X-34 binding site, these molecules provide new tools to selectively probe the PIB binding site that is found in human AD brain, but not in brains of AD pathology animal models. Our chalcone derivatives also provide important information on the effects of fibril polymorphism on ligand binding.

  7. Insulin Promotes Survival of Amyloid-Beta Oligomers Neuroblastoma Damaged Cells via Caspase 9 Inhibition and Hsp70 Upregulation

    Directory of Open Access Journals (Sweden)

    M. Di Carlo

    2010-01-01

    Full Text Available Alzheimer's disease (AD and type 2 diabetes are connected in a way that is still not completely understood, but insulin resistance has been implicated as a risk factor for developing AD. Here we show an evidence that insulin is capable of reducing cytotoxicity induced by Amyloid-beta peptides (A-beta in its oligomeric form in a dose-dependent manner. By TUNEL and biochemical assays we demonstrate that the recovery of the cell viability is obtained by inhibition of intrinsic apoptotic program, triggered by A-beta and involving caspase 9 and 3 activation. A protective role of insulin on mitochondrial damage is also shown by using Mito-red vital dye. Furthermore, A-beta activates the stress inducible Hsp70 protein in LAN5 cells and an overexpression is detectable after the addition of insulin, suggesting that this major induction is the necessary condition to activate a cell survival program. Together, these results may provide opportunities for the design of preventive and therapeutic strategies against AD.

  8. Acetylcholineestarase-inhibiting alkaloids from Lycoris radiata delay paralysis of amyloid beta-expressing transgenic C. elegans CL4176.

    Directory of Open Access Journals (Sweden)

    Lijuan Xin

    Full Text Available The limited symptom relief and side effects of current Alzheimer's disease (AD medications warrant urgent discovery and study of new anti-AD agents. The "cholinergic hypothesis" of AD prompts us to search for plant-derived acetylcholineesterase (AChE inhibitors such as galanthamine that has been licensed in Europe for AD treatment. We used the unique amyloid β-expressing transgenic C. elegans CL4176, which exhibits paralysis when human Aβ1-42 is induced, to study two natural benzylphenethylamine alkaloids isolated from Lycoris radiata (L' Her. Herb, galanthamine and haemanthidine, and their synthetic derivatives 1,2-Di-O-acetyllycorine and 1-O-acetyllycorine for their anti-paralysis effects. Our data indicate that these Lycoris compounds effectively delay the paralysis of CL4176 worms upon temperature up-shift, and prolong the lives of these transgenic worms. Lycoris compounds were shown to significantly inhibit the gene expression of ace-1 and ace-2. Additionally, the Lycoris compounds may modulate inflammatory and stress-related gene expressions to combat the Aβ-toxicity in C. elegans.

  9. Acetylcholineestarase-inhibiting alkaloids from Lycoris radiata delay paralysis of amyloid beta-expressing transgenic C. elegans CL4176.

    Science.gov (United States)

    Xin, Lijuan; Yamujala, Ritupriya; Wang, Yuehu; Wang, Huan; Wu, Wen-Hsuan; Lawton, Michael A; Long, Chunlin; Di, Rong

    2013-01-01

    The limited symptom relief and side effects of current Alzheimer's disease (AD) medications warrant urgent discovery and study of new anti-AD agents. The "cholinergic hypothesis" of AD prompts us to search for plant-derived acetylcholineesterase (AChE) inhibitors such as galanthamine that has been licensed in Europe for AD treatment. We used the unique amyloid β-expressing transgenic C. elegans CL4176, which exhibits paralysis when human Aβ1-42 is induced, to study two natural benzylphenethylamine alkaloids isolated from Lycoris radiata (L' Her.) Herb, galanthamine and haemanthidine, and their synthetic derivatives 1,2-Di-O-acetyllycorine and 1-O-acetyllycorine for their anti-paralysis effects. Our data indicate that these Lycoris compounds effectively delay the paralysis of CL4176 worms upon temperature up-shift, and prolong the lives of these transgenic worms. Lycoris compounds were shown to significantly inhibit the gene expression of ace-1 and ace-2. Additionally, the Lycoris compounds may modulate inflammatory and stress-related gene expressions to combat the Aβ-toxicity in C. elegans.

  10. Serum amyloid P component inhibits influenza A virus infections: in vitro and in vivo studies

    DEFF Research Database (Denmark)

    Horvath, A; Andersen, I; Junker, K

    2001-01-01

    . These studies were extended to comprise five mouse-adapted influenza A strains, two swine influenza A strains, a mink influenza A virus, a ferret influenza A reassortant virus, a influenza B virus and a parainfluenza 3 virus. The HA activity of all these viruses was inhibited by SAP. Western blotting showed...

  11. Superoxide dismutase 1 and tgSOD1 mouse spinal cord seed fibrils, suggesting a propagative cell death mechanism in amyotrophic lateral sclerosis.

    Directory of Open Access Journals (Sweden)

    Ruth Chia

    2010-05-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a neurodegenerative disease that specifically affects motor neurons and leads to a progressive and ultimately fatal loss of function, resulting in death typically within 3 to 5 years of diagnosis. The disease starts with a focal centre of weakness, such as one limb, and appears to spread to other parts of the body. Mutations in superoxide dismutase 1 (SOD1 are known to cause disease and it is generally accepted they lead to pathology not by loss of enzymatic activity but by gain of some unknown toxic function(s. Although different mutations lead to varying tendencies of SOD1 to aggregate, we suggest abnormal proteins share a common misfolding pathway that leads to the formation of amyloid fibrils.Here we demonstrate that misfolding of superoxide dismutase 1 leads to the formation of amyloid fibrils associated with seeding activity, which can accelerate the formation of new fibrils in an autocatalytic cascade. The time limiting event is nucleation to form a stable protein "seed" before a rapid linear polymerisation results in amyloid fibrils analogous to other protein misfolding disorders. This phenomenon was not confined to fibrils of recombinant protein as here we show, for the first time, that spinal cord homogenates obtained from a transgenic mouse model that overexpresses mutant human superoxide dismutase 1 (the TgSOD1(G93A mouse also contain amyloid seeds that accelerate the formation of new fibrils in both wildtype and mutant SOD1 protein in vitro.These findings provide new insights into ALS disease mechanism and in particular a mechanism that could account for the spread of pathology throughout the nervous system. This model of disease spread, which has analogies to other protein misfolding disorders such as prion disease, also suggests it may be possible to design assays for therapeutics that can inhibit fibril propagation and hence, possibly, disease progression.

  12. The effect of limited proteolysis by different proteases on the formation of whey protein fibrils.

    Science.gov (United States)

    Gao, Yu-Zhe; Xu, Hong-Hua; Ju, Ting-Ting; Zhao, Xin-Huai

    2013-01-01

    Four proteases: trypsin, protease A, pepsin, and protease M were selected to modify whey protein concentrate (WPC) at a low degree of hydrolysis (0.1, 0.2, and 0.3%) before adjusting to pH 2.0 and heating at 90°C to gain insight into the influence of proteolysis on fibril formation. The kinetics of fibril formation were performed on native and modified WPC using the fluorescent dye thioflavin T in conjunction with transmission electron microscopy and far-UV circular dichroism spectroscopy for the morphological and secondary structural analyses. The change in surface hydrophobicity and content of free sulfhydryl groups were also observed during the formation of fibrils for the native and modified WPC. The content of aggregation and thioflavin T kinetic data indicated that the ability of fibril formation was apparently different for WPC modified by the 4 proteases. Whey protein concentrate modified by trypsin aggregated more during heating and the fibril formation rate was faster than that of the native WPC. Whey protein concentrate modified by the other proteases showed slower aggregation with worse amyloid fibril morphology. Compared with the native WPC, the structure of WPC changed differently after being modified by proteases. The state of α-helix structure for modified WPC played the most important role in the formation of fibrils. Under the mild conditions used in this work, the α-helix structure of WPC modified by trypsin caused little destruction and resulted in fibrils with good morphology; the content of α-helices for WPC modified by other proteases decreased to 36.19 to 50.94%; thus, fibril formation was inhibited. In addition, it was beneficial for the modified WPC to form fibrils such that the surface hydrophobicity increased and the content of free sulfhydryl groups slightly decreased during heating. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  13. Modulation of human IAPP fibrillation: cosolutes, crowders and chaperones.

    Science.gov (United States)

    Gao, Mimi; Estel, Kathrin; Seeliger, Janine; Friedrich, Ralf P; Dogan, Susanne; Wanker, Erich E; Winter, Roland; Ebbinghaus, Simon

    2015-04-07

    The cellular environment determines the structure and function of proteins. Marginal changes of the environment can severely affect the energy landscape of protein folding. However, despite the important role of chaperones on protein folding, less is known about chaperonal modulation of protein aggregation and fibrillation considering different classes of chaperones. We find that the pharmacological chaperone O4, the chemical chaperone proline as well as the protein chaperone serum amyloid P component (SAP) are inhibitors of the type 2 diabetes mellitus-related aggregation process of islet amyloid polypeptide (IAPP). By applying biophysical methods such as thioflavin T fluorescence spectroscopy, fluorescence anisotropy, total reflection Fourier-transform infrared spectroscopy, circular dichroism spectroscopy and atomic force microscopy we analyse and compare their inhibition mechanism. We demonstrate that the fibrillation reaction of human IAPP is strongly inhibited by formation of globular, amorphous assemblies by both, the pharmacological and the protein chaperones. We studied the inhibition mechanism under cell-like conditions by using the artificial crowding agents Ficoll 70 and sucrose. Under such conditions the suppressive effect of proline was decreased, whereas the pharmacological chaperone remains active.

  14. Fenofibrate inhibits atrial metabolic remodelling in atrial fibrillation through PPAR-α/sirtuin 1/PGC-1α pathway.

    Science.gov (United States)

    Liu, Guang-Zhong; Hou, Ting-Ting; Yuan, Yue; Hang, Peng-Zhou; Zhao, Jing-Jing; Sun, Li; Zhao, Guan-Qi; Zhao, Jing; Dong, Jing-Mei; Wang, Xiao-Bing; Shi, Hang; Liu, Yong-Wu; Zhou, Jing-Hua; Dong, Zeng-Xiang; Liu, Yang; Zhan, Cheng-Chuang; Li, Yue; Li, Wei-Min

    2016-03-01

    Atrial metabolic remodelling is critical for the process of atrial fibrillation (AF). The PPAR-α/sirtuin 1 /PPAR co-activator α (PGC-1α) pathway plays an important role in maintaining energy metabolism. However, the effect of the PPAR-α agonist fenofibrate on AF is unclear. Therefore, the aim of this study was to determine the effect of fenofibrate on atrial metabolic remodelling in AF and explore its possible mechanisms of action. The expression of metabolic proteins was examined in the left atria of AF patients. Thirty-two rabbits were divided into sham, AF (pacing with 600 beats·min(-1) for 1 week), fenofibrate treated (pretreated with fenofibrate before pacing) and fenofibrate alone treated (for 2 weeks) groups. HL-1 cells were subjected to rapid pacing in the presence or absence of fenofibrate, the PPAR-α antagonist GW6471 or sirtuin 1-specific inhibitor EX527. Metabolic factors, circulating biochemical metabolites, atrial electrophysiology, adenine nucleotide levels and accumulation of glycogen and lipid droplets were assessed. The PPAR-α/sirtuin 1/PGC-1α pathway was significantly inhibited in AF patients and in the rabbit/HL-1 cell models, resulting in a reduction of key downstream metabolic factors; this effect was significantly restored by fenofibrate. Fenofibrate prevented the alterations in circulating biochemical metabolites, reduced the level of adenine nucleotides and accumulation of glycogen and lipid droplets, reversed the shortened atrial effective refractory period and increased risk of AF. Fenofibrate inhibited atrial metabolic remodelling in AF by regulating the PPAR-α/sirtuin 1/PGC-1α pathway. The present study may provide a novel therapeutic strategy for AF. © 2016 The British Pharmacological Society.

  15. The effect of resveratrol on beta amyloid-induced memory impairment involves inhibition of phosphodiesterase-4 related signaling.

    Science.gov (United States)

    Wang, Gang; Chen, Ling; Pan, Xiaoyu; Chen, Jiechun; Wang, Liqun; Wang, Weijie; Cheng, Ruochuan; Wu, Fan; Feng, Xiaoqing; Yu, Yingcong; Zhang, Han-Ting; O'Donnell, James M; Xu, Ying

    2016-04-05

    Resveratrol, a natural polyphenol found in red wine, has wide spectrum of pharmacological properties including antioxidative and antiaging activities. Beta amyloid peptides (Aβ) are known to involve cognitive impairment, neuroinflammatory and apoptotic processes in Alzheimer's disease (AD). Activation of cAMP and/or cGMP activities can improve memory performance and decrease the neuroinflammation and apoptosis. However, it remains unknown whether the memory enhancing effect of resveratrol on AD associated cognitive disorders is related to the inhibition of phosphodiesterase 4 (PDE4) subtypes and subsequent increases in intracellular cAMP and/or cGMP activities. This study investigated the effect of resveratrol on Aβ1-42-induced cognitive impairment and the participation of PDE4 subtypes related cAMP or cGMP signaling. Mice microinfused with Aβ1-42 into bilateral CA1 subregions displayed learning and memory impairment, as evidenced by reduced memory acquisition and retrieval in the water maze and retention in the passive avoidance tasks; it was also significant that neuroinflammatory and pro-apoptotic factors were increased in Aβ1-42-treated mice. Aβ1-42-treated mice also increased in PDE4A, 4B and 4D expression, and decreased in PKA level. However, PKA inhibitor H89, but not PKG inhibitor KT5823, prevented resveratrol's effects on these parameters. Resveratrol also reversed Aβ1-42-induced decreases in phosphorylated cAMP response-element binding protein (pCREB), brain derived neurotrophic factor (BDNF) and anti-apoptotic factor BCl-2 expression, which were reversed by H89. These findings suggest that resveratrol reversing Aβ-induced learning and memory disorder may involve the regulation of neuronal inflammation and apoptosis via PDE4 subtypes related cAMP-CREB-BDNF signaling.

  16. Inhibiting amyloid β-protein assembly: Size-activity relationships among grape seed-derived polyphenols.

    Science.gov (United States)

    Hayden, Eric Y; Yamin, Ghiam; Beroukhim, Shiela; Chen, Benson; Kibalchenko, Mikhail; Jiang, Lin; Ho, Lap; Wang, Jun; Pasinetti, Giulio M; Teplow, David B

    2015-10-01

    Epidemiological evidence that red wine consumption negatively correlates with risk of Alzheimer's disease has led to experimental studies demonstrating that grape seed extracts inhibit the aggregation and oligomerization of Aβ in vitro and ameliorate neuropathology and behavioral deficits in a mouse model of Alzheimer's disease. The active agent in the extracts is a mixed population of polyphenolic compounds. To evaluate the relative potency of each of these compounds, HPLC was used to fractionate the mixture into monomers, dimers, and oligomers. Each fraction was analyzed for its effect on Aβ conformational dynamics (circular dichroism), oligomerization (zero-length photochemical cross-linking), aggregation kinetics (Thioflavin T fluorescence), and morphology (electron microscopy). The relative activities of each fraction were determined on the basis of molar concentration (mol/L) or mass concentration (g/L). When molar concentration, the number concentration of each polyphenolic compound, was considered, the oligomer fraction was the most potent inhibitor of Aβ oligomerization and aggregation. However, when mass concentration, the number concentration of phenolic groups, was considered, monomers were the most potent inhibitors. To understand these ostensibly contradictory results, a model of polyphenol:Aβ complexation was developed. This model, which was found to be consistent with published X-ray crystallographic studies, offers an explanation for the effects of functional group polyvalency on inhibitor activity. Our data emphasize the importance of an in-depth understanding of the mechanism(s) underlying 'concentration dependence' in inhibitor systems involving polyfunctional agents. © 2015 International Society for Neurochemistry.

  17. Rescue of amyloid-Beta-induced inhibition of nicotinic acetylcholine receptors by a peptide homologous to the nicotine binding domain of the alpha 7 subtype.

    Directory of Open Access Journals (Sweden)

    Arthur A Nery

    Full Text Available Alzheimer's disease (AD is characterized by brain accumulation of the neurotoxic amyloid-β peptide (Aβ and by loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs. Recent evidence indicates that memory loss and cognitive decline in AD correlate better with the amount of soluble Aβ than with the extent of amyloid plaque deposits in affected brains. Inhibition of nAChRs by soluble Aβ40 is suggested to contribute to early cholinergic dysfunction in AD. Using phage display screening, we have previously identified a heptapeptide, termed IQ, homologous to most nAChR subtypes, binding with nanomolar affinity to soluble Aβ40 and blocking Aβ-induced inhibition of carbamylcholine-induced currents in PC12 cells expressing α7 nAChRs. Using alanine scanning mutagenesis and whole-cell current recording, we have now defined the amino acids in IQ essential for reversal of Aβ40 inhibition of carbamylcholine-induced responses in PC12 cells, mediated by α7 subtypes and other endogenously expressed nAChRs. We further investigated the effects of soluble Aβ, IQ and analogues of IQ on α3β4 nAChRs recombinantly expressed in HEK293 cells. Results show that nanomolar concentrations of soluble Aβ40 potently inhibit the function of α3β4 nAChRs, and that subsequent addition of IQ or its analogues does not reverse this effect. However, co-application of IQ makes the inhibition of α3β4 nAChRs by Aβ40 reversible. These findings indicate that Aβ40 inhibits different subtypes of nAChRs by interacting with specific receptor domains homologous to the IQ peptide, suggesting that IQ may be a lead for novel drugs to block the inhibition of cholinergic function in AD.

  18. Interaction of the amyloid β peptide with sodium dodecyl sulfate as a membrane-mimicking detergent.

    NARCIS (Netherlands)

    Hashemi, Shabestari M.; Meeuwenoord, N.J.; Filippov, D.V.; Huber, M.I.

    2016-01-01

    The amyloid β (A β) peptide is important in the context of Alzheimer's disease, since it is one of the major components of the fibrils that constitute amyloid plaques. Agents that can influence fibril formation are important, and of those, membrane mimics are particularly relevant, because the

  19. The Role of Functional Amyloids in Multicellular Growth and Development of Gram-Positive Bacteria

    NARCIS (Netherlands)

    Dragoš, A.; Kovács, Á.T.; Claessen, D.

    2017-01-01

    Amyloid fibrils play pivotal roles in all domains of life. In bacteria, these fibrillar structures are often part of an extracellular matrix that surrounds the producing organism and thereby provides protection to harsh environmental conditions. Here, we discuss the role of amyloid fibrils in the

  20. Amyloid beta dimers/trimers potently induce cofilin-actin rods that are inhibited by maintaining cofilin-phosphorylation

    Directory of Open Access Journals (Sweden)

    Podlisny Marcia

    2011-01-01

    Full Text Available Abstract Background Previously we reported 1 μM synthetic human amyloid beta1-42 oligomers induced cofilin dephosphorylation (activation and formation of cofilin-actin rods within rat hippocampal neurons primarily localized to the dentate gyrus. Results Here we demonstrate that a gel filtration fraction of 7PA2 cell-secreted SDS-stable human Aβ dimers and trimers (Aβd/t induces maximal neuronal rod response at ~250 pM. This is 4,000-fold more active than traditionally prepared human Aβ oligomers, which contain SDS-stable trimers and tetramers, but are devoid of dimers. When incubated under tyrosine oxidizing conditions, synthetic human but not rodent Aβ1-42, the latter lacking tyrosine, acquires a marked increase (620 fold for EC50 in rod-inducing activity. Gel filtration of this preparation yielded two fractions containing SDS-stable dimers, trimers and tetramers. One, eluting at a similar volume to 7PA2 Aβd/t, had maximum activity at ~5 nM, whereas the other, eluting at the void volume (high-n state, lacked rod inducing activity at the same concentration. Fractions from 7PA2 medium containing Aβ monomers are not active, suggesting oxidized SDS-stable Aβ1-42 dimers in a low-n state are the most active rod-inducing species. Aβd/t-induced rods are predominantly localized to the dentate gyrus and mossy fiber tract, reach significance over controls within 2 h of treatment, and are reversible, disappearing by 24 h after Aβd/t washout. Overexpression of cofilin phosphatases increase rod formation when expressed alone and exacerbate rod formation when coupled with Aβd/t, whereas overexpression of a cofilin kinase inhibits Aβd/t-induced rod formation. Conclusions Together these data support a mechanism by which Aβd/t alters the actin cytoskeleton via effects on cofilin in neurons critical to learning and memory.

  1. Long-term treatment of thalidomide ameliorates amyloid-like pathology through inhibition of β-secretase in a mouse model of Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Ping He

    Full Text Available Thalidomide is a tumor necrosis factor alpha (TNFα inhibitor which has been found to have abilities against tumor growth, angiogenesis and inflammation. Recently, it has been applied in clinic for the treatment of multiple myeloma as well as some inflammatory diseases. However, whether thalidomide has any therapeutic effects on neurodegenerative disorders, i.e. Alzheimer's disease (AD is not clear. AD is characterized by excessive amount of amyloid β peptides (Aβ, which results in a significant release of inflammatory factors, including TNFα in the brain. Studies have shown that inhibition of TNFα reduces amyloid-associated pathology, prevents neuron loss and improves cognition. Our recent report showed that genetic inhibition of TNFα/TNF receptor signal transduction down-regulates β amyloid cleavage enzyme 1 (BACE1 activity, reduces Aβ generation and improves learning and memory deficits. However, the mechanism of thalidomide involving in the mitigation of AD neuropathological features remains unclear. Here, we chronically administrated thalidomide on human APPswedish mutation transgenic (APP23 mice from 9 months old (an onset of Aβ deposits and early stage of AD-like changes to 12 months old. We found that, in addition of dramatic decrease in the activation of both astrocytes and microglia, thalidomide significantly reduces Aβ load and plaque formation. Furthermore, we found a significant decrease in BACE1 level and activity with long-term thalidomide application. Interestingly, these findings cannot be observed in the brains of 12-month-old APP23 mice with short-term treatment of thalidomide (3 days. These results suggest that chronic thalidomide administration is an alternative approach for AD prevention and therapeutics.

  2. Collapsed state of polyglutamic acid results in amyloid spherulite formation

    Science.gov (United States)

    Stehli, Daniel; Mulaj, Mentor; Miti, Tatiana; Traina, Joshua; Foley, Joseph; Muschol, Martin

    2015-01-01

    Self-assembly of proteins and peptides into amyloid fibrils involves multiple distinct intermediates and late-stage fibrillar polymorphs. Understanding the conditions and mechanisms that promote the formation of one type of intermediate and polymorph over the other represents a fundamental challenge. Answers to this question are also of immediate biomedical relevance since different amyloid aggregate species have been shown to have distinct pathogenic potencies. One amyloid polymorph that has received comparatively little attention are amyloid spherulites. Here we report that self-assembly of the intrinsically disordered polymer poly(L-glutamic) acid (PLE) can generate amyloid spherulites. We characterize spherulite growth kinetics, as well as the morphological, optical and tinctorial features of this amyloid polymorph previously unreported for PLE. We find that PLE spherulites share both tinctorial and structural characteristics with their amyloid fibril counterparts. Differences in PLE's molecular weight, polydispersity or chemistry could not explain the selective propensity toward either fibril or spherulite formation. Instead, we provide evidence that PLE polymers can exist in either a collapsed globule or an extended random coil conformation. The collapsed globule consistently produces spherulites while the extended coil assembles into disordered fibril bundles. This results suggests that these 2 PLE conformers directly affect the morphology of the resulting macroscopic amyloid assembly. PMID:28232889

  3. Whole body amyloid deposition imaging by 123I-SAP scintigraphy

    NARCIS (Netherlands)

    van Rheenen, Ronald; Glaudemans, Andor; Hazenberg, Bouke

    2011-01-01

    Amyloidosis is the name of a group of diseases characterized by extracellular deposition of amyloid fibrils. Deposition of amyloid can be localized or systemic. The 123I-SAP-scan can be used to image extent and distribution of amyloid deposition in patients with systemic AA, AL and ATTR amyloidosis.

  4. Trifluoroethanol modulates α-synuclein amyloid-like aggregate formation, stability and dissolution

    DEFF Research Database (Denmark)

    Di Carlo, Maria Giovanna; Vetri, Valeria; Buscarino, Gianpiero

    2016-01-01

    The conversion of proteins into amyloid fibrils and other amyloid-like aggregates is closely connected to the onset of a series of age-related pathologies. Upon changes in environmental conditions, amyloid-like aggregates may also undergo disassembly into oligomeric aggregates, the latter being r...

  5. Amyloid β oligomers induce interleukin-1β production in primary microglia in a cathepsin B- and reactive oxygen species-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Taneo, Jun; Adachi, Takumi [Department of Animal Development and Physiology, Kyoto University, Yoshida-Konoe, Sakyo, Kyoto 606-8501 (Japan); Yoshida, Aiko; Takayasu, Kunio [Responses to Environmental Signals and Stresses, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe, Sakyo, Kyoto, Kyoto 606-8501 (Japan); Takahara, Kazuhiko, E-mail: ktakahar@zoo.zool.kyoto-u.ac.jp [Department of Animal Development and Physiology, Kyoto University, Yoshida-Konoe, Sakyo, Kyoto 606-8501 (Japan); Japan Science and Technology Agency, Core Research for Evolutional Science and Technology (CREST), Tokyo 102-0081 (Japan); Inaba, Kayo [Department of Animal Development and Physiology, Kyoto University, Yoshida-Konoe, Sakyo, Kyoto 606-8501 (Japan); Japan Science and Technology Agency, Core Research for Evolutional Science and Technology (CREST), Tokyo 102-0081 (Japan)

    2015-03-13

    Amyloid β (Aβ) peptide, a causative agent of Alzheimer's disease, forms two types of aggregates: oligomers and fibrils. These aggregates induce inflammatory responses, such as interleukin-1β (IL-1β) production by microglia, which are macrophage-like cells located in the brain. In this study, we examined the effect of the two forms of Aβ aggregates on IL-1β production in mouse primary microglia. We prepared Aβ oligomer and fibril from Aβ (1–42) peptide in vitro. We analyzed the characteristics of these oligomers and fibrils by electrophoresis and atomic force microscopy. Interestingly, Aβ oligomers but not Aβ monomers or fibrils induced robust IL-1β production in the presence of lipopolysaccharide. Moreover, Aβ oligomers induced endo/phagolysosome rupture, which released cathepsin B into the cytoplasm. Aβ oligomer-induced IL-1β production was inhibited not only by the cathepsin B inhibitor CA-074-Me but also by the reactive oxygen species (ROS) inhibitor N-acetylcysteine. Random chemical crosslinking abolished the ability of the oligomers to induce IL-1β. Thus, multimerization and fibrillization causes Aβ oligomers to lose the ability to induce IL-1β. These results indicate that Aβ oligomers, but not fibrils, induce IL-1β production in primary microglia in a cathepsin B- and ROS-dependent manner. - Highlights: • We prepared amyloid β (Aβ) fibrils with minimum contamination of Aβ oligomers. • Primary microglia (MG) produced IL-1β in response to Aβ oligomers, but not fibrils. • Only Aβ oligomers induced leakage of cathepsin B from endo/phagolysosomes. • IL-1β production in response to Aβ oligomers depended on both cathepsin B and ROS. • Crosslinking reduced the ability of the Aβ oligomers to induce IL-1β from MG.

  6. Aloe arborescens Extract Protects IMR-32 Cells against Alzheimer Amyloid Beta Peptide via Inhibition of Radical Peroxide Production.

    Science.gov (United States)

    Clementi, Maria Elisabetta; Tringali, Giuseppe; Triggiani, Doriana; Giardina, Bruno

    2015-11-01

    Aloe arborescens is commonly used as a pharmaceutical ingredient for its effect in burn treatment and ability to increase skin wound healing properties. Besides, it is well known to have beneficial phytotherapeutic, anticancer, and radio-protective properties. In this study, we first provided evidence that A. arborescens extract protects IMR32, a neuroblastoma human cellular line, from toxicity induced by beta amyloid, the peptide responsible for Alzheimer's disease. In particular, pretreatment with A. arborescens maintains an elevated cell viability and exerts a protective effect on mitochondrial functionality, as evidenced by oxygen consumption experiments. The protective mechanism exerted by A. arborescens seems be related to lowering of oxidative potential of the cells, as demonstrated by the ROS measurement compared with the results obtained in the presence of amyloid beta (1-42) peptide alone. Based on these preliminary observations we suggest that use ofA. arborescens extract could be developed as agents for the management of AD.

  7. Acetylcholineestarase-Inhibiting Alkaloids from Lycoris radiata Delay Paralysis of Amyloid Beta-Expressing Transgenic C. elegans CL4176

    OpenAIRE

    Xin, Lijuan; Yamujala, Ritupriya; Wang, Yuehu; Wang, Huan; Wu, Wen-Hsuan; Lawton, Michael A.; Long, Chunlin; Di, Rong

    2013-01-01

    The limited symptom relief and side effects of current Alzheimer's disease (AD) medications warrant urgent discovery and study of new anti-AD agents. The "cholinergic hypothesis" of AD prompts us to search for plant-derived acetylcholineesterase (AChE) inhibitors such as galanthamine that has been licensed in Europe for AD treatment. We used the unique amyloid β-expressing transgenic C. elegans CL4176, which exhibits paralysis when human Aβ1-42 is induced, to study two natural benzylphenethyl...

  8. Calpain inhibition prevents pacing-induced cellular remodeling in a HL-1 myocyte model for atrial fibrillation

    NARCIS (Netherlands)

    Brundel, BJJM; Kampinga, HH; Henning, RH

    2004-01-01

    Objective: Atrial fibrillation (AF) is a progressive disease. Previously, clinical and animal experimental studies in AF revealed a variety of myocyte remodeling processes including L-type Ca(2+) channel reduction and structural changes, which finally result in electrical remodeling and contractile

  9. Termination of Vernakalant-Resistant Atrial Fibrillation by Inhibition of Small-Conductance Ca2+-Activated K+ Channels in Pigs

    DEFF Research Database (Denmark)

    Diness, Jonas Goldin; Skibsbye, Lasse; Simó-Vicens, Rafel

    2017-01-01

    Background Evidence has emerged that small-conductance Ca2+-activated K+ (SK) channels constitute a new target for treatment of atrial fibrillation (AF). SK channels are predominantly expressed in the atria as compared with the ventricles. Various marketed antiarrhythmic drugs are limited......-resistant porcine model of AF. These results suggest SK channel blockers as potentially interesting anti-AF drugs....

  10. Pitx2-microRNA pathway that delimits sinoatrial node development and inhibits predisposition to atrial fibrillation.

    Science.gov (United States)

    Wang, Jun; Bai, Yan; Li, Na; Ye, Wenduo; Zhang, Min; Greene, Stephanie B; Tao, Ye; Chen, Yiping; Wehrens, Xander H T; Martin, James F

    2014-06-24

    The molecular mechanisms underlying atrial fibrillation, the most common sustained cardiac arrhythmia, remain poorly understood. Genome-wide association studies uncovered a major atrial fibrillation susceptibility locus on human chromosome 4q25 in close proximity to the paired-like homeodomain transcription factor 2 (Pitx2) homeobox gene. Pitx2, a target of the left-sided Nodal signaling pathway that initiates early in development, represses the sinoatrial node program and pacemaker activity on the left side. To address the mechanisms underlying this repressive activity, we hypothesized that Pitx2 regulates microRNAs (miRs) to repress the sinoatrial node genetic program. MiRs are small noncoding RNAs that regulate gene expression posttranscriptionally. Using an integrated genomic approach, we discovered that Pitx2 positively regulates miR-17-92 and miR-106b-25. Intracardiac electrical stimulation revealed that both miR-17-92 and miR-106b-25 deficient mice exhibit pacing-induced atrial fibrillation. Furthermore electrocardiogram telemetry revealed that mice with miR-17-92 cardiac-specific inactivation develop prolonged PR intervals whereas mice with miR-17-92 cardiac-specific inactivation and miR-106b-25 heterozygosity develop sinoatrial node dysfunction. Both arrhythmias are risk factors for atrial fibrillation in humans. Importantly, miR-17-92 and miR-106b-25 directly repress genes, such as Shox2 and Tbx3, that are required for sinoatrial node development. Together, to our knowledge, these findings provide the first genetic evidence for an miR loss-of-function that increases atrial fibrillation susceptibility.

  11. NMR reveals two-step association of Congo Red to amyloid ß in low-molecular-weight aggregates

    DEFF Research Database (Denmark)

    Pedersen, Marie Ø; Mikkelsen, Katrine; Behrens, Manja Annette

    2010-01-01

    Aggregation of the Amyloid ß peptide into amyloid fibrils is closely related to development of Alzheimer's disease. Many small aromatic compounds have been found to act as inhibitors of fibril formation, and have inspired the search for new drug candidates. However, the detailed mechanisms...

  12. Amyloid-β oligomers are sequestered by both intracellular and extracellular chaperones.

    Science.gov (United States)

    Narayan, Priyanka; Meehan, Sarah; Carver, John A; Wilson, Mark R; Dobson, Christopher M; Klenerman, David

    2012-11-20

    The aberrant aggregation of the amyloid-β peptide into β-sheet rich, fibrillar structures proceeds via a heterogeneous ensemble of oligomeric intermediates that have been associated with neurotoxicity in Alzheimer's disease (AD). Of particular interest in this context are the mechanisms by which molecular chaperones, part of the primary biological defenses against protein misfolding, influence Aβ aggregation. We have used single-molecule fluorescence techniques to compare the interactions between distinct aggregation states (monomers, oligomers, and amyloid fibrils) of the AD-associated amyloid-β(1-40) peptide, and two molecular chaperones, both of which are upregulated in the brains of patients with AD and have been found colocalized with Aβ in senile plaques. One of the chaperones, αB-crystallin, is primarily found inside cells, while the other, clusterin, is predominantly located in the extracellular environment. We find that both chaperones bind to misfolded oligomeric species and form long-lived complexes, thereby preventing both their further growth into fibrils and their dissociation. From these studies, we conclude that these chaperones have a common mechanism of action based on sequestering Aβ oligomers. This conclusion suggests that these chaperones, both of which are ATP-independent, are able to inhibit potentially pathogenic Aβ oligomer-associated processes whether they occur in the extracellular or intracellular environment.

  13. A mimotope peptide of Aβ42 fibril-specific antibodies with Aβ42 fibrillation inhibitory activity induces anti-Aβ42 conformer antibody response by a displayed form on an M13 phage in mice.

    Science.gov (United States)

    Tanaka, Koichi; Nishimura, Masaaki; Yamaguchi, Yuya; Hashiguchi, Shuhei; Takiguchi, Sho; Yamaguchi, Makoto; Tahara, Haruna; Gotanda, Takuma; Abe, Risa; Ito, Yuji; Sugimura, Kazuhisa

    2011-07-01

    In Alzheimer's disease (AD), amyloid-β (Aβ) peptides accumulate in the brain in different forms, including fibrils and oligomers. Recently, we established three distinct conformation-dependent human single-chain Fv (scFv) antibodies, including B6 scFv, which bound to Aβ42 fibril but not to soluble-form Aβ, inhibiting Aβ42 fibril formation. In this study, we determined the mimotopes of these antibodies and found a common mimotope sequence, B6-C15, using the Ph.D.-C7C phage library. The B6-C15 showed weak homology to the C-terminus of Aβ42 containing GXXXG dimerization motifs. We synthesized the peptide of B6-C15 fused with biotinylated TAT at the N-terminus (TAT-B6-C15) and characterized its biochemical features on an Aβ42-fibrillation reaction in vitro. We demonstrated that, first, TAT-B6-C15 inhibited Aβ42 fibril formation; secondly, TAT-B6-C15 bound to prefibril Aβ42 oligomers but not to monomers, trimers, tetramers, fibrils, or ultrasonicated fragments; thirdly, TAT-B6-C15 inhibited Aβ42-induced cytotoxicity against human SH-SY5Y neuroblastoma cells; and, fourthly, when mice were administered B6-C15-phages dissolved in phosphate-buffered saline, the anti-Aβ42 conformer IgG antibody response was induced. These results suggested that the B6-C15 peptide might provide unique opportunities to analyze the Aβ42 fibrillation pathway and develop a vaccine vehicle for Alzheimer's disease. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Insulin fibrillation: The influence and coordination of Zn2+

    DEFF Research Database (Denmark)

    Frankær, Christian Grundahl; Sønderby, Pernille; Bang, Maria Blanner

    2017-01-01

    Protein amyloid fibrillation is obtaining much focus because it is connected with amyloid-related human diseases such as Alzheimer's disease, diabetes mellitus type 2, or Parkinson's disease. The influence of metal ions on the fibrillation process and whether it is implemented in the amyloid...... Electron Microscopy and Thioflavin T fluorescence measurements. It is well-known that Zn2+ ions coordinate and stabilize the hexameric forms of insulin. However, this study is the first to show that zinc indeed binds to the insulin fibrils. Furthermore, zinc influences the kinetics and the morphology...... of the fibrils. It also shows that zinc coordinates to histidine residues in an environment, which is similar to the coordination seen in the insulin R6 hexamers, where three histidine residues and a chloride ion is coordinating the zinc....

  15. Architecture of polyglutamine-containing fibrils from time-resolved fluorescence decay.

    Science.gov (United States)

    Röthlein, Christoph; Miettinen, Markus S; Borwankar, Tejas; Bürger, Jörg; Mielke, Thorsten; Kumke, Michael U; Ignatova, Zoya

    2014-09-26

    The disease risk and age of onset of Huntington disease (HD) and nine other repeat disorders strongly depend on the expansion of CAG repeats encoding consecutive polyglutamines (polyQ) in the corresponding disease protein. PolyQ length-dependent misfolding and aggregation are the hallmarks of CAG pathologies. Despite intense effort, the overall structure of these aggregates remains poorly understood. Here, we used sensitive time-dependent fluorescent decay measurements to assess the architecture of mature fibrils of huntingtin (Htt) exon 1 implicated in HD pathology. Varying the position of the fluorescent labels in the Htt monomer with expanded 51Q (Htt51Q) and using structural models of putative fibril structures, we generated distance distributions between donors and acceptors covering all possible distances between the monomers or monomer dimensions within the polyQ amyloid fibril. Using Monte Carlo simulations, we systematically scanned all possible monomer conformations that fit the experimentally measured decay times. Monomers with four-stranded 51Q stretches organized into five-layered β-sheets with alternating N termini of the monomers perpendicular to the fibril axis gave the best fit to our data. Alternatively, the core structure of the polyQ fibrils might also be a zipper layer with antiparallel four-stranded stretches as this structure showed the next best fit. All other remaining arrangements are clearly excluded by the data. Furthermore, the assessed dimensions of the polyQ stretch of each monomer provide structural evidence for the observed polyQ length threshold in HD pathology. Our approach can be used to validate the effect of pharmacological substances that inhibit or alter amyloid growth and structure. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Phosphodiesterase 5 inhibition improves synaptic function, memory, and amyloid-beta load in an Alzheimer's disease mouse model.

    Science.gov (United States)

    Puzzo, Daniela; Staniszewski, Agnieszka; Deng, Shi Xian; Privitera, Lucia; Leznik, Elena; Liu, Shumin; Zhang, Hong; Feng, Yan; Palmeri, Agostino; Landry, Donald W; Arancio, Ottavio

    2009-06-24

    Memory loss, synaptic dysfunction, and accumulation of amyloid beta-peptides (A beta) are major hallmarks of Alzheimer's disease (AD). Downregulation of the nitric oxide/cGMP/cGMP-dependent protein kinase/c-AMP responsive element-binding protein (CREB) cascade has been linked to the synaptic deficits after A beta elevation. Here, we report that the phosphodiesterase 5 inhibitor (PDE5) sildenafil (Viagra), a molecule that enhances phosphorylation of CREB, a molecule involved in memory, through elevation of cGMP levels, is beneficial against the AD phenotype in a mouse model of amyloid deposition. We demonstrate that the inhibitor produces an immediate and long-lasting amelioration of synaptic function, CREB phosphorylation, and memory. This effect is also associated with a long-lasting reduction of A beta levels. Given that side effects of PDE5 inhibitors are widely known and do not preclude their administration to a senile population, these drugs have potential for the treatment of AD and other diseases associated with elevated A beta levels.

  17. Aggregation and fibrillation of bovine serum albumin

    DEFF Research Database (Denmark)

    Holm, NK; Jespersen, SK; Thomassen, LV

    2007-01-01

    The all-alpha helix multi-domain protein bovine serum albumin (BSA) aggregates at elevated temperatures. Here we show that these thermal aggregates have amyloid properties. They bind the fibril-specific dyes Thioflavin T and Congo Red, show elongated although somewhat worm-like morphology...

  18. The Role of Functional Amyloids in Multicellular Growth and Development of Gram-Positive Bacteria

    DEFF Research Database (Denmark)

    Dragoš, Anna; Kovács, Ákos T.; Claessen, Dennis

    2017-01-01

    Amyloid fibrils play pivotal roles in all domains of life. In bacteria, these fibrillar structures are often part of an extracellular matrix that surrounds the producing organism and thereby provides protection to harsh environmental conditions. Here, we discuss the role of amyloid fibrils...... in the two distant Gram-positive bacteria, Streptomyces coelicolor and Bacillus subtilis. We describe how amyloid fibrils contribute to a multitude of developmental processes in each of these systems, including multicellular growth and community development. Despite this variety of tasks, we know...

  19. Electrostatics promotes molecular crowding and selects the aggregation pathway in fibril-forming protein solutions

    International Nuclear Information System (INIS)

    Raccosta, S.; Martorana, V.; Manno, M.; Blanco, M.; Roberts, C.J.

    2016-01-01

    The role of intermolecular interaction in fibril-forming protein solutions and its relation with molecular conformation are crucial aspects for the control and inhibition of amyloid structures. Here, we study the fibril formation and the protein-protein interactions for two proteins at acidic ph, lysozyme and α-chymotrypsinogen. By using light scattering experiments and the Kirkwood-Buff integral approach, we show how concentration fluctuations are damped even at moderate protein concentrations by the dominant long-ranged electrostatic repulsion, which determines an effective crowded environment. In denaturing conditions, electrostatic repulsion keeps the monomeric solution in a thermodynamically metastable state, which is escaped through kinetically populated conformational sub-states. This explains how electrostatics acts as a gatekeeper in selecting a specific aggregation pathway.

  20. Electrostatics promotes molecular crowding and selects the aggregation pathway in fibril-forming protein solutions

    Science.gov (United States)

    Raccosta, S.; Blanco, M.; J. Roberts, C.; Martorana, V.; Manno, M.

    2016-05-01

    The role of intermolecular interaction in fibril-forming protein solutions and its relation with molecular conformation are crucial aspects for the control and inhibition of amyloid structures. Here, we study the fibril formation and the protein-protein interactions for two proteins at acidic p H, lysozyme and α -chymotrypsinogen. By using light scattering experiments and the Kirkwood-Buff integral approach, we show how concentration fluctuations are damped even at moderate protein concentrations by the dominant long-ranged electrostatic repulsion, which determines an effective crowded environment. In denaturing conditions, electrostatic repulsion keeps the monomeric solution in a thermodynamically metastable state, which is escaped through kinetically populated conformational sub-states. This explains how electrostatics acts as a gatekeeper in selecting a specific aggregation pathway.

  1. Alzheimer's Disease Brain-Derived Amyloid-{beta}-Mediated Inhibition of LTP In Vivo Is Prevented by Immunotargeting Cellular Prion Protein.

    LENUS (Irish Health Repository)

    Barry, Andrew E

    2011-05-18

    Synthetic amyloid-β protein (Aβ) oligomers bind with high affinity to cellular prion protein (PrP(C)), but the role of this interaction in mediating the disruption of synaptic plasticity by such soluble Aβ in vitro is controversial. Here we report that intracerebroventricular injection of Aβ-containing aqueous extracts of Alzheimer\\'s disease (AD) brain robustly inhibits long-term potentiation (LTP) without significantly affecting baseline excitatory synaptic transmission in the rat hippocampus in vivo. Moreover, the disruption of LTP was abrogated by immunodepletion of Aβ. Importantly, intracerebroventricular administration of antigen-binding antibody fragment D13, directed to a putative Aβ-binding site on PrP(C), prevented the inhibition of LTP by AD brain-derived Aβ. In contrast, R1, a Fab directed to the C terminus of PrP(C), a region not implicated in binding of Aβ, did not significantly affect the Aβ-mediated inhibition of LTP. These data support the pathophysiological significance of SDS-stable Aβ dimer and the role of PrP(C) in mediating synaptic plasticity disruption by soluble Aβ.

  2. Erroneous Magnet Positioning Leads to Failure of Inhibition of Inappropriate Shock during Fast Conducting Atrial Fibrillation Episodes.

    Science.gov (United States)

    Römers, Hans; VAN Dijk, Vincent; Balt, Jippe

    2017-06-01

    We present the case of a 75-year-old patient with a single-chamber St. Jude Medical internal cardioverter defibrillator (ICD; St. Jude Medical, St. Paul, MN, USA) for primary prevention, who was admitted with 39 inappropriate ICD shocks because of atrial fibrillation with rapid ventricular frequention, despite magnet placement. Review of the device manual and literature revealed that apart from different responses to magnet placement programmed for the various manufacturers, the type of magnet and the positioning can be of specific interest. In the case presented, the donut-shaped magnet should have been placed off-center instead of directly over the device. © 2017 Wiley Periodicals, Inc.

  3. Amyloid Structure and Assembly: Insights from Scanning Transmission Electron Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Goldsbury, C.; Wall, J.; Baxa, U.; Simon, M. N.; Steven, A. C.; Engel, A.; Aebi, U.; Muller, S. A.

    2011-01-01

    Amyloid fibrils are filamentous protein aggregates implicated in several common diseases such as Alzheimer's disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR).

  4. Engineered aggregation inhibitor fusion for production of highly amyloidogenic human islet amyloid polypeptide.

    Science.gov (United States)

    Mirecka, Ewa Agnieszka; Gremer, Lothar; Schiefer, Stephanie; Oesterhelt, Filipp; Stoldt, Matthias; Willbold, Dieter; Hoyer, Wolfgang

    2014-12-10

    Human islet amyloid polypeptide (IAPP) is the major component of pancreatic amyloid deposits in type 2 diabetes. The structural conversion of IAPP from a monomeric state into amyloid assemblies is the subject of intense research. Recombinant production of IAPP is, however, difficult due to its extreme aggregation propensity. Here we describe a novel strategy for expression of IAPP in Escherichia coli, based on an engineered protein tag, which sequesters IAPP monomers and prevents IAPP aggregation. The IAPP-binding protein HI18 was selected by phage display from a β-wrapin library. Fusion of HI18 to IAPP enabled the soluble expression of the construct. IAPP was cleaved from the fusion construct and purified to homogeneity with a yield of 3mg of isotopically labeled peptide per liter of culture. In the monomeric state, IAPP was largely disordered as evidenced by far-UV CD and liquid-state NMR spectroscopy but competent to form amyloid fibrils according to atomic force microscopy. These results demonstrate the ability of the engineered β-wrapin HI18 for shielding the hydrophobic sequence of IAPP during expression and purification. Fusion of aggregation-inhibiting β-wrapins is a suitable approach for the recombinant production of aggregation-prone proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Phosphate and HEPES buffers potently affect the fibrillation and oligomerization mechanism of Alzheimer's Aβ peptide

    International Nuclear Information System (INIS)

    Garvey, Megan; Tepper, Katharina; Haupt, Caroline; Knuepfer, Uwe; Klement, Karolin; Meinhardt, Jessica; Horn, Uwe; Balbach, Jochen; Faendrich, Marcus

    2011-01-01

    Highlights: → Sodium phosphate buffer accelerated Aβ(1-40) nucleation relative to HEPES. → Aβ(1-40) fibrils formed in the two buffers show only minor structural differences. → NMR revealed that Aβ(1-40) histidine residues mediate buffer dependent changes. -- Abstract: The oligomerization of Aβ peptide into amyloid fibrils is a hallmark of Alzheimer's disease. Due to its biological relevance, phosphate is the most commonly used buffer system for studying the formation of Aβ and other amyloid fibrils. Investigation into the characteristics and formation of amyloid fibrils frequently relies upon material formed in vitro, predominantly in phosphate buffers. Herein, we examine the effects on the fibrillation and oligomerization mechanism of Aβ peptide that occur due solely to the influence of phosphate buffer. We reveal that significant differences in amyloid fibrillation are observed due to fibrillation being initiated in phosphate or HEPES buffer (at physiological pH and temperature). Except for the differing buffer ions, all experimental parameters were kept constant. Fibril formation was assessed using fluorescently monitored kinetic studies, microscopy, X-ray fiber diffraction and infrared and nuclear magnetic resonance spectroscopies. Based on this set up, we herein reveal profound effects on the mechanism and speed of Aβ fibrillation. The three histidine residues at positions 6, 13 and 14 of Aβ(1-40) are instrumental in these mechanistic changes. We conclude that buffer plays a more significant role in fibril formation than has been generally acknowledged.

  6. The E1 copper binding domain of full-length amyloid precursor protein mitigates copper-induced growth inhibition in brain metastatic prostate cancer DU145 cells

    Energy Technology Data Exchange (ETDEWEB)

    Gough, Mallory, E-mail: m.gough1@lancaster.ac.uk; Blanthorn-Hazell, Sophee, E-mail: s.blanthorn-hazell@lancaster.ac.uk; Delury, Craig, E-mail: c.delury@lancaster.ac.uk; Parkin, Edward, E-mail: e.parkin@lancaster.ac.uk

    2014-10-31

    Highlights: • Copper levels are elevated in the tumour microenvironment. • APP mitigates copper-induced growth inhibition of DU145 prostate cancer (PCa) cells. • The APP intracellular domain is a prerequisite; soluble forms have no effect. • The E1 CuBD of APP is also a prerequisite. • APP copper binding potentially mitigates copper-induced PCa cell growth inhibition. - Abstract: Copper plays an important role in the aetiology and growth of tumours and levels of the metal are increased in the serum and tumour tissue of patients affected by a range of cancers including prostate cancer (PCa). The molecular mechanisms that enable cancer cells to proliferate in the presence of elevated copper levels are, therefore, of key importance in our understanding of tumour growth progression. In the current study, we have examined the role played by the amyloid precursor protein (APP) in mitigating copper-induced growth inhibition of the PCa cell line, DU145. A range of APP molecular constructs were stably over-expressed in DU145 cells and their effects on cell proliferation in the presence of copper were monitored. Our results show that endogenous APP expression was induced by sub-toxic copper concentrations in DU145 cells and over-expression of the wild-type protein was able to mitigate copper-induced growth inhibition via a mechanism involving the cytosolic and E1 copper binding domains of the full-length protein. APP likely represents one of a range of copper binding proteins that PCa cells employ in order to ensure efficient proliferation despite elevated concentrations of the metal within the tumour microenvironment. Targeting the expression of such proteins may contribute to therapeutic strategies for the treatment of cancers.

  7. Serum amyloid P component binds to influenza A virus haemagglutinin and inhibits the virus infection in vitro

    DEFF Research Database (Denmark)

    Andersen, Ove; Vilsgaard Ravn, K; Juul Sørensen, I

    1997-01-01

    that SAP can bind to influenza A virus and inhibit agglutination of erythrocytes mediated by the virus subtypes H1N1, H2N2 and H3N2. SAP also inhibits the production of haemagglutinin (HA) an the cytopathogenic effect of influenza A virus in MDCK cells. The binding of SAP to the virus requires...

  8. Differential effect of amyloid beta peptides on mitochondrial axonal trafficking depends on their state of aggregation and binding to the plasma membrane.

    Science.gov (United States)

    Zhang, Liang; Trushin, Sergey; Christensen, Trace A; Tripathi, Utkarsh; Hong, Courtney; Geroux, Rachel E; Howell, Kyle G; Poduslo, Joseph F; Trushina, Eugenia

    2018-02-26

    Inhibition of mitochondrial axonal trafficking by amyloid beta (Aβ) peptides has been implicated in early pathophysiology of Alzheimer's Disease (AD). Yet, it remains unclear whether the loss of motility inevitably induces the loss of mitochondrial function, and whether restoration of axonal trafficking represents a valid therapeutic target. Moreover, while some investigations identify Aβ oligomers as the culprit of trafficking inhibition, others propose that fibrils play the detrimental role. We have examined the effect of a panel of Aβ peptides with different mutations found in familial AD on mitochondrial motility in primary cortical mouse neurons. Peptides with higher propensity to aggregate inhibit mitochondrial trafficking to a greater extent with fibrils inducing the strongest inhibition. Binding of Aβ peptides to the plasma membrane was sufficient to induce trafficking inhibition where peptides with reduced plasma membrane binding and internalization had lesser effect on mitochondrial motility. We also found that Aβ peptide with Icelandic mutation A673T affects axonal trafficking of mitochondria but has very low rates of plasma membrane binding and internalization in neurons, which could explain its relatively low toxicity. Inhibition of mitochondrial dynamics caused by Aβ peptides or fibrils did not instantly affect mitochondrial bioenergetic and function. Our results support a mechanism where inhibition of axonal trafficking is initiated at the plasma membrane by soluble low molecular weight Aβ species and is exacerbated by fibrils. Since trafficking inhibition does not coincide with the loss of mitochondrial function, restoration of axonal transport could be beneficial at early stages of AD progression. However, strategies designed to block Aβ aggregation or fibril formation alone without ensuring the efficient clearance of soluble Aβ may not be sufficient to alleviate the trafficking phenotype. Copyright © 2018 The Authors. Published by

  9. Morphological Effects of CuO Nanostructures on Fibrillation of Human Serum Albumin.

    Science.gov (United States)

    Konar, Suraj; Sen, Shubhatam; Pathak, Amita

    2017-12-28

    The influence of different morphologies of nanostructures on amyloid fibrillation has been investigated by monitoring the fibrillation of human serum albumin (HSA) in the presence of rod-, sphere-, flower-, and star-shaped copper oxide (CuO) nanostructures. The different morphologies of CuO have been synthesized from an aqueous solution-based precipitation method using various organic acids, viz., acetic acid, citric acid, and tartaric acid. The fibrillation process of HSA has been examined using various biophysical techniques, e.g., Thioflavin T fluorescence, Congo red binding studies through UV spectroscopy, circular dichroism spectroscopy, and fluorescence microscopy. The monolayer protein coverage on the CuO nanostructures has been established through DLS studies, and the well-fitted Langmuir isotherm model has been used to interpret the differential adsorption behavior of HSA molecules on the CuO nanostructures. The nanostar-shaped CuO, by virtue of their higher specific surface area (94.45 m 2 g -1 ), presence of high indexed facets {211} and high positive surface charge potential (+16.2 mV at pH 7.0) was found to show the highest adsorption of the HSA monomers and thus was more competent to inhibit the formation of HSA fibrils compared to the other nanostructures of CuO.

  10. Triptolide Inhibited Cytotoxicity of Differentiated PC12 Cells Induced by Amyloid-Beta₂₅₋₃₅ via the Autophagy Pathway.

    Science.gov (United States)

    Xu, Pengjuan; Li, Zhigui; Wang, Hui; Zhang, Xiaochen; Yang, Zhuo

    2015-01-01

    Evidence shows that an abnormal deposition of amyloid beta-peptide25-35 (Aβ25-35) was the primary cause of the pathogenesis of Alzheimer's disease (AD). And the elimination of Aβ25-35 is considered an important target for the treatment of AD. Triptolide (TP), isolated from Tripterygium wilfordii Hook.f. (TWHF), has been shown to possess a broad spectrum of biological profiles, including neurotrophic and neuroprotective effects. In our study investigating the effect and potential mechanism of triptolide on cytotoxicity of differentiated rat pheochromocytoma cell line (the PC12 cell line is often used as a neuronal developmental model) induced by Amyloid-Beta25-35 (Aβ25-35), we used 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) assay, flow cytometry, Western blot, and acridine orange staining to detect whether triptolide could inhibit Aβ25-35-induced cell apoptosis. We focused on the potential role of the autophagy pathway in Aβ25-35-treated differentiated PC12 cells. Our experiments show that cell viability is significantly decreased, and the apoptosis increased in Aβ25-35-treated differentiated PC12 cells. Meanwhile, Aβ25-35 treatment increased the expression of microtubule-associated protein light chain 3 II (LC3 II), which indicates an activation of autophagy. However, triptolide could protect differentiated PC12 cells against Aβ25-35-induced cytotoxicity and attenuate Aβ25-35-induced differentiated PC12 cell apoptosis. Triptolide could also suppress the level of autophagy. In order to assess the effect of autophagy on the protective effects of triptolide in differentiated PC12 cells treated with Aβ25-35, we used 3-Methyladenine (3-MA, an autophagy inhibitor) and rapamycin (an autophagy activator). MTT assay showed that 3-MA elevated cell viability compared with the Aβ25-35-treated group and rapamycin inhibits the protection of triptolide. These results suggest that triptolide will repair the neurological damage in AD caused by

  11. Functional bacterial amyloid increases Pseudomonas biofilm hydrophobicity and stiffness

    DEFF Research Database (Denmark)

    Zeng, Guanghong; Vad, Brian S; Dueholm, Morten S

    2015-01-01

    The success of Pseudomonas species as opportunistic pathogens derives in great part from their ability to form stable biofilms that offer protection against chemical and mechanical attack. The extracellular matrix of biofilms contains numerous biomolecules, and it has recently been discovered...... that in Pseudomonas one of the components includes β-sheet rich amyloid fibrils (functional amyloid) produced by the fap operon. However, the role of the functional amyloid within the biofilm has not yet been investigated in detail. Here we investigate how the fap-based amyloid produced by Pseudomonas affects biofilm...... hydrophobicity and mechanical properties. Using atomic force microscopy imaging and force spectroscopy, we show that the amyloid renders individual cells more resistant to drying and alters their interactions with hydrophobic probes. Importantly, amyloid makes Pseudomonas more hydrophobic and increases biofilm...

  12. Inhibitions of late INa and CaMKII act synergistically to prevent ATX-II-induced atrial fibrillation in isolated rat right atria.

    Science.gov (United States)

    Liang, Faquan; Fan, Peidong; Jia, Jessie; Yang, Suya; Jiang, Zhan; Karpinski, Serge; Kornyeyev, Dmytro; Pagratis, Nikos; Belardinelli, Luiz; Yao, Lina

    2016-05-01

    Increases in late Na(+) current (late INa) and activation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII) are associated with atrial arrhythmias. CaMKII also phosphorylates Nav1.5, further increasing late INa. The combination of a CaMKII inhibitor with a late INa inhibitor may be superior to each compound alone to suppress atrial arrhythmias. Therefore, we investigated the effect of a CaMKII inhibitor in combination with a late INa inhibitor on anemone toxin II (ATX-II, a late INa enhancer)-induced atrial arrhythmias. Rat right atrial tissue was isolated and preincubated with either the CaMKII inhibitor autocamtide-2-related inhibitory peptide (AIP), the late INa inhibitor GS458967, or both, and then exposed to ATX-II. ATX-II increased diastolic tension and caused fibrillation of isolated right atrial tissue. AIP (0.3μmol/L) and 0.1μmol/L GS458967 alone inhibited ATX-II-induced arrhythmias by 20±3% (mean±SEM, n=14) and 34±5% (n=13), respectively, whereas the two compounds in combination inhibited arrhythmias by 81±4% (n=10, pATX-induced increase of diastolic tension. Consistent with the mechanical and electrical data, 0.3μmol/L AIP and 0.1μmol/L GS458967 each inhibited ATX-II-induced CaMKII phosphorylation by 23±3% and 32±4%, whereas the combination of both compounds inhibited CaMKII phosphorylation completely. The effects of an enhanced late INa to induce arrhythmic activity and activation of CaMKII in atria are attenuated synergistically by inhibitors of late INa and CaMKII. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Carbon nanospecies affecting amyloid formation

    Czech Academy of Sciences Publication Activity Database

    Holubová, Monika; Konefal, Rafal; Morávková, Zuzana; Zhigunov, Alexander; Svoboda, Jan; Pop-Georgievski, Ognen; Hromádková, Jiřina; Groborz, Ondřej; Štěpánek, Petr; Hrubý, Martin

    2017-01-01

    Roč. 7, č. 85 (2017), s. 53887-53898 ISSN 2046-2069 R&D Projects: GA MŠk(CZ) LM2015064; GA MZd(CZ) NV16-30544A; GA ČR(CZ) GA16-03156S; GA TA ČR(CZ) TE01020118; GA MŠk(CZ) LO1507 Grant - others:OPPK(XE) CZ.2.16/3.1.00/21545 Program:OPPK Institutional support: RVO:61389013 Keywords : amyloid fibril * nanodiamond * fullerene Subject RIV: CD - Macromolecular Chemistry OBOR OECD: Polymer science Impact factor: 3.108, year: 2016

  14. SAS-Based Studies of Protein Fibrillation

    DEFF Research Database (Denmark)

    Marasini, Carlotta; Vestergaard, Bente

    2017-01-01

    .Small angle scattering is an optimal method for structural studies of the fibrillation process in order to further the knowledge of the associated diseases. The recorded scattering data include the scattering contribution of all the species in solution and must be decomposed to enable structural modeling...... of the individual components involved during the fibrillation, notably without physical separation of the species. In this chapter we explain how to optimize a small angle scattering analysis of the fibrillation process and the basic principles behind analysis of the data. We include several practical tips......Protein fibrillation is associated with a number of fatal amyloid diseases (e.g. Alzheimer's and Parkinson's diseases). From a structural point of view, the aggregation process starts from an ensemble of native states that convert into transiently formed oligomers, higher order assemblies...

  15. Atrial Fibrillation: Diagnosis

    Science.gov (United States)

    ... of this page please turn JavaScript on. Feature: Atrial Fibrillation Atrial Fibrillation: Diagnosis Past Issues / Winter 2015 Table of Contents ... of your body's cells and organs. Read More "Atrial Fibrillation" Articles Atrial Fibrillation / Who Is at Risk for ...

  16. The effect of tachykinin neuropeptides on amyloid β aggregation.

    Science.gov (United States)

    Flashner, Efrat; Raviv, Uri; Friedler, Assaf

    2011-04-01

    A hallmark of Alzheimer's disease is production of amyloid β peptides resulting from aberrant cleavage of the amyloid precursor protein. Amyloid β assembles into fibrils under physiological conditions, through formation of neurotoxic intermediate oligomers. Tachykinin peptides are known to affect amyloid β neurotoxicity in cells. To understand the mechanism of this effect, we studied how tachykinins affect Aβ(1-40) aggregation in vitro. Fibrils grown in the presence of tachykinins exhibited reduced thioflavin T (ThT) fluorescence, while their morphology, observed in transmission electron microscopy (TEM), did not alter. Cross linking studies revealed that the distribution of low molecular weight species was not affected by tachykinins. Our results suggest that there may be a specific interaction between tachykinins and Aβ(1-40) that allows them to co-assemble. This effect may explain the reduction of Aβ(1-40) neurotoxicity in cells treated with tachykinins. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Amyloid structure exhibits polymorphism on multiple length scales in human brain tissue

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jiliang; Costantino, Isabel; Venugopalan, Nagarajan; Fischetti, Robert F.; Hyman, Bradley; Frosch, Matthew; Gomez-Isla, Teresa; Makowski, Lee

    2016-09-15

    Although aggregation of Aβ amyloid fibrils into plaques in the brain is a hallmark of Alzheimer's Disease (AD), the correlation between amyloid burden and severity of symptoms is weak. One possible reason is that amyloid fibrils are structurally polymorphic and different polymorphs may contribute differentially to disease. However, the occurrence and distribution of amyloid polymorphisms in human brain is poorly documented. Here we seek to fill this knowledge gap by using X-ray microdiffraction of histological sections of human tissue to map the abundance, orientation and structural heterogeneities of amyloid within individual plaques; among proximal plaques and in subjects with distinct clinical histories. A 5 µ x-ray beam was used to generate diffraction data with each pattern arising from a scattering volume of only ~ 450 µ3 , making possible collection of dozens to hundreds of diffraction patterns from a single amyloid plaque. X-ray scattering from these samples exhibited all the properties expected for scattering from amyloid. Amyloid distribution was mapped using the intensity of its signature 4.7 Å reflection which also provided information on the orientation of amyloid fibrils across plaques. Margins of plaques exhibited a greater degree of orientation than cores and orientation around blood vessels frequently appeared tangential. Variation in the structure of Aβ fibrils is reflected in the shape of the 4.7 Å peak which usually appears as a doublet. Variations in this peak correspond to differences between the structure of amyloid within cores of plaques and at their periphery. Examination of tissue from a mismatch case - an individual with high plaque burden but no overt signs of dementia at time of death - revealed a diversity of structure and spatial distribution of amyloid that is distinct from typical AD cases. We demonstrate the existence of structural polymorphisms among amyloid within and among plaques of a single individual and suggest

  18. Surface Mediated Self-Assembly of Amyloid Peptides

    Science.gov (United States)

    Fakhraai, Zahra

    2015-03-01

    Amyloid fibrils have been considered as causative agents in many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, type II diabetes and amyloidosis. Amyloid fibrils form when proteins or peptides misfold into one dimensional crystals of stacked beta-sheets. In solution, amyloid fibrils form through a nucleation and growth mechanism. The rate limiting nucleation step requires a critical concentration much larger than those measured in physiological conditions. As such the exact origins of the seeds or oligomers that result in the formation of fully mature fibrils in the body remain topic intense studies. It has been suggested that surfaces and interfaces can enhance the fibrillization rate. However, studies of the mechanism and kinetics of the surface-mediated fibrillization are technologically challenging due to the small size of the oligomer and protofibril species. Using smart sample preparation technique to dry the samples after various incubation times we are able to study the kinetics of fibril formation both in solution and in the vicinity of various surfaces using high-resolution atomic force microscopy. These studies elucidate the role of surfaces in catalyzing amyloid peptide formation through a nucleation-free process. The nucleation free self-assembly is rapid and requires much smaller concentrations of peptides or proteins. We show that this process resembles diffusion limited aggregation and is governed by the peptide adhesion rate, two -dimensional diffusion of the peptides on the surface, and preferential interactions between the peptides. These studies suggest an alternative pathway for amyloid formation may exist, which could lead to new criteria for disease prevention and alternative therapies. Research was partially supported by a seed grant from the National Institute of Aging of the National Institutes of Health (NIH) under Award Number P30AG010124 (PI: John Trojanowski) and the University of Pennsylvania.

  19. Some commonly used brominated flame retardants cause Ca2+-ATPase inhibition, beta-amyloid peptide release and apoptosis in SH-SY5Y neuronal cells.

    Directory of Open Access Journals (Sweden)

    Fawaz Al-Mousa

    Full Text Available Brominated flame retardants (BFRs are chemicals commonly used to reduce the flammability of consumer products and are considered pollutants since they have become widely dispersed throughout the environment and have also been shown to bio-accumulate within animals and man. This study investigated the cytotoxicity of some of the most commonly used groups of BFRs on SH-SY5Y human neuroblastoma cells. The results showed that of the BFRs tested, hexabromocyclododecane (HBCD, tetrabromobisphenol-A (TBBPA and decabromodiphenyl ether (DBPE, all are cytotoxic at low micromolar concentrations (LC(50 being 2.7 ± 0.7 µM, 15 ± 4 µM and 28 ± 7 µM, respectively. They induced cell death, at least in part, by apoptosis through activation of caspases. They also increased intracellular [Ca(2+] levels and reactive-oxygen-species within these neuronal cells. Furthermore, these BFRs also caused rapid depolarization of the mitochondria and cytochrome c release in these neuronal cells. Elevated intracellular [Ca(2+] levels appear to occur through a mechanism involving microsomal Ca(2+-ATPase inhibition and this maybe responsible for Ca(2+-induced mitochondrial dysfunction. In addition, µM levels of these BFRs caused β-amyloid peptide (Aβ-42 processing and release from these cells with a few hours of exposure. These results therefore shows that these pollutants are both neurotoxic and amyloidogenic in-vitro.

  20. Inhibition of β-amyloid1-42 internalization attenuates neuronal death by stabilizing the endosomal-lysosomal system in rat cortical cultured neurons.

    Science.gov (United States)

    Song, M S; Baker, G B; Todd, K G; Kar, S

    2011-03-31

    A number of recent studies have indicated that accumulation of β amyloid (Aβ) peptides within neurons is an early event which may trigger degeneration of neurons and subsequent development of Alzheimer's disease (AD) pathology. However, very little is known about the internalization and/or subcellular sites involved in trafficking of Aβ peptides into the neurons that are vulnerable in AD pathology. To address this issue we evaluated internalization of fluoroscein conjugated Aβ1-42 (FAβ1-42) and subsequent alteration of endosomal-lysosomal (EL) markers such as cathepsin D, Rab5 and Rab7 in rat cortical cultured neurons. It is evident from our results that internalization of FAβ1-42, which occurred in a dose- and time-dependent manner, triggered degeneration of neurons along with increased levels and/or altered distribution of cathepsin D, Rab5 and Rab7. Our results further revealed that FAβ1-42 internalization was attenuated by phenylarsine oxide (a general inhibitor of endocytosis) and sucrose (an inhibitor of clathrin-mediated endocytosis) but not by antagonists of N-methyl-d-aspartate (NMDA) glutamate receptors. Additionally, inhibition of FAβ1-42 endocytosis not only protected neurons against toxicity but also reversed the altered levels/distributions of EL markers. These results, taken together, suggest that internalization of exogenous Aβ1-42, which is partly mediated via a clathrin-dependent process, can lead to degeneration of neurons, possibly by activating the EL system. Inhibition of FAβ endocytosis attenuated toxicity, thus suggesting a potential strategy for preventing loss of neurons in AD pathology. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

  1. Hydrogen-Deuterium Exchange Profiles of Polyubiquitin Fibrils

    Directory of Open Access Journals (Sweden)

    Daichi Morimoto

    2018-02-01

    Full Text Available Ubiquitin and its polymeric forms are conjugated to intracellular proteins to regulate diverse intracellular processes. Intriguingly, polyubiquitin has also been identified as a component of pathological protein aggregates associated with Alzheimer’s disease and other neurodegenerative disorders. We recently found that polyubiquitin can form amyloid-like fibrils, and that these fibrillar aggregates can be degraded by macroautophagy. Although the structural properties appear to function in recognition of the fibrils, no structural information on polyubiquitin fibrils has been reported so far. Here, we identify the core of M1-linked diubiquitin fibrils from hydrogen-deuterium exchange experiments using solution nuclear magnetic resonance (NMR spectroscopy. Intriguingly, intrinsically flexible regions became highly solvent-protected in the fibril structure. These results indicate that polyubiquitin fibrils are formed by inter-molecular interactions between relatively flexible structural components, including the loops and edges of secondary structure elements.

  2. Fibril aggregates formed by a glatiramer-mimicking random copolymer of amino acids.

    Science.gov (United States)

    Lai, Jingjing; Fu, Wenxin; Zhu, Lin; Guo, Ruohai; Liang, Dehai; Li, Zhibo; Huang, Yanbin

    2014-06-24

    Amyloid formation is now considered a universal and intrinsic property of all proteins, irrespective of their sequences. Therefore, it is interesting to see whether random copolymers of amino acids can also form amyloid aggregates. Here we use a copolymer of 4 amino acids, mimicking the clinically used drug Glatiramer, and demonstrate that it does form amyloid-like fibrils in the aqueous solution despite its random sequence structure. The fibrillar aggregates show an alanine-rich β-sheet secondary structure, proving the high tolerance of amyloid aggregates to the sequence irregularity in poly(amino acid)s, and suggesting the potential application of random copolymers as amyloid materials.

  3. AURAMINE O AS POTENTIAL AMYLOID MARKER: FLUORESCENCE AND MOLECULAR DOCKING STUDIES

    Directory of Open Access Journals (Sweden)

    K. Vus

    2017-10-01

    Full Text Available The applicability of Auramine O to the detection and characterization of lysozyme and serum albumin amyloid fibrils has been assessed using the fluorimetric titration and molecular docking. The parameters of the dye binding to native and fibrillar proteins were estimated in terms of the Langmuir adsorption model. It was found that Auramine O displays the high affinity for amyloid fibrils, being of the same order of magnitude as that of the classical amyloid markers. The dye also showed greater fluorescence response to lysozyme fibrils and lower sensitivity to the native protein, than Thioflavin T. Furthermore, unlike Thioflavin T, Auramine O was able to detect the morphological differences between lysozyme and albumin fibrils due to the shifts in the position of the emission maxima of the fibril-incorporated fluorophore. The molecular docking studies revealed that Auramine O and Thioflavin T form the most stable complexes with the G54_L56/S60_W62 groove of lysozyme fibrils, running parallel to the fibril axis. The results obtained suggest the contribution of both hydrophobic and electrostatic interactions to the stabilization of the dye complexes with amyloid fibrils.

  4. Heat of supersaturation-limited amyloid burst directly monitored by isothermal titration calorimetry.

    Science.gov (United States)

    Ikenoue, Tatsuya; Lee, Young-Ho; Kardos, József; Yagi, Hisashi; Ikegami, Takahisa; Naiki, Hironobu; Goto, Yuji

    2014-05-06

    Amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. Although the structural features of amyloid fibrils have become increasingly clearer, knowledge on the thermodynamics of fibrillation is limited. Furthermore, protein aggregation is not a target of calorimetry, one of the most powerful approaches used to study proteins. Here, with β2-microglobulin, a protein responsible for dialysis-related amyloidosis, we show direct heat measurements of the formation of amyloid fibrils using isothermal titration calorimetry (ITC). The spontaneous fibrillation after a lag phase was accompanied by exothermic heat. The thermodynamic parameters of fibrillation obtained under various protein concentrations and temperatures were consistent with the main-chain dominated structural model of fibrils, in which overall packing was less than that of the native structures. We also characterized the thermodynamics of amorphous aggregation, enabling the comparison of protein folding, amyloid fibrillation, and amorphous aggregation. These results indicate that ITC will become a promising approach for clarifying comprehensively the thermodynamics of protein folding and misfolding.

  5. Amyloid-hydroxyapatite bone biomimetic composites.

    Science.gov (United States)

    Li, Chaoxu; Born, Anne-Kathrin; Schweizer, Thomas; Zenobi-Wong, Marcy; Cerruti, Marta; Mezzenga, Raffaele

    2014-05-28

    A "bottom up" strategy is proposed to synthesize high aspect ratio hydroxyapatite (and brushite) platelets, and combine them with amyloid fibrils into layered hybrid nanocomposites. Their hierarchical structure, despite the differences from natural bone, confers to the nanocomposites a density and elastic modulus matching those of cancellous bone. Evidence of good adhesion and spreading of human trabecular bone-derived pre-osteoblasts cells on these nanocomposites is provided. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Herpes simplex virus type I induces the accumulation of intracellular β-amyloid in autophagic compartments and the inhibition of the non-amyloidogenic pathway in human neuroblastoma cells.

    Science.gov (United States)

    Santana, Soraya; Recuero, Maria; Bullido, Maria Jesús; Valdivieso, Fernando; Aldudo, Jesus

    2012-02-01

    Mounting evidence suggests that herpes simplex virus type 1 (HSV-1) is involved in the pathogenesis of Alzheimer's disease (AD). Epidemiological analyses have shown that HSV-1 is a risk factor for AD in people with at least 1 type 4 allele of the apolipoprotein E gene. Recent studies have also suggested that HSV-1 contributes to the appearance of the biochemical anomalies characteristic of AD brains. In addition, autophagic activity appears to be reduced with aging, and the final stages of autophagy in neurodegenerative process appear to be impaired. The present work reports that HSV-1 provokes the strong intracellular accumulation of both the main species of β-amyloid (Aβ) in the autophagic compartments and that it is associated with a marked inhibition of Aβ secretion. Autophagosomes containing Aβ failed to fuse with lysosomes in HSV-1-infected cells, indicating the impaired degradation of Aβ localized in the autophagic vesicles. In addition, HSV-1 infection was associated with the inhibition of the nonamyloidogenic pathway of amyloid precursor protein (APP) processing without significantly affecting the activity of the secretases involved in the amyloidogenic pathway. Taken together, these data suggest that HSV-1 infection modulates autophagy and amyloid precursor protein processing, contributing to the accumulation of Aβ characteristic of AD. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Synthesis and evaluation of a (18)F-curcumin derivate for β-amyloid plaque imaging.

    Science.gov (United States)

    Rokka, Johanna; Snellman, Anniina; Zona, Cristiano; La Ferla, Barbara; Nicotra, Francesco; Salmona, Mario; Forloni, Gianluigi; Haaparanta-Solin, Merja; Rinne, Juha O; Solin, Olof

    2014-05-01

    Curcumin is a neuroprotective compound that inhibits the formation of amyloid oligomers and fibrils and binds to β-amyloid plaques in Alzheimer's disease (AD). We aimed to synthesize an (18)F-labeled curcumin derivate ([(18)F]4) and to characterize its positron emission tomography (PET) tracer-binding properties to β-amyloid plaques in a transgenic APP23 mouse model of AD. We utilized facile one-pot synthesis of [(18)F]4 using nucleophilic (18)F-fluorination and click chemistry. Binding of [(18)F]4 to β-amyloid plaques in the transgenic APP23 mouse brain cryosections was studied in vitro using heterologous competitive binding against PIB. [(18)F]4 uptake was studied ex vivo in rodents and in vivo using PET/computed tomography of transgenic APP23 and wild-type control mice. The radiochemical yield of [(18)F]4 was 21 ± 11%, the specific activity exceeded 1TBq/μmol, and the radiochemical purity exceeded 99.3% at the end of synthesis. In vitro studies of [(18)F]4 with the transgenic APP23 mouse revealed high β-amyloid plaque binding. In vivo and ex vivo studies demonstrated that [(18)F]4 has fast clearance from the blood, moderate metabolism but low blood-brain barrier (BBB) penetration. [(18)F]4 was synthesized in high yield and excellent quality. In vitro studies, metabolite profile, and fast clearance from the blood indicated a promising tracer for Aβ imaging. However, [(18)F]4 has low in vivo BBB penetration and thus further studies are needed to reveal the reason for this and to possibly overcome this issue. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. [Amyloid goiter].

    Science.gov (United States)

    Hrívó, A; Péter, I; Bánkúti, B; Péley, G; Baska, F; Besznyák, I

    1999-03-21

    Amyloid goitre is at an extremely rare occurrence. Authors review the origin of disease and its symptoms, diagnostic and therapeutic tools. The disease may be due to either primary or secondary systemic or local amyloidosis. Diagnosis may be made even before surgery on anamnestic data, on very rapid growth of thyroid glands, on diffuse appearance, on other symptoms of systemic amyloidosis, on findings of iconographic procedures and on detection of amyloid in aspirates. Final diagnosis is based on histology. Surgical therapy is aiming at avoidance of the existing and the threatening consequences of expanding mass. The outcome is independent from thyroid surgery, it is related to other manifestations of amyloidosis. Concerning with the present case the chronic superior vena cava syndrome and chylous pleural effusion as first described symptoms and asymptomatic hyperthyroxinaemia is emphasised. Neither other organ involvement, nor primary amyloidogenous molecula was found during the 18 months follow up, so patient has secondary and localised amyloidosis.

  9. Amyloid Beta Peptide Folding in Reverse Micelles.

    Science.gov (United States)

    Eskici, Gözde; Axelsen, Paul H

    2017-07-19

    Previously published experimental studies have suggested that when the 40-residue amyloid beta peptide is encapsulated in a reverse micelle, it folds into a structure that may nucleate amyloid fibril formation (Yeung, P. S.-W.; Axelsen, P. H. J. Am. Chem. Soc. 2012, 134, 6061 ). The factors that induce the formation of this structure have now been identified in a multi-microsecond simulation of the same reverse micelle system that was studied experimentally. Key features of the polypeptide-micelle interaction include the anchoring of a hydrophobic residue cluster into gaps in the reverse micelle surface, the formation of a beta turn at the anchor point that brings N- and C-terminal segments of the polypeptide into proximity, high ionic strength that promotes intramolecular hydrogen bond formation, and deformation of the reverse micelle surface to facilitate interactions with the surface along the entire length of the polypeptide. Together, these features cause the simulation-derived vibrational spectrum to red shift in a manner that reproduces the red-shift previously reported experimentally. On the basis of these findings, a new mechanism is proposed whereby membranes nucleate fibril formation and facilitate the in-register alignment of polypeptide strands that is characteristic of amyloid fibrils.

  10. Polymorphism, metastable species and interconversion: the many states of glucagon fibrils

    DEFF Research Database (Denmark)

    Ghodke, Shirin D; Jensen, Grethe Vestergaard; Svane, Anna Sigrid P.

    2014-01-01

    The natively unfolded peptide hormone glucagon forms fibrillar structures with amyloid properties. Here, we summarize past advances in glucagon fibrillation and combine them with recent new unpublished data to provide some more general conclusions on how glucagon fibrillation adapts to different...

  11. Polymorphic structures of Alzheimer's β-amyloid globulomers.

    Directory of Open Access Journals (Sweden)

    Xiang Yu

    Full Text Available BACKGROUND: Misfolding and self-assembly of Amyloid-β (Aβ peptides into amyloid fibrils is pathologically linked to the development of Alzheimer's disease. Polymorphic Aβ structures derived from monomers to intermediate oligomers, protofilaments, and mature fibrils have been often observed in solution. Some aggregates are on-pathway species to amyloid fibrils, while the others are off-pathway species that do not evolve into amyloid fibrils. Both on-pathway and off-pathway species could be biologically relevant species. But, the lack of atomic-level structural information for these Aβ species leads to the difficulty in the understanding of their biological roles in amyloid toxicity and amyloid formation. METHODS AND FINDINGS: Here, we model a series of molecular structures of Aβ globulomers assembled by monomer and dimer building blocks using our peptide-packing program and explicit-solvent molecular dynamics (MD simulations. Structural and energetic analysis shows that although Aβ globulomers could adopt different energetically favorable but structurally heterogeneous conformations in a rugged energy landscape, they are still preferentially organized by dynamic dimeric subunits with a hydrophobic core formed by the C-terminal residues independence of initial peptide packing and organization. Such structural organizations offer high structural stability by maximizing peptide-peptide association and optimizing peptide-water solvation. Moreover, curved surface, compact size, and less populated β-structure in Aβ globulomers make them difficult to convert into other high-order Aβ aggregates and fibrils with dominant β-structure, suggesting that they are likely to be off-pathway species to amyloid fibrils. These Aβ globulomers are compatible with experimental data in overall size, subunit organization, and molecular weight from AFM images and H/D amide exchange NMR. CONCLUSIONS: Our computationally modeled Aβ globulomers provide useful

  12. Inhibition of IK,ACh current may contribute to clinical efficacy of class I and class III antiarrhythmic drugs in patients with atrial fibrillation.

    Science.gov (United States)

    Voigt, Niels; Rozmaritsa, Nadiia; Trausch, Anne; Zimniak, Thomasz; Christ, Torsten; Wettwer, Erich; Matschke, Klaus; Dobrev, Dobromir; Ravens, Ursula

    2010-03-01

    Inward rectifier potassium currents I(K1) and acetylcholine activated I(K,ACh) are implicated in atrial fibrillation (AF) pathophysiology. In chronic AF (cAF), I(K,ACh) develops a receptor-independent, constitutively active component that together with increased I(K1) is considered to support maintenance of AF. Here, we tested whether class I (propafenone, flecainide) and class III (dofetilide, AVE0118) antiarrhythmic drugs inhibit atrial I(K1) and I(K,ACh) in patients with and without cAF. I(K1) and I(K,ACh) were measured with voltage clamp technique in atrial myocytes from 58 sinus rhythm (SR) and 35 cAF patients. The M-receptor agonist carbachol (CCh; 2 microM) was employed to activate I(K,ACh). In SR, basal current was not affected by either drug indicating no effect of these compounds on I(K1). In contrast, all tested drugs inhibited CCh-activated I(K,ACh) in a concentration-dependent manner. In cAF, basal current was confirmed to be larger than in SR (at -80 mV, -15.2 +/- 1.2 pA/pF, n = 88/35 vs. -6.5 +/- 0.4 pA/pF, n = 194/58 [myocytes/patients]; P ACh) was smaller (-4.1 +/- 0.5 pA/pF vs. -9.5 +/- 0.6 pA/pF; P ACh) contributes to increased basal current, which was reduced by flecainide and AVE0118 only. This may be due to inhibition of constitutively active I(K,ACh) channels. In cAF, all tested drugs reduced CCh-activated I(K,ACh). We conclude that in cAF, flecainide and AVE0118 reduce receptor-independent, constitutively active I(K,ACh), suggesting that they may block I(K,ACh) channels, whereas propafenone and dofetilide likely inhibit M-receptors. The efficacy of flecainide to terminate AF may in part result from blockade of I(K,ACh).

  13. Design and synthesis of curcumin analogues for in vivo fluorescence imaging and inhibiting copper-induced cross-linking of amyloid beta species in Alzheimer's disease.

    Science.gov (United States)

    Zhang, Xueli; Tian, Yanli; Li, Zeng; Tian, Xiaoyu; Sun, Hongbin; Liu, Hong; Moore, Anna; Ran, Chongzhao

    2013-11-06

    In this article, we first designed and synthesized curcumin-based near-infrared (NIR) fluorescence imaging probes for detecting both soluble and insoluble amyloid beta (Aβ) species and then an inhibitor that could attenuate cross-linking of Aβ induced by copper. According to our previous results and the possible structural stereohindrance compatibility of the Aβ peptide and the hydrophobic/hydrophilic property of the Aβ13-20 (HHQKLVFF) fragment, NIR imaging probe CRANAD-58 was designed and synthesized. As expected CRANAD-58 showed significant fluorescence property changes upon mixing with both soluble and insoluble Aβ species in vitro. In vivo NIR imaging revealed that CRANAD-58 was capable of differentiating transgenic and wild-type mice as young as 4 months old, the age that lacks apparently visible Aβ plaques and Aβ is likely in its soluble forms. According to our limited studies on the interaction mechanism between CRANAD-58 and Aβ, we also designed CRANAD-17 to attenuate the cross-linking of Aβ42 induced by copper. It is well-known that the coordination of copper with imidazoles on Histidine-13 and 14 (H13, H14) of Aβ peptides could initialize covalent cross-linking of Aβ. In CRANAD-17, a curcumin scaffold was used as an anchoring moiety to usher the designed compound to the vicinity of H13 and H14 of Aβ, and imidazole rings were incorporated to compete with H13/H14 for copper binding. The results of SDS-PAGE gel and Western blot indicated that CRANAD-17 was capable of inhibiting Aβ42 cross-linking induced by copper. This raises a potential for CRANAD-17 to be considered for AD therapy.

  14. Fish β-parvalbumin acquires allergenic properties by amyloid assembly.

    Science.gov (United States)

    Martínez, Javier; Sánchez, Rosa; Castellanos, Milagros; Fernández-Escamilla, Ana M; Vázquez-Cortés, Sonia; Fernández-Rivas, Montserrat; Gasset, María

    2015-01-01

    Amyloids are highly cross-β-sheet-rich aggregated states that confer protease resistance, membrane activity and multivalence properties to proteins, all essential features for the undesired preservation of food proteins transiting the gastrointestinal tract and causing type I allergy. Amyloid propensity of β-parvalbumin, the major fish allergen, was theoretically analysed and assayed under gastrointestinal-relevant conditions using the binding of thioflavin T, the formation of sodium dodecyl sulphate- (SDS-) resistant aggregates, circular dichroism spectroscopy and atomic force microscopy fibril imaging. Impact of amyloid aggregates on allergenicity was assessed with dot blot. Sequences of β-parvalbumin from species with commercial value contain several adhesive hexapeptides capable of driving amyloid formation. Using Atlantic cod β-parvalbumin (rGad m 1) displaying high IgE cross-reactivity, we found that formation of amyloid fibres under simulated gastrointestinal conditions accounts for the resistance to acid and neutral proteases, for the presence of membrane active species under gastrointestinal relevant conditions and for the IgE-recognition in the sera of allergic patients. Incorporation of the anti-amyloid compound epigallocatechin gallate prevents rGad m 1 fibrillation, facilitates its protease digestion and impairs its recognition by IgE. the formation of amyloid by rGad m 1 explains its degradation resistance, its facilitated passage across the intestinal epithelial barrier and its epitope architecture as allergen.

  15. Rivaroxaban in atrial fibrillation

    Directory of Open Access Journals (Sweden)

    Giorgi MA

    2012-08-01

    Full Text Available Mariano A Giorgi,1,2 Lucas San Miguel31Cardiology Service, Centro de Educación Médica e Investigaciones Clínicas “Norberto Quirno”, 2Department of Pharmacology, School of Medicine, Universidad Austral, 3Department of Cardiology and Cardiovascular Surgery, FLENI, Buenos Aires, ArgentinaAbstract: Warfarin is the traditional therapeutic option available to manage thromboembolic risk in atrial fibrillation. The hemorrhagic risk with warfarin depends mainly on the international normalized ratio (INR. Data from randomized controlled trials show that patients have a therapeutic INR (2.00–3.00 only 61%–68% of the time while taking warfarin, and this target is sometimes hard to establish. Many compounds have been developed in order to optimize the profile of oral anticoagulants. We focus on one of them, rivaroxaban, comparing it with novel alternatives, ie, dabigatran and apixaban. The indication for rivaroxaban in nonvalvular atrial fibrillation was evaluated in ROCKET-AF (Rivaroxaban-once daily, Oral, direct factor Xa inhibition Compared with vitamin K antagonism for prevention of stroke and Embolism Trial in Atrial Fibrillation. In this trial, rivaroxaban was associated with a 12% reduction in the incidence of the primary endpoint compared with warfarin (hazard ratio 0.88; 95% confidence interval [CI] 0.74–1.03; P < 0.001 for noninferiority and P = 0.12 for superiority. However, patients remained in the therapeutic range for INR only 55% of the time, which is less than that in RE-LY (the Randomized Evaluation of Long-Term Anticoagulation Therapy, 64% and in the ARISTOTLE trial (Apixaban for Reduction in Stroke and Other Thromboembolic Events in Atrial Fibrillation, 66%. This shorter time spent in the therapeutic range has been one of the main criticisms of the ROCKET-AF trial, but could actually reflect what happens in real life. In addition, rivaroxaban exhibits good pharmacokinetic and pharmacoeconomic properties. Novel anticoagulants

  16. Atrial fibrillation

    African Journals Online (AJOL)

    ABEOLUGBENGAS

    optimal. Keywords: Atrial fibrillation, thrombosis, CHADS2 Score, stroke risk, hypertensive heart disease, ... (3,4). In developing countries, AF is a growing public health problem due to the epidemiologic transition from communicable to non- communicable diseases. However in ... ECG laboratory and was interpreted by two.

  17. Atrial fibrillation

    DEFF Research Database (Denmark)

    Olesen, Morten S; Nielsen, Morten W; Haunsø, Stig

    2014-01-01

    Atrial fibrillation (AF) is the most common cardiac arrhythmia affecting 1-2% of the general population. A number of studies have demonstrated that AF, and in particular lone AF, has a substantial genetic component. Monogenic mutations in lone and familial AF, although rare, have been recognized...

  18. β-Hairpin of Islet Amyloid Polypeptide Bound to an Aggregation Inhibitor

    Science.gov (United States)

    Mirecka, Ewa A.; Feuerstein, Sophie; Gremer, Lothar; Schröder, Gunnar F.; Stoldt, Matthias; Willbold, Dieter; Hoyer, Wolfgang

    2016-01-01

    In type 2 diabetes, the formation of islet amyloid consisting of islet amyloid polypeptide (IAPP) is associated with reduction in β-cell mass and contributes to the failure of islet cell transplantation. Rational design of inhibitors of IAPP amyloid formation has therapeutic potential, but is hampered by the lack of structural information on inhibitor complexes of the conformationally flexible, aggregation-prone IAPP. Here we characterize a β-hairpin conformation of IAPP in complex with the engineered binding protein β-wrapin HI18. The β-strands correspond to two amyloidogenic motifs, 12-LANFLVH-18 and 22-NFGAILS-28, which are connected by a turn established around Ser-20. Besides backbone hydrogen bonding, the IAPP:HI18 interaction surface is dominated by non-polar contacts involving hydrophobic side chains of the IAPP β-strands. Apart from monomers, HI18 binds oligomers and fibrils and inhibits IAPP aggregation and toxicity at low substoichiometric concentrations. The IAPP β-hairpin can serve as a molecular recognition motif enabling control of IAPP aggregation. PMID:27641459

  19. Identification of a Unique Amyloid Sequence in AA Amyloidosis of a Pig Associated With Streptococcus Suis Infection.

    Science.gov (United States)

    Kamiie, J; Sugahara, G; Yoshimoto, S; Aihara, N; Mineshige, T; Uetsuka, K; Shirota, K

    2017-01-01

    Here we report a pig with amyloid A (AA) amyloidosis associated with Streptococcus suis infection and identification of a unique amyloid sequence in the amyloid deposits in the tissue. Tissues from the 180-day-old underdeveloped pig contained foci of necrosis and suppurative inflammation associated with S. suis infection. Congo red stain, immunohistochemistry, and electron microscopy revealed intense AA deposition in the spleen and renal glomeruli. Mass spectrometric analysis of amyloid material extracted from the spleen showed serum AA 2 (SAA2) peptide as well as a unique peptide sequence previously reported in a pig with AA amyloidosis. The common detection of the unique amyloid sequence in the current and past cases of AA amyloidosis in pigs suggests that this amyloid sequence might play a key role in the development of porcine AA amyloidosis. An in vitro fibrillation assay demonstrated that the unique AA peptide formed typically rigid, long amyloid fibrils (10 nm wide) and the N-terminus peptide of SAA2 formed zigzagged, short fibers (7 nm wide). Moreover, the SAA2 peptide formed long, rigid amyloid fibrils in the presence of sonicated amyloid fibrils formed by the unique AA peptide. These findings indicate that the N-terminus of SAA2 as well as the AA peptide mediate the development of AA amyloidosis in pigs via cross-seeding polymerization.

  20. Raman optical activity study on insulin amyloid- and prefibril intermediate

    Czech Academy of Sciences Publication Activity Database

    Yamamoto, Shigeki; Watarai, H.

    2012-01-01

    Roč. 24, č. 2 (2012), s. 97-103 ISSN 0899-0042 Institutional research plan: CEZ:AV0Z40550506 Keywords : raman optical activity * amyloid * fibril * intermediate * insulin Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 1.718, year: 2012

  1. Mechanical Deformation Mechanisms and Properties of Prion Fibrils Probed by Atomistic Simulations

    Science.gov (United States)

    Choi, Bumjoon; Kim, Taehee; Ahn, Eue Soo; Lee, Sang Woo; Eom, Kilho

    2017-03-01

    Prion fibrils, which are a hallmark for neurodegenerative diseases, have recently been found to exhibit the structural diversity that governs disease pathology. Despite our recent finding concerning the role of the disease-specific structure of prion fibrils in determining their elastic properties, the mechanical deformation mechanisms and fracture properties of prion fibrils depending on their structures have not been fully characterized. In this work, we have studied the tensile deformation mechanisms of prion and non-prion amyloid fibrils by using steered molecular dynamics simulations. Our simulation results show that the elastic modulus of prion fibril, which is formed based on left-handed β-helical structure, is larger than that of non-prion fibril constructed based on right-handed β-helix. However, the mechanical toughness of prion fibril is found to be less than that of non-prion fibril, which indicates that infectious prion fibril is more fragile than non-infectious (non-prion) fibril. Our study sheds light on the role of the helical structure of amyloid fibrils, which is related to prion infectivity, in determining their mechanical deformation mechanisms and properties.

  2. How Glycosaminoglycans Promote Fibrillation of Salmon Calcitonin*

    Science.gov (United States)

    Malmos, Kirsten Gade; Bjerring, Morten; Jessen, Christian Moestrup; Nielsen, Erik Holm Toustrup; Poulsen, Ebbe T.; Christiansen, Gunna; Vosegaard, Thomas; Skrydstrup, Troels; Enghild, Jan J.; Pedersen, Jan Skov; Otzen, Daniel E.

    2016-01-01

    Glycosaminoglycans (GAGs) bind all known amyloid plaques and help store protein hormones in (acidic) granular vesicles, but the molecular mechanisms underlying these important effects are unclear. Here we investigate GAG interactions with the peptide hormone salmon calcitonin (sCT). GAGs induce fast sCT fibrillation at acidic pH and only bind monomeric sCT at acidic pH, inducing sCT helicity. Increasing GAG sulfation expands the pH range for binding. Heparin, the most highly sulfated GAG, binds sCT in the pH interval 3–7. Small angle x-ray scattering indicates that sCT monomers densely decorate and pack single heparin chains, possibly via hydrophobic patches on helical sCT. sCT fibrillates without GAGs, but heparin binding accelerates the process by decreasing the otherwise long fibrillation lag times at low pH and accelerates fibril growth rates at neutral pH. sCT·heparin complexes form β-sheet-rich heparin-covered fibrils. Solid-state NMR reveals that heparin does not alter the sCT fibrillary core around Lys11 but makes changes to Val8 on the exterior side of the β-strand, possibly through contacts to Lys18. Thus GAGs significantly modulate sCT fibrillation in a pH-dependent manner by interacting with both monomeric and aggregated sCT. PMID:27281819

  3. An in vitro screening assay based on synthetic prion protein peptides for identification of fibril-interfering compounds

    NARCIS (Netherlands)

    Boshuizen, R.S.; Langeveld, J.P.M.; Salmona, M.; Williams, A.; Meloen, R.H.; Langendijk, J.P.

    2004-01-01

    Transmissible spongiform encephalopathies are neurodegenerative diseases and are considered to be caused by malformed prion proteins accumulated into fibrillar structures that can then aggregate to form larger deposits or amyloid plaques. The identification of fibril-interfering compounds is of

  4. Molecular and Biological Compatibility with Host Alpha-Synuclein Influences Fibril Pathogenicity

    Directory of Open Access Journals (Sweden)

    Kelvin C. Luk

    2016-09-01

    Full Text Available The accumulation and propagation of misfolded α-synuclein (α-Syn is a central feature of Parkinson’s disease and other synucleinopathies. Molecular compatibility between a fibrillar seed and its native protein state is a major determinant of amyloid self-replication. We show that cross-seeded aggregation of human (Hu and mouse (Ms α-Syn is bidirectionally restricted. Although fibrils formed by Hu-Ms-α-Syn chimeric mutants can overcome this inhibition in cell-free systems, sequence homology poorly predicts their efficiency in inducing α-Syn pathology in primary neurons or after intracerebral injection into wild-type mice. Chimeric α-Syn fibrils demonstrate enhanced or reduced pathogenicities compared with wild-type Hu- or Ms-α-Syn fibrils. Furthermore, α-Syn mutants induced to polymerize by fibrillar seeds inherit the functional properties of their template, suggesting that transferable pathogenic and non-pathogenic states likely influence the initial engagement between exogenous α-Syn seeds and endogenous neuronal α-Syn. Thus, transmission of synucleinopathies is regulated by biological processes in addition to molecular compatibility.

  5. Adverse postresuscitation myocardial effects elicited by buffer-induced alkalemia ameliorated by NHE-1 inhibition in a rat model of ventricular fibrillation.

    Science.gov (United States)

    Lamoureux, Lorissa; Radhakrishnan, Jeejabai; Mason, Thomas G; Kraut, Jeffrey A; Gazmuri, Raúl J

    2016-11-01

    Major myocardial abnormalities occur during cardiac arrest and resuscitation including intracellular acidosis-partly caused by CO 2 accumulation-and activation of the Na + -H + exchanger isoform-1 (NHE-1). We hypothesized that a favorable interaction may result from NHE-1 inhibition during cardiac resuscitation followed by administration of a CO 2 -consuming buffer upon return of spontaneous circulation (ROSC). Ventricular fibrillation was electrically induced in 24 male rats and left untreated for 8 min followed by defibrillation after 8 min of cardiopulmonary resuscitation (CPR). Rats were randomized 1:1:1 to the NHE-1 inhibitor zoniporide or vehicle during CPR and disodium carbonate/sodium bicarbonate buffer or normal saline (30 ml/kg) after ROSC. Survival at 240 min declined from 100% with Zoniporide/Saline to 50% with Zoniporide/Buffer and 25% with Vehicle/Buffer (P = 0.004), explained by worsening postresuscitation myocardial dysfunction. Marked alkalemia occurred after buffer administration along with lactatemia that was maximal after Vehicle/Buffer, attenuated by Zoniporide/Buffer, and minimal with Zoniporide/Saline [13.3 ± 4.8 (SD), 9.2 ± 4.6, and 2.7 ± 1.0 mmol/l; P ≤ 0.001]. We attributed the intense postresuscitation lactatemia to enhanced glycolysis consequent to severe buffer-induced alkalemia transmitted intracellularly by an active NHE-1. We attributed the worsened postresuscitation myocardial dysfunction also to severe alkalemia intensifying Na + entry via NHE-1 with consequent Ca 2+ overload injuring mitochondria, evidenced by increased plasma cytochrome c Both buffer-induced effects were ameliorated by zoniporide. Accordingly, buffer-induced alkalemia after ROSC worsened myocardial function and survival, likely through enhancing NHE-1 activity. Zoniporide attenuated these effects and uncovered a complex postresuscitation acid-base physiology whereby blood pH drives NHE-1 activity and compromises mitochondrial function and integrity along

  6. Curcumin Binding to Beta Amyloid: A Computational Study.

    Science.gov (United States)

    Rao, Praveen P N; Mohamed, Tarek; Teckwani, Karan; Tin, Gary

    2015-10-01

    Curcumin, a chemical constituent present in the spice turmeric, is known to prevent the aggregation of amyloid peptide implicated in the pathophysiology of Alzheimer's disease. While curcumin is known to bind directly to various amyloid aggregates, no systematic investigations have been carried out to understand its ability to bind to the amyloid aggregates including oligomers and fibrils. In this study, we constructed computational models of (i) Aβ hexapeptide (16) KLVFFA(21) octamer steric-zipper β-sheet assembly and (ii) full-length Aβ fibril β-sheet assembly. Curcumin binding in these models was evaluated by molecular docking and molecular dynamics (MD) simulation studies. In both the models, curcumin was oriented in a linear extended conformation parallel to fiber axis and exhibited better stability in the Aβ hexapeptide (16) KLVFFA(21) octamer steric-zipper model (Ebinding  = -10.05 kcal/mol) compared to full-length Aβ fibril model (Ebinding  = -3.47 kcal/mol). Analysis of MD trajectories of curcumin bound to full-length Aβ fibril shows good stability with minimum Cα-atom RMSD shifts. Interestingly, curcumin binding led to marked fluctuations in the (14) HQKLVFFA(21) region that constitute the fibril spine with RMSF values ranging from 1.4 to 3.6 Å. These results show that curcumin binding to Aβ shifts the equilibrium in the aggregation pathway by promoting the formation of non-toxic aggregates. © 2015 John Wiley & Sons A/S.

  7. A continuum model for hierarchical fibril assembly

    Science.gov (United States)

    van Lith, B. S.; Muntean, A.; Storm, C.

    2014-06-01

    Most of the biological polymers that make up our cells and tissues are hierarchically structured. For biopolymers ranging from collagen, to actin, to fibrin and amyloid fibrils this hierarchy provides vitally important versatility. The structural hierarchy must be encoded in the self-assembly process, from the earliest stages onward, in order to produce the appropriate substructures. In this letter, we explore the kinetics of multistage self-assembly processes in a model system which allows comparison to bulk probes such as light scattering. We apply our model to recent turbidimetry data on the self-assembly of collagen fibrils. Our analysis suggests a connection between diffusion-limited aggregation kinetics and fibril growth, supported by slow, power-law growth at very long time scales.

  8. Atrial Fibrillation: Treatment

    Science.gov (United States)

    ... of this page please turn JavaScript on. Feature: Atrial Fibrillation Atrial Fibrillation: Treatment Past Issues / Winter 2015 Table of Contents Treatment for atrial fibrillation depends on how often you have symptoms, how ...

  9. Atrial fibrillation

    OpenAIRE

    Munger, Thomas M.; Wu, Li-Qun; Shen, Win K.

    2013-01-01

    Atrial fibrillation is the most common arrhythmia affecting patients today. Disease prevalence is increasing at an alarming rate worldwide, and is associated with often catastrophic and costly consequences, including heart failure, syncope, dementia, and stroke. Therapies including anticoagulants, anti-arrhythmic medications, devices, and non-pharmacologic procedures in the last 30 years have improved patients' functionality with the disease. Nonetheless, it remains imperative that further re...

  10. Difference in aggregation between functional and toxic amyloids studied by atomistic simulations

    Science.gov (United States)

    Carballo Pacheco, Martin; Ismail, Ahmed E.; Strodel, Birgit

    Amyloids are highly structured protein aggregates, normally associated with neurodegenerative diseases such as Alzheimer's disease. In recent years, a number of nontoxic amyloids with physiologically normal functions, called functional amyloids, have been found. It is known that soluble small oligomers are more toxic than large fibrils. Thus, we study with atomistic explicit-solvent molecular dynamics simulations the oligomer formation of the amyloid- β peptide Aβ25 - 35, associated with Alzheimer's disease, and two functional amyloid-forming tachykinin peptides: kassinin and neuromedin K. Our simulations show that monomeric peptides in extended conformations aggregate faster than those in collapsed hairpin-like conformations. In addition, we observe faster aggregation by functional amyloids than toxic amyloids, which could explain their lack of toxicity.

  11. Thermal Stability Threshold for Amyloid Formation in Light Chain Amyloidosis

    Directory of Open Access Journals (Sweden)

    Tanya L. Poshusta

    2013-11-01

    Full Text Available Light chain (AL amyloidosis is a devastating disease characterized by amyloid deposits formed by immunoglobulin light chains. Current available treatments involve conventional chemotherapy and autologous stem cell transplant. We have recently concluded a phase III trial comparing these two treatments. AL amyloidosis patients who achieve hematological complete response (CR do not necessarily achieve organ response regardless of the treatment they received. In order to investigate the possible correlation between amyloid formation kinetics and organ response, we selected AL amyloidosis patients from the trial with kidney involvement and CR after treatment. Six patients were selected and their monoclonal immunoglobulin light chains were characterized. The proteins showed differences in their stability and their kinetics of amyloid formation. A correlation was detected at pH 7.4, showing that less stable proteins are more likely to form amyloid fibrils. AL-T03 is too unstable to form amyloid fibrils at pH 7.4. This protein was found in the only patient in the study that had organ response, suggesting that partially folded species are required for amyloid formation to occur in AL amyloidosis.

  12. [Clinical Laboratory Test Using Proteomics: The Usefulness of Proteomic Techniques for Amyloid Typing].

    Science.gov (United States)

    Tasaki, Masayoshi; Obayashi, Konen; Ando, Yukio

    2015-08-01

    Amyloidosis is a heterogeneous group of disorders characterized by the deposition of amyloid fibrils. To diagnose amyloidosis, it is important to detect amyloid deposits and identify the amyloid precursor protein in specimens, such as tissues and serum. Mass spectrometry is a powerful tool to measure the molecular weight and identify the protein. Recently, mass spectrometries such as liquid chromatography/tandem mass spectrometry and surface-enhanced laser desorption/ionization time of flight mass spectrometry, have made a contribution to amyloid typing. In the paper, we describe the usefulness of mass spectrometric analyses for the typing of amyloidosis.

  13. A Peptide-Fc Opsonin with Pan-Amyloid Reactivity

    Directory of Open Access Journals (Sweden)

    James S. Foster

    2017-09-01

    Full Text Available There is a continuing need for therapeutic interventions for patients with the protein misfolding disorders that result in systemic amyloidosis. Recently, specific antibodies have been employed to treat AL amyloidosis by opsonizing tissue amyloid deposits thereby inducing cell-mediated dissolution and organ improvement. To develop a pan-amyloid therapeutic agent, we have produced an Fc-fusion product incorporating a peptide, p5, which binds many if not all forms of amyloid. This protein, designated Fcp5, expressed in mammalian cells, forms the desired bivalent dimer structure and retains pan-amyloid reactivity similar to the p5 peptide as measured by immunosorbent assays, immunohistochemistry, surface plasmon resonance, and pulldown assays using radioiodinated Fcp5. Additionally, Fcp5 was capable of opsonizing amyloid fibrils in vitro using a pH-sensitive fluorescence assay of phagocytosis. In mice,125 I-labeled Fcp5 exhibited an extended serum circulation time, relative to the p5 peptide. It specifically bound AA amyloid deposits in diseased mice, as evidenced by biodistribution and microautoradiographic methods, which coincided with an increase in active, Iba-1-positive macrophages in the liver at 48 h postinjection of Fcp5. In healthy mice, no specific tissue accumulation was observed. The data indicate that polybasic, pan-amyloid-targeting peptides, in the context of an Fc fusion, can yield amyloid reactive, opsonizing reagents that may serve as next-generation immunotherapeutics.

  14. Expression and purification of 15N- and 13C-isotope labeled 40-residue human Alzheimer’s β-amyloid peptide for NMR-based structural analysis

    OpenAIRE

    Long, Fei; Cho, Wonhwa; Ishii, Yoshitaka

    2011-01-01

    Amyloid fibrils of Alzheimer’s β-amyloid peptide (Aβ) are a primary component of amyloid plaques, a hallmark of Alzheimer’s disease (AD). Enormous attention has been given to the structural features and functions of Aβ in amyloid fibrils and other type of aggregates in associated with development of AD. This report describes an efficient protocol to express and purify high-quality 40-residue Aβ(1–40), the most abundant Aβ in brains, for structural studies by NMR spectroscopy. Over-expression ...

  15. The role of stable α-synuclein oligomers in the molecular events underlying amyloid formation

    DEFF Research Database (Denmark)

    Lorenzen, Nikolai; Nielsen, Søren Bang; Buell, Alexander K.

    2014-01-01

    α-synuclein (αSN), whose aggregation is strongly implicated in the development of Parkinson’s disease (PD). The two types of oligomers are both formed under conditions where amyloid fibril formation is observed but differ in molecular weight by an order of magnitude. Both possess a degree of β......, either as precursors of fibrils or as species involved in the fibril elongation process or instead if they are associated with an aggregation process that is distinct from that generating mature fibrils. Here we describe and characterize in detail two well-defined oligomeric species formed by the protein...

  16. Functional Amyloids in Reproduction.

    Science.gov (United States)

    Hewetson, Aveline; Do, Hoa Quynh; Myers, Caitlyn; Muthusubramanian, Archana; Sutton, Roger Bryan; Wylie, Benjamin J; Cornwall, Gail A

    2017-06-29

    Amyloids are traditionally considered pathological protein aggregates that play causative roles in neurodegenerative disease, diabetes and prionopathies. However, increasing evidence indicates that in many biological systems nonpathological amyloids are formed for functional purposes. In this review, we will specifically describe amyloids that carry out biological roles in sexual reproduction including the processes of gametogenesis, germline specification, sperm maturation and fertilization. Several of these functional amyloids are evolutionarily conserved across several taxa, including human, emphasizing the critical role amyloids perform in reproduction. Evidence will also be presented suggesting that, if altered, some functional amyloids may become pathological.

  17. Chiral recognition in amyloid fiber growth.

    Science.gov (United States)

    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. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  18. Exploring the early steps of aggregation of amyloid-forming peptide KFFE

    International Nuclear Information System (INIS)

    Wei Guanghong; Mousseau, Normand; Derreumaux, Philippe

    2004-01-01

    It has been shown recently that even a tetrapeptide can form amyloid fibrils sharing all the characteristics of amyloid fibrils built from large proteins. Recent experimental studies also suggest that the toxicity observed in several neurodegenerative diseases, such as Alzheimer's disease and Creutzfeldt-Jakob disease, is not only related to the mature fibrils themselves, but also to the soluble oligomers formed early in the process of fibrillogenesis. This raises the interest in studying the early steps of the aggregation process. Although fibril formation follows the nucleation-condensation process, characterized by the presence of lag phase, the exact pathways remain to be determined. In this study, we used the activation-relaxation technique and a generic energy model to explore the process of self-assembly and the structures of the resulting aggregates of eight KFFE peptides. Our simulations show, starting from different states with a preformed antiparallel dimer, that eight chains can self-assemble to adopt, with various orientations, four possible distant oligomeric well-aligned structures of similar energy. Two of these structures show a double-layer β-sheet organization, in agreement with the structure of amyloid fibrils as observed by x-ray diffraction; another two are mixtures of dimers and trimers. Our results also suggest that octamers are likely to be below the critical size for nucleation of amyloid fibrils for small peptides

  19. Exploring the early steps of aggregation of amyloid-forming peptide KFFE

    Energy Technology Data Exchange (ETDEWEB)

    Wei Guanghong [Departement de Physique and Regroupement Quebecois sur les Materiaux de Pointe, Universite de Montreal, CP 6128, succursale centre-ville, Montreal, QC, H3C 3J7 (Canada); Mousseau, Normand [Departement de Physique and Regroupement Quebecois sur les Materiaux de Pointe, Universite de Montreal, CP 6128, succursale centre-ville, Montreal, QC, H3C 3J7 (Canada); Derreumaux, Philippe [Laboratoire de Biochimie, Theorique, UPR 9080 CNRS, IBPC, Universite Paris 7 Denis-Diderot, 13 rue Pierre et Marie Curie, 75005 Paris (France)

    2004-11-10

    It has been shown recently that even a tetrapeptide can form amyloid fibrils sharing all the characteristics of amyloid fibrils built from large proteins. Recent experimental studies also suggest that the toxicity observed in several neurodegenerative diseases, such as Alzheimer's disease and Creutzfeldt-Jakob disease, is not only related to the mature fibrils themselves, but also to the soluble oligomers formed early in the process of fibrillogenesis. This raises the interest in studying the early steps of the aggregation process. Although fibril formation follows the nucleation-condensation process, characterized by the presence of lag phase, the exact pathways remain to be determined. In this study, we used the activation-relaxation technique and a generic energy model to explore the process of self-assembly and the structures of the resulting aggregates of eight KFFE peptides. Our simulations show, starting from different states with a preformed antiparallel dimer, that eight chains can self-assemble to adopt, with various orientations, four possible distant oligomeric well-aligned structures of similar energy. Two of these structures show a double-layer {beta}-sheet organization, in agreement with the structure of amyloid fibrils as observed by x-ray diffraction; another two are mixtures of dimers and trimers. Our results also suggest that octamers are likely to be below the critical size for nucleation of amyloid fibrils for small peptides.

  20. Structural model for alpha-synuclein fibrils derived from high resolution imaging and nanomechanical studies using atomic force microscopy

    NARCIS (Netherlands)

    Sweers, K.K.M.; Segers-Nolten, Gezina M.J.; Bennink, Martin L.; Subramaniam, Vinod

    2012-01-01

    A number of proteins form supramolecular protein aggregates called amyloid fibrils which self-assemble under appropriate conditions. We have used high-resolution atomic force microscopy to obtain detailed ultrastructural information on fibrils formed from the E46K mutant of the human α-synuclein

  1. Concentration dependence of alpha-synuclein fibril length assessed by quantitative atomic force microscopy and statistical-mechanical theory

    NARCIS (Netherlands)

    van Raaij, Martijn E; van Gestel, Jeroen; Segers-Nolten, Ine M J; de Leeuw, Simon W; Subramaniam, Vinod

    2008-01-01

    The initial concentration of monomeric amyloidogenic proteins is a crucial factor in the in vitro formation of amyloid fibrils. We use quantitative atomic force microscopy to study the effect of the initial concentration of human alpha-synuclein on the mean length of mature alpha-synuclein fibrils,

  2. Glycosaminoglycans enhance the fibrillation propensity of the ß2-microglobulin cleavage variant--¿K58-ß2m

    DEFF Research Database (Denmark)

    Corlin, Dorthe B; Johnsen, Christina K; Nissen, Mogens H

    2010-01-01

    when heparan sulfate is present have increased length and diameter, and possess enhanced stability and seeding properties. However, when copper ions are present the fibrils are short, thin and less stable, and form at a slower rate. We suggest that heparan sulfate stabilizes the cleaved monomers...... in the early aggregates, hereby promoting the assembly of these into fibrils, whereas the copper ions appear to have a destabilizing effect on the monomers. This keeps them in a structure forming amorphous aggregates for a longer period of time, leading to the formation of spherical bodies followed...... by the assembly of fibrils. Hence, the in vivo formation of amyloid fibrils in DRA could be initiated by the generation of ¿K58-ß2m which spontaneously aggregate and form fibrils. The fibrillogenesis is enhanced by the involvement of GAGs and/or metal ions, and results in amyloid-like fibrils able to promote...

  3. Atomistic mechanism of polyphenol amyloid aggregation inhibitors: molecular dynamics study of Curcumin, Exifone, and Myricetin interaction with the segment of tau peptide oligomer.

    Science.gov (United States)

    Berhanu, Workalemahu M; Masunov, Artëm E

    2015-01-01

    Amyloid fibrils are highly ordered protein aggregates associated with many diseases affecting millions of people worldwide. Polyphenols such as Curcumin, Exifone, and Myricetin exhibit modest inhibition toward fibril formation of tau peptide which is associated with Alzheimer's disease. However, the molecular mechanisms of this inhibition remain elusive. We investigated the binding of three polyphenol molecules to the protofibrils of an amyloidogenic fragment VQIVYK of tau peptide by molecular dynamics simulations in explicit solvent. We find that polyphenols induce conformational changes in the oligomer aggregate. These changes disrupt the amyloid H bonding, perturbing the aggregate. While the structural evolution of the control oligomer with no ligand is limited to the twisting of the β-sheets without their disassembly, the presence of polyphenol molecule pushes the β-sheets apart, and leads to a loosely packed structure where two of four β-sheets dissociate in each of the three cases considered here. The H-bonding capacity of polyphenols is responsible for the observed behavior. The calculated binding free energies and its individual components enabled better understanding of the binding. Results indicated that the contribution from Van der Waals interactions is more significant than electrostatic contribution to the binding. The findings from this study are expected to assist in the development of aggregation inhibitors. Significant binding between polyphenols and aggregate oligomer identified in our simulations confirms the previous experimental observations in which polyphenols refold the tau peptide without forming covalent bonds.

  4. Imaging amyloid beta peptide oligomeric particles in solution.

    Science.gov (United States)

    Dong, Jijun; Apkarian, Robert P; Lynn, David G

    2005-09-01

    While all protein misfolding diseases are characterized by fibrous amyloid deposits, the favorable free energy and strongly cooperative nature of the self-assembly have complicated the development of therapeutic strategies aimed at preventing their formation. As structural models for the amyloid fibrils approach atomic resolution, increasing evidence suggests that early folding intermediates, rather than the final structure, are more strongly associated with the loss of neuronal function. For that reason we now demonstrate the use of cryo-etch high-resolution scanning electron microscopy (cryo-HRSEM) for the direct observation of pathway intermediates in amyloid assembly. A congener of the Abeta peptide of Alzheimer's disease, Abeta(13-21), samples a variety of time-dependent self-assembles in a manner similar to those seen for larger proteins. A morphological description of these intermediates is the first step towards their structural characterization and the definition of their role in both amyloid assembly and neurotoxicity.

  5. The interaction with gold suppresses fiber-like conformations of the amyloid β (16-22) peptide

    Science.gov (United States)

    Bellucci, Luca; Ardèvol, Albert; Parrinello, Michele; Lutz, Helmut; Lu, Hao; Weidner, Tobias; Corni, Stefano

    2016-04-01

    Inorganic surfaces and nanoparticles can accelerate or inhibit the fibrillation process of proteins and peptides, including the biomedically relevant amyloid β peptide. However, the microscopic mechanisms that determine such an effect are still poorly understood. By means of large-scale, state-of-the-art enhanced sampling molecular dynamics simulations, here we identify an interaction mechanism between the segments 16-22 of the amyloid β peptide, known to be fibrillogenic by itself, and the Au(111) surface in water that leads to the suppression of fiber-like conformations from the peptide conformational ensemble. Moreover, thanks to advanced simulation analysis techniques, we characterize the conformational selection vs. induced fit nature of the gold effect. Our results disclose an inhibition mechanism that is rooted in the details of the microscopic peptide-surface interaction rather than in general phenomena such as peptide sequestration from the solution.Inorganic surfaces and nanoparticles can accelerate or inhibit the fibrillation process of proteins and peptides, including the biomedically relevant amyloid β peptide. However, the microscopic mechanisms that determine such an effect are still poorly understood. By means of large-scale, state-of-the-art enhanced sampling molecular dynamics simulations, here we identify an interaction mechanism between the segments 16-22 of the amyloid β peptide, known to be fibrillogenic by itself, and the Au(111) surface in water that leads to the suppression of fiber-like conformations from the peptide conformational ensemble. Moreover, thanks to advanced simulation analysis techniques, we characterize the conformational selection vs. induced fit nature of the gold effect. Our results disclose an inhibition mechanism that is rooted in the details of the microscopic peptide-surface interaction rather than in general phenomena such as peptide sequestration from the solution. Electronic supplementary information (ESI

  6. The first step of hen egg white lysozyme fibrillation, irreversible partial unfolding, is a two-state transition

    OpenAIRE

    Xu, Ming; Shashilov, Victor A.; Ermolenkov, Vladimir V.; Fredriksen, Laura; Zagorevski, Dmitri; Lednev, Igor K.

    2007-01-01

    Amyloid fibril depositions are associated with many neurodegenerative diseases as well as amyloidosis. The detailed molecular mechanism of fibrillation is still far from complete understanding. In our previous study of in vitro fibrillation of hen egg white lysozyme, an irreversible partially unfolded intermediate was characterized. A similarity of unfolding kinetics found for the secondary and tertiary structure of lysozyme using deep UV resonance Raman (DUVRR) and tryptophan fluorescence sp...

  7. Chronic treatment with amyloid beta(1-42) inhibits non-cholinergic high-affinity choline transport in NG108-15 cells through protein kinase C signaling

    Czech Academy of Sciences Publication Activity Database

    Nováková, Jana; Mikasová, Lenka; Machová, Eva; Lisá, Věra; Doležal, Vladimír

    2005-01-01

    Roč. 1062, č. 1-2 (2005), s. 101-110 ISSN 0006-8993 R&D Projects: GA AV ČR(CZ) IAA5011206; GA MŠk(CZ) LC554 Grant - others:Lipidiet(XE) QLK1-CT-2002-00172 Institutional research plan: CEZ:AV0Z50110509 Keywords : choline transporter * beta-amyloid * protein kinase C Subject RIV: ED - Physiology Impact factor: 2.296, year: 2005

  8. Crowding alone cannot account for cosolute effect on amyloid aggregation.

    Directory of Open Access Journals (Sweden)

    Shahar Sukenik

    Full Text Available Amyloid fiber formation is a specific form of protein aggregation, often resulting from the misfolding of native proteins. Aimed at modeling the crowded environment of the cell, recent experiments showed a reduction in fibrillation halftimes for amyloid-forming peptides in the presence of cosolutes that are preferentially excluded from proteins and peptides. The effect of excluded cosolutes has previously been attributed to the large volume excluded by such inert cellular solutes, sometimes termed "macromolecular crowding". Here, we studied a model peptide that can fold to a stable monomeric β-hairpin conformation, but under certain solution conditions aggregates in the form of amyloid fibrils. Using Circular Dichroism spectroscopy (CD, we found that, in the presence of polyols and polyethylene glycols acting as excluded cosolutes, the monomeric β-hairpin conformation was stabilized with respect to the unfolded state. Stabilization free energy was linear with cosolute concentration, and grew with molecular volume, as would also be predicted by crowding models. After initiating the aggregation process with a pH jump, fibrillation in the presence and absence of cosolutes was followed by ThT fluorescence, transmission electron microscopy, and CD spectroscopy. Polyols (glycerol and sorbitol increased the lag time for fibril formation and elevated the amount of aggregated peptide at equilibrium, in a cosolute size and concentration dependent manner. However, fibrillation rates remained almost unaffected by a wide range of molecular weights of soluble polyethylene glycols. Our results highlight the importance of other forces beyond the excluded volume interactions responsible for crowding that may contribute to the cosolute effects acting on amyloid formation.

  9. Atrial Fibrillation

    DEFF Research Database (Denmark)

    Staerk, Laila; Sherer, Jason A; Ko, Darae

    2017-01-01

    that advanced age, male sex, and European ancestry are prominent AF risk factors. Other modifiable risk factors include sedentary lifestyle, smoking, obesity, diabetes mellitus, obstructive sleep apnea, and elevated blood pressure predispose to AF, and each factor has been shown to induce structural......The past 3 decades have been characterized by an exponential growth in knowledge and advances in the clinical treatment of atrial fibrillation (AF). It is now known that AF genesis requires a vulnerable atrial substrate and that the formation and composition of this substrate may vary depending...... and electric remodeling of the atria. Both heart failure and myocardial infarction increase risk of AF and vice versa creating a feed-forward loop that increases mortality. Other cardiovascular outcomes attributed to AF, including stroke and thromboembolism, are well established, and epidemiology studies have...

  10. Hydrogen sulfide inhibits A2A adenosine receptor agonist induced β-amyloid production in SH-SY5Y neuroblastoma cells via a cAMP dependent pathway.

    Directory of Open Access Journals (Sweden)

    Bhushan Vijay Nagpure

    Full Text Available Alzheimer's disease (AD is the leading cause of senile dementia in today's society. Its debilitating symptoms are manifested by disturbances in many important brain functions, which are influenced by adenosine. Hence, adenosinergic system is considered as a potential therapeutic target in AD treatment. In the present study, we found that sodium hydrosulfide (NaHS, an H2S donor, 100 µM attenuated HENECA (a selective A2A receptor agonist, 10-200 nM induced β-amyloid (1-42 (Aβ42 production in SH-SY5Y cells. NaHS also interfered with HENECA-stimulated production and post-translational modification of amyloid precursor protein (APP by inhibiting its maturation. Measurement of the C-terminal APP fragments generated from its enzymatic cleavage by β-site amyloid precursor protein cleaving enzyme 1 (BACE1 showed that NaHS did not have any significant effect on β-secretase activity. However, the direct measurements of HENECA-elevated γ-secretase activity and mRNA expressions of presenilins suggested that the suppression of Aβ42 production in NaHS pretreated cells was mediated by inhibiting γ-secretase. NaHS induced reductions were accompanied by similar decreases in intracellular cAMP levels and phosphorylation of cAMP responsive element binding protein (CREB. NaHS significantly reduced the elevated cAMP and Aβ42 production caused by forskolin (an adenylyl cyclase, AC agonist alone or forskolin in combination with IBMX (a phosphodiesterase inhibitor, but had no effect on those caused by IBMX alone. Moreover, pretreatment with NaHS significantly attenuated HENECA-elevated AC activity and mRNA expressions of various AC isoforms. These data suggest that NaHS may preferentially suppress AC activity when it was stimulated. In conclusion, H2S attenuated HENECA induced Aβ42 production in SH-SY5Y neuroblastoma cells through inhibiting γ-secretase via a cAMP dependent pathway.

  11. The physical chemistry of the amyloid phenomenon: thermodynamics and kinetics of filamentous protein aggregation.

    Science.gov (United States)

    Buell, Alexander K; Dobson, Christopher M; Knowles, Tuomas P J

    2014-01-01

    In this chapter, we present an overview of the kinetics and thermodynamics of protein aggregation into amyloid fibrils. The perspective we adopt is largely experimental, but we also discuss recent developments in data analysis and we show that only a combination of well-designed experiments with appropriate theoretical modelling is able to provide detailed mechanistic insight into the complex pathways of amyloid formation. In the first part of the chapter, we describe measurements of the thermodynamic stability of the amyloid state with respect to the soluble state of proteins, as well as the magnitude and origin of this stability. In the second part, we discuss in detail the kinetics of the individual molecular steps in the overall mechanism of the conversion of soluble protein into amyloid fibrils. Finally, we highlight the effects of external factors, such as salt type and concentration, chemical denaturants and molecular chaperones on the kinetics of aggregation.

  12. Amyloid and immune homeostasis.

    Science.gov (United States)

    Wang, Ying-Hui; Zhang, Yu-Gen

    2018-03-01

    Extracellular amyloid deposition defines a range of amyloidosis and amyloid-related disease. Addition to primary and secondary amyloidosis, amyloid-related disease can be observed in different tissue/organ that sharing the common pathogenesis based on the formation of amyloid deposition. Currently, both Alzheimer's disease and type 2 diabetes can be diagnosed with certainly only based on the autopsy results, by which amyloidosis of the associative tissue/organ is observed. Intriguingly, since it demonstrated that amyloid deposits trigger inflammatory reaction through the activation of cascaded immune response, wherein several lines of evidence implies a protective role of amyloid in preventing autoimmunity. Furthermore, attempts for preventing amyloid formation and/or removing amyloid deposits from the brain have caused meningoencephalitis and consequent deaths among the subjects. Hence, it is important to note that amyloid positively participates in maintaining immune homeostasis and contributes to irreversible inflammatory response. In this review, we will focus on the interactive relationship between amyloid and the immune system, discussing the potential functional roles of amyloid in immune tolerance and homeostasis. Copyright © 2017 Elsevier GmbH. All rights reserved.

  13. Multimodal imaging Gd-nanoparticles functionalized with Pittsburgh compound B or a nanobody for amyloid plaques targeting.

    Science.gov (United States)

    Pansieri, Jonathan; Plissonneau, Marie; Stransky-Heilkron, Nathalie; Dumoulin, Mireille; Heinrich-Balard, Laurence; Rivory, Pascaline; Morfin, Jean-François; Toth, Eva; Saraiva, Maria Joao; Allémann, Eric; Tillement, Olivier; Forge, Vincent; Lux, François; Marquette, Christel

    2017-07-01

    Gadolinium-based nanoparticles were functionalized with either the Pittsburgh compound B or a nanobody (B10AP) in order to create multimodal tools for an early diagnosis of amyloidoses. The ability of the functionalized nanoparticles to target amyloid fibrils made of β-amyloid peptide, amylin or Val30Met-mutated transthyretin formed in vitro or from pathological tissues was investigated by a range of spectroscopic and biophysics techniques including fluorescence microscopy. Nanoparticles functionalized by both probes efficiently interacted with the three types of amyloid fibrils, with K D values in 10 micromolar and 10 nanomolar range for, respectively, Pittsburgh compound B and B10AP nanoparticles. Moreover, they allowed the detection of amyloid deposits on pathological tissues. Such functionalized nanoparticles could represent promising flexible and multimodal imaging tools for the early diagnostic of amyloid diseases, in other words, Alzheimer's disease, Type 2 diabetes mellitus and the familial amyloidotic polyneuropathy.

  14. Aβ(39–42) Modulates Aβ Oligomerization but Not Fibril Formation

    Science.gov (United States)

    Gessel, Megan Murray; Wu, Chun; Li, Huiyuan; Bitan, Gal; Shea, Joan-Emma; Bowers, Michael T.

    2012-01-01

    Recently, certain C-terminal fragments (CTFs) of Aβ42 have been shown to be effective inhibitors of Aβ42 toxicity. Here, we examine the interactions between the shortest CTF in the original series, Aβ(39–42) and full-length Aβ. Mass spectrometry results indicate that Aβ(39–42) binds directly to Aβ monomers and to the n=2,4, and 6 oligomers. The Aβ42:Aβ(39–42) complex is further probed using in molecular dynamics simulations. Although the CTF was expected to bind to the hydrophobic C-terminus of Aβ42, the simulations show that Aβ(39–42) binds at several locations on Aβ42, including the C-terminus, other hydrophobic regions, and preferentially in the N-terminus. Ion mobility-mass spectrometry (IM-MS) and electron microscopy experiments indicate that Aβ(39–42) disrupts the early assembly of full-length Aβ. Specifically, the ion-mobility results show that Aβ(39–42) prevents the formation of large decamer/dodecamer Aβ42 species and, moreover, can remove these structures from solution. At the same time, thioflavin T fluorescence and electron microscopy results show that the CTF does not inhibit fibril formation, lending strong support to the hypothesis that oligomers and not amyloid fibrils are the Aβ form responsible for toxicity. The results emphasize the role of small, soluble assemblies in Aβ-induced toxicity and suggest that Aβ(39–42) inhibits Aβ-induced toxicity by a unique mechanism, modulating early assembly into non-toxic heterooligomers, without preventing fibril formation. PMID:22129303

  15. Increased plasma concentration of serum amyloid P component in centenarians with impaired cognitive performance

    DEFF Research Database (Denmark)

    Nybo, M; Olsen, H; Jeune, B

    1998-01-01

    these to the cognitive performance evaluated by Mini Mental State Examination (MMSE). We observed a significantly (p ... performance had significantly increased plasma concentrations of SAP, while the values for cognitive intact centenarians were within the normal range.......Serum amyloid P component (SAP) binds to all amyloid fibrils including those in the plaques and tangles of Alzheimer patients. To investigate whether the plasma SAP concentration correlated to cognitive impairment, we measured SAP levels in blood samples from 41 centenarians and compared...

  16. Amyloid formation and disaggregation of α-synuclein and its tandem repeat (α-TR)

    International Nuclear Information System (INIS)

    Bae, Song Yi; Kim, Seulgi; Hwang, Heejin; Kim, Hyun-Kyung; Yoon, Hyun C.; Kim, Jae Ho; Lee, SangYoon; Kim, T. Doohun

    2010-01-01

    Research highlights: → Formation of the α-synuclein amyloid fibrils by [BIMbF 3 Im]. → Disaggregation of amyloid fibrils by epigallocatechin gallate (EGCG) and baicalein. → Amyloid formation of α-synuclein tandem repeat (α-TR). -- Abstract: The aggregation of α-synuclein is clearly related to the pathogenesis of Parkinson's disease. Therefore, detailed understanding of the mechanism of fibril formation is highly valuable for the development of clinical treatment and also of the diagnostic tools. Here, we have investigated the interaction of α-synuclein with ionic liquids by using several biochemical techniques including Thioflavin T assays and transmission electron microscopy (TEM). Our data shows a rapid formation of α-synuclein amyloid fibrils was stimulated by 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [BIMbF 3 Im], and these fibrils could be disaggregated by polyphenols such as epigallocatechin gallate (EGCG) and baicalein. Furthermore, the effect of [BIMbF 3 Im] on the α-synuclein tandem repeat (α-TR) in the aggregation process was studied.

  17. Outcomes of temporary interruption of rivaroxaban compared with warfarin in patients with nonvalvular atrial fibrillation: results from the rivaroxaban once daily, oral, direct factor Xa inhibition compared with vitamin K antagonism for prevention of stroke and embolism trial in atrial fibrillation (ROCKET AF).

    Science.gov (United States)

    Sherwood, Matthew W; Douketis, James D; Patel, Manesh R; Piccini, Jonathan P; Hellkamp, Anne S; Lokhnygina, Yuliya; Spyropoulos, Alex C; Hankey, Graeme J; Singer, Daniel E; Nessel, Christopher C; Mahaffey, Kenneth W; Fox, Keith A A; Califf, Robert M; Becker, Richard C

    2014-05-06

    During long-term anticoagulation in atrial fibrillation, temporary interruptions (TIs) of therapy are common, but the relationship between patient outcomes and TIs has not been well studied. We sought to determine reasons for TI, the characteristics of patients undergoing TI, and the relationship between anticoagulant and outcomes among patients with TI. In the Rivaroxaban Once Daily, Oral, Direct Factor Xa Inhibition Compared With Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF), a randomized, double-blind, double-dummy study of rivaroxaban and warfarin in nonvalvular atrial fibrillation, baseline characteristics, management, and outcomes, including stroke, non-central nervous system systemic embolism, death, myocardial infarction, and bleeding, were reported in participants who experienced TI (3-30 days) for any reason. The at-risk period for outcomes associated with TI was from TI start to 30 days after resumption of study drug. In 14 236 participants who received at least 1 dose of study drug, 4692 (33%) experienced TI. Participants with TI were similar to the overall ROCKET AF population in regard to baseline clinical characteristics. Only 6% (n=483) of TI incidences involved bridging therapy. Stroke/systemic embolism rates during the at-risk period were similar in rivaroxaban-treated and warfarin-treated participants (0.30% versus 0.41% per 30 days; hazard ratio [confidence interval]=0.74 [0.36-1.50]; P=0.40). Risk of major bleeding during the at-risk period was also similar in rivaroxaban-treated and warfarin-treated participants (0.99% versus 0.79% per 30 days; hazard ratio [confidence interval]=1.26 [0.80-2.00]; P=0.32). TI of oral anticoagulation is common and is associated with substantial stroke risks and bleeding risks that were similar among patients treated with rivaroxaban or warfarin. Further investigation is needed to determine the optimal management strategy in patients with atrial fibrillation

  18. Rational design of amyloid beta peptide-binding proteins: pseudo-Abeta beta-sheet surface presented in green fluorescent protein binds tightly and preferentially to structured Abeta.

    Science.gov (United States)

    Takahashi, Tsuyoshi; Ohta, Kenichi; Mihara, Hisakazu

    2010-02-01

    Some neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson disease are caused by protein misfolding. In AD, amyloid beta-peptide (Abeta) is thought to be a toxic agent by self-assembling into a variety of aggregates involving soluble oligomeric intermediates and amyloid fibrils. Here, we have designed several green fluorescent protein (GFP) variants that contain pseudo-Abeta beta-sheet surfaces and evaluated their abilities to bind to Abeta and inhibit Abeta oligomerization. Two GFP variants P13H and AP93Q bound tightly to Abeta, K(d) = 260 nM and K(d) = 420 nM, respectively. Moreover, P13H and AP93Q were capable of efficiently suppressing the generation of toxic Abeta oligomers as shown by a cell viability assay. By combining the P13H and AP93Q mutations, a super variant SFAB4 comprising four strands of Abeta-derived sequences was designed and bound more tightly to Abeta (K(d) = 100 nM) than those having only two pseudo-Abeta strands. The SFAB4 protein preferentially recognized the soluble oligomeric intermediates of Abeta more than both unstructured monomer and mature amyloid fibrils. Thus, the design strategy for embedding pseudo-Abeta beta-sheet structures onto a protein surface arranged in the beta-barrel structure is useful to construct molecules capable of binding tightly to Abeta and inhibiting its aggregation. This strategy may provide implication for the diagnostic and therapeutic development in the treatment of AD. (c) 2009 Wiley-Liss, Inc.

  19. Navβ2 knockdown improves cognition in APP/PS1 mice by partially inhibiting seizures and APP amyloid processing.

    Science.gov (United States)

    Hu, Tao; Xiao, Zhangang; Mao, Rui; Chen, Bo; Lu, Min-Nan; Tong, Jun; Mei, Rong; Li, Shan-Shan; Xiao, Zhi-Cheng; Zhang, Lian-Feng; Xiyang, Yan-Bin

    2017-11-21

    Voltage-gated sodium channels beta 2 (Navβ2, encoded by SCN2B) is a substrate of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and regulates cell surface expression of channels in neurons. Previous studies reported enhanced Navβ2 processing by BACE1 in Alzheimer's disease (AD) model and patients. We investigated whether changes in Navβ2 expression affect neuronal seizure and amyloid precursor protein (APP) processing in an AD mouse model. Our study used eight-month-old APP/presenilin 1 (PS1) mice and transgenic Navβ2 knockdown [by 61% vs. wild type (WT)] APP/PS1 mice (APP/PS1/Navβ2-kd), with age-matched WT and Navβ2 knockdown (Navβ2-kd) mice as controls. We found that Navβ2 knockdown in APP/PS1 mice partially reversed the abnormal Navβ2 cleavage and the changes in intracellular and total Nav1.1α expression. It also restored sodium currents density in hippocampal neurons and neuronal activity, as indicated by EEG tracing; improved Morris water maze performance; and shifted APP amyloidogenic metabolism towards non-amyloidogenic processing. There were no differences in these indicators between WT and Navβ2-kd mice. These results suggest Navβ2 knockdown may be a promising strategy for treating AD.

  20. Atrial fibrillation - discharge

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/patientinstructions/000237.htm Atrial fibrillation - discharge To use the sharing features on this ... have been in the hospital because you have atrial fibrillation . This condition occurs when your heart beats faster ...

  1. Comparison of the aggregation of homologous β2-microglobulin variants reveals protein solubility as a key determinant of amyloid formation.

    Science.gov (United States)

    Pashley, Clare L; Hewitt, Eric W; Radford, Sheena E

    2016-02-13

    The mouse and human β2-microglobulin protein orthologs are 70% identical in sequence and share 88% sequence similarity. These proteins are predicted by various algorithms to have similar aggregation and amyloid propensities. However, whilst human β2m (hβ2m) forms amyloid-like fibrils in denaturing conditions (e.g. pH2.5) in the absence of NaCl, mouse β2m (mβ2m) requires the addition of 0.3M NaCl to cause fibrillation. Here, the factors which give rise to this difference in amyloid propensity are investigated. We utilise structural and mutational analyses, fibril growth kinetics and solubility measurements under a range of pH and salt conditions, to determine why these two proteins have different amyloid propensities. The results show that, although other factors influence the fibril growth kinetics, a striking difference in the solubility of the proteins is a key determinant of the different amyloidogenicity of hβ2m and mβ2m. The relationship between protein solubility and lag time of amyloid formation is not captured by current aggregation or amyloid prediction algorithms, indicating a need to better understand the role of solubility on the lag time of amyloid formation. The results demonstrate the key contribution of protein solubility in determining amyloid propensity and lag time of amyloid formation, highlighting how small differences in protein sequence can have dramatic effects on amyloid formation. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. Sequence dependent aggregation of peptides and fibril formation

    Science.gov (United States)

    Hung, Nguyen Ba; Le, Duy-Manh; Hoang, Trinh X.

    2017-09-01

    Deciphering the links between amino acid sequence and amyloid fibril formation is key for understanding protein misfolding diseases. Here we use Monte Carlo simulations to study the aggregation of short peptides in a coarse-grained model with hydrophobic-polar (HP) amino acid sequences and correlated side chain orientations for hydrophobic contacts. A significant heterogeneity is observed in the aggregate structures and in the thermodynamics of aggregation for systems of different HP sequences and different numbers of peptides. Fibril-like ordered aggregates are found for several sequences that contain the common HPH pattern, while other sequences may form helix bundles or disordered aggregates. A wide variation of the aggregation transition temperatures among sequences, even among those of the same hydrophobic fraction, indicates that not all sequences undergo aggregation at a presumable physiological temperature. The transition is found to be the most cooperative for sequences forming fibril-like structures. For a fibril-prone sequence, it is shown that fibril formation follows the nucleation and growth mechanism. Interestingly, a binary mixture of peptides of an aggregation-prone and a non-aggregation-prone sequence shows the association and conversion of the latter to the fibrillar structure. Our study highlights the role of a sequence in selecting fibril-like aggregates and also the impact of a structural template on fibril formation by peptides of unrelated sequences.

  3. Replica Exchange Simulations of the Thermodynamics of Aβ Fibril Growth

    Science.gov (United States)

    Takeda, Takako; Klimov, Dmitri K.

    2009-01-01

    Abstract Replica exchange molecular dynamics and an all-atom implicit solvent model are used to probe the thermodynamics of deposition of Alzheimer's Aβ monomers on preformed amyloid fibrils. Consistent with the experiments, two deposition stages have been identified. The docking stage occurs over a wide temperature range, starting with the formation of the first peptide-fibril interactions at 500 K. Docking is completed when a peptide fully adsorbs on the fibril edge at the temperature of 380 K. The docking transition appears to be continuous, and occurs without free energy barriers or intermediates. During docking, incoming Aβ monomer adopts a disordered structure on the fibril edge. The locking stage occurs at the temperature of ≈360 K and is characterized by the rugged free energy landscape. Locking takes place when incoming Aβ peptide forms a parallel β-sheet structure on the fibril edge. Because the β-sheets formed by locked Aβ peptides are typically off-registry, the structure of the locked phase differs from the structure of the fibril interior. The study also reports that binding affinities of two distinct fibril edges with respect to incoming Aβ peptides are different. The peptides bound to the concave edge have significantly lower free energy compared to those bound on the convex edge. Comparison with the available experimental data is discussed. PMID:19167295

  4. Atrial Fibrillation: Complications

    Science.gov (United States)

    ... of this page please turn JavaScript on. Feature: Atrial Fibrillation Atrial Fibrillation: Complications Past Issues / Winter 2015 Table of Contents ... has two major complications—stroke and heart failure. Atrial Fibrillation and Stroke Click to enlarge image This illustration ...

  5. Atrial Fibrillation Medications

    Science.gov (United States)

    ... Peripheral Artery Disease Venous Thromboembolism Aortic Aneurysm More Atrial Fibrillation Medications Updated:Jun 28,2017 Understand medications and ... you. This content was last reviewed July 2016. Atrial Fibrillation • Introduction • What is Atrial Fibrillation? • Why AFib Matters ...

  6. Atrial Fibrillation in Children

    Science.gov (United States)

    ... Peripheral Artery Disease Venous Thromboembolism Aortic Aneurysm More Atrial Fibrillation in Children Updated:Oct 18,2016 Does your ... arrhythmia. This content was last reviewed July 2016. Atrial Fibrillation • Introduction • What is Atrial Fibrillation? • Why AFib Matters ...

  7. Depletion of spleen macrophages delays AA amyloid development: a study performed in the rapid mouse model of AA amyloidosis.

    Directory of Open Access Journals (Sweden)

    Katarzyna Lundmark

    Full Text Available AA amyloidosis is a systemic disease that develops secondary to chronic inflammatory diseases Macrophages are often found in the vicinity of amyloid deposits and considered to play a role in both formation and degradation of amyloid fibrils. In spleen reside at least three types of macrophages, red pulp macrophages (RPM, marginal zone macrophages (MZM, metallophilic marginal zone macrophages (MMZM. MMZM and MZM are located in the marginal zone and express a unique collection of scavenger receptors that are involved in the uptake of blood-born particles. The murine AA amyloid model that resembles the human form of the disease has been used to study amyloid effects on different macrophage populations. Amyloid was induced by intravenous injection of amyloid enhancing factor and subcutaneous injections of silver nitrate and macrophages were identified with specific antibodies. We show that MZMs are highly sensitive to amyloid and decrease in number progressively with increasing amyloid load. Total area of MMZMs is unaffected by amyloid but cells are activated and migrate into the white pulp. In a group of mice spleen macrophages were depleted by an intravenous injection of clodronate filled liposomes. Subsequent injections of AEF and silver nitrate showed a sustained amyloid development. RPMs that constitute the majority of macrophages in spleen, appear insensitive to amyloid and do not participate in amyloid formation.

  8. Natural product-based amyloid inhibitors.

    Science.gov (United States)

    Velander, Paul; Wu, Ling; Henderson, Frances; Zhang, Shijun; Bevan, David R; Xu, Bin

    2017-09-01

    Many chronic human diseases, including multiple neurodegenerative diseases, are associated with deleterious protein aggregates, also called protein amyloids. One common therapeutic strategy is to develop protein aggregation inhibitors that can slow down, prevent, or remodel toxic amyloids. Natural products are a major class of amyloid inhibitors, and several dozens of natural product-based amyloid inhibitors have been identified and characterized in recent years. These plant- or microorganism-extracted compounds have shown significant therapeutic potential from in vitro studies as well as in vivo animal tests. Despite the technical challenges of intrinsic disordered or partially unfolded amyloid proteins that are less amenable to characterizations by structural biology, a significant amount of research has been performed, yielding biochemical and pharmacological insights into how inhibitors function. This review aims to summarize recent progress in natural product-based amyloid inhibitors and to analyze their mechanisms of inhibition in vitro. Major classes of natural product inhibitors and how they were identified are described. Our analyses comprehensively address the molecular interactions between the inhibitors and relevant amyloidogenic proteins. These interactions are delineated at molecular and atomic levels, which include covalent, non-covalent, and metal-mediated mechanisms. In vivo animal studies and clinical trials have been summarized as an extension. To enhance natural product bioavailability in vivo, emerging work using nanocarriers for delivery has also been described. Finally, issues and challenges as well as future development of such inhibitors are envisioned. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Sugar-Terminated Nanoparticle Chaperones Are 102-105Times Better Than Molecular Sugars in Inhibiting Protein Aggregation and Reducing Amyloidogenic Cytotoxicity.

    Science.gov (United States)

    Pradhan, Nibedita; Shekhar, Shashi; Jana, Nihar R; Jana, Nikhil R

    2017-03-29

    Sugar-based osmolyte molecules are known to stabilize proteins under stress, but usually they have poor chaperone performance in inhibiting protein aggregation. Here, we show that the nanoparticle form of sugars molecule can enhance their chaperone performance typically by 10 2 -10 5 times, compared to molecular sugar. Sugar-based plate-like nanoparticles of 20-40 nm hydrodynamic size have been synthesized by simple heating of acidic aqueous solution of glucose/sucrose/maltose/trehalose. These nanoparticles have excitation-dependent green/yellow/orange emission and surface chemistry identical to the respective sugar molecule. Fibrillation of lysozyme/insulin/amyloid beta in extracellular space, aggregation of mutant huntingtin protein inside model neuronal cell, and cytotoxic effect of fibrils are investigated in the presence of these sugar nanoparticles. We found that sugar nanoparticles are 10 2 -10 5 times efficient than respective sugar molecules in inhibiting protein fibrillation and preventing cytotoxicity arising of fibrils. We propose that better performance of the nanoparticle form is linked to its stronger binding with fibril structure and enhanced cell uptake. This result suggests that nanoparticle form of osmolyte can be an attractive option in prevention and curing of protein aggregation-derived diseases.

  10. Extended analysis of AL-amyloid protein from abdominal wall subcutaneous fat biopsy

    DEFF Research Database (Denmark)

    Olsen, K E; Sletten, K; Westermark, Per

    1998-01-01

    In AL-amyloidosis the cause of amyloid fibril formation in beta-pleated sheets from the precursor protein immunoglobulin light chain is not established, but studies of AL-proteins indicate that amino acid substitutions are important in the pathogenesis. Amyloid material was extracted from...... a subcutaneous fat tissue biopsy and submitted to extended protein separation, typing and amino acid sequence analyses. The AL-protein belonged to the rare immunoglobulin light chain kappa, subtype kappa IV and contained unique amino acid substitutions, mostly in the highly preserved framework regions. The study...... shows that subcutaneous fat biopsies are useful sources of amyloid material for biochemical studies....

  11. MetAmyl: a METa-predictor for AMYLoid proteins.

    Directory of Open Access Journals (Sweden)

    Mathieu Emily

    Full Text Available The aggregation of proteins or peptides in amyloid fibrils is associated with a number of clinical disorders, including Alzheimer's, Huntington's and prion diseases, medullary thyroid cancer, renal and cardiac amyloidosis. Despite extensive studies, the molecular mechanisms underlying the initiation of fibril formation remain largely unknown. Several lines of evidence revealed that short amino-acid segments (hot spots, located in amyloid precursor proteins act as seeds for fibril elongation. Therefore, hot spots are potential targets for diagnostic/therapeutic applications, and a current challenge in bioinformatics is the development of methods to accurately predict hot spots from protein sequences. In this paper, we combined existing methods into a meta-predictor for hot spots prediction, called MetAmyl for METapredictor for AMYLoid proteins. MetAmyl is based on a logistic regression model that aims at weighting predictions from a set of popular algorithms, statistically selected as being the most informative and complementary predictors. We evaluated the performances of MetAmyl through a large scale comparative study based on three independent datasets and thus demonstrated its ability to differentiate between amyloidogenic and non-amyloidogenic polypeptides. Compared to 9 other methods, MetAmyl provides significant improvement in prediction on studied datasets. We further show that MetAmyl is efficient to highlight the effect of point mutations involved in human amyloidosis, so we suggest this program should be a useful complementary tool for the diagnosis of these diseases.

  12. Atrial fibrillation.

    Science.gov (United States)

    Bang, Casper N

    2013-10-01

    Atrial fibrillation (AF) is a common complication after myocardial infarction (MI) and new-onset AF has been demonstrated to be associated with adverse outcome and a large excess risk of death in both MI and aortic stenosis (AS) patients. Prevention of new-onset AF is therefore a potential therapeutic target in AS and MI patients. Lipid-lowering drugs, particularly statins, have anti-inflammatory and antioxidant properties that may prevent AF. Accordingly, statins are recommended as a class IIa recommendation for prevention of new-onset AF after coronary artery bypass grafting (CABG). However, this preventive effect has not been investigated on new-onset AF in asymptomatic patients with AS or a large scale first-time MI patient sample and data in patients not undergoing invasive cardiac interventions are limited. This PhD thesis was conducted at the Heart Centre, Rigshospitalet, Denmark, with the aim to investigate the three aforementioned questions and to add to the existing evidence of AF prevention with statins. This was done using three different settings: 1) a randomized patients sample of 1,873 from the Simvastatin and Ezetimibe in Aortic Stenosis (SEAS) study, 2) a register patient sample of 97,499 with first-time MI, and 3) all published studies until beginning of June 2011 examining statin treatment on new-onset and recurrent AF in patients not undergoing cardiac surgery. This thesis revealed that statins did not lower the incidence or the time to new-onset AF in patients with asymptomatic AS. However, statin treatment showed an independently preventive effect on new-onset AF, including type-dependent effect and a trend to dosage-dependent effect. In addition, this thesis showed that good compliance to statin treatment was important to prevent new-onset AF. Finally, the meta-analysis in this PhD thesis showed a preventive effect in the observational studies although this effect was absent in the randomized controlled trials. Based on this PhD thesis

  13. Renal dysfunction as a predictor of stroke and systemic embolism in patients with nonvalvular atrial fibrillation: validation of the R(2)CHADS(2) index in the ROCKET AF (Rivaroxaban Once-daily, oral, direct factor Xa inhibition Compared with vitamin K antagonism for prevention of stroke and Embolism Trial in Atrial Fibrillation) and ATRIA (AnTicoagulation and Risk factors In Atrial fibrillation) study cohorts.

    Science.gov (United States)

    Piccini, Jonathan P; Stevens, Susanna R; Chang, YuChiao; Singer, Daniel E; Lokhnygina, Yuliya; Go, Alan S; Patel, Manesh R; Mahaffey, Kenneth W; Halperin, Jonathan L; Breithardt, Günter; Hankey, Graeme J; Hacke, Werner; Becker, Richard C; Nessel, Christopher C; Fox, Keith A A; Califf, Robert M

    2013-01-15

    We sought to define the factors associated with the occurrence of stroke and systemic embolism in a large, international atrial fibrillation (AF) trial. In ROCKET AF (Rivaroxaban Once-daily, oral, direct factor Xa inhibition Compared with vitamin K antagonism for prevention of stroke and Embolism Trial in Atrial Fibrillation), 14 264 patients with nonvalvular AF and creatinine clearance ≥30 mL/min were randomized to rivaroxaban or dose-adjusted warfarin. Cox proportional hazards modeling was used to identify factors at randomization independently associated with the occurrence of stroke or non-central nervous system embolism based on intention-to-treat analysis. A risk score was developed in ROCKET AF and validated in ATRIA (AnTicoagulation and Risk factors In Atrial fibrillation), an independent AF patient cohort. Over a median follow-up of 1.94 years, 575 patients (4.0%) experienced primary end-point events. Reduced creatinine clearance was a strong, independent predictor of stroke and systemic embolism, second only to prior stroke or transient ischemic attack. Additional factors associated with stroke and systemic embolism included elevated diastolic blood pressure and heart rate, as well as vascular disease of the heart and limbs (C-index 0.635). A model that included creatinine clearance (R(2)CHADS(2)) improved net reclassification index by 6.2% compared with CHA(2)DS(2)VASc (C statistic=0.578) and by 8.2% compared with CHADS(2) (C statistic=0.575). The inclusion of creatinine clearance <60 mL/min and prior stroke or transient ischemic attack in a model with no other covariates led to a C statistic of 0.590.Validation of R(2)CHADS(2) in an external, separate population improved net reclassification index by 17.4% (95% confidence interval, 12.1%-22.5%) relative to CHADS(2). In patients with nonvalvular AF at moderate to high risk of stroke, impaired renal function is a potent predictor of stroke and systemic embolism. Stroke risk stratification in patients

  14. Lack of evidence for protein AA reactivity in amyloid deposits of lattice corneal dystrophy and amyloid corneal degeneration.

    Science.gov (United States)

    Gorevic, P D; Rodrigues, M M; Krachmer, J H; Green, C; Fujihara, S; Glenner, G G

    1984-08-15

    Amyloid fibrils occurring in primary and myeloma-associated (AL), secondary (AA), and certain neuropathic hereditary forms of systemic amyloidosis can be distinguished biochemically or immunohistologically as being composed of immunoglobulin light chain, protein AA, or prealbumin respectively. All types of systemic and several localized forms of amyloidosis contain amyloid P component (protein AP). We studied formalin-fixed tissue from eight cases of lattice corneal dystrophy by the immunoperoxidase method using antisera to proteins AA and AP, to normal serum prealbumin and prealbumin isolated from a case of hereditary amyloidosis, and to light-chain determinants; additional cases were examined by indirect immunofluorescence of fresh-frozen material. We found weak (1:10 dilution) staining with anti-AP, but no reactivity with other antisera. Congo red staining was resistant to pretreatment of sections with potassium permanganate, a characteristic of non-AA amyloid. Two-dimensional gels of solubilized proteins from frozen tissue from two cases of lattice corneal dystrophy resembled those obtained from normal human cornea. Western blots of two cases of polymorphous amyloid degeneration and solubilized protein from normal cornea did not react with radioactive iodine-labeled anti-AA or anti-AP with purified protein AP and unfixed protein AA amyloid tissue as controls. We were unable to corroborate the presence of protein AA in the amyloid deposits of lattice corneal dystrophy. Although staining with antiserum to protein AP was demonstrable, the molecular configuration of this protein in stromal deposits remains to be defined.

  15. Heterogeneous amylin fibril growth mechanisms imaged by total internal reflection fluorescence microscopy.

    Science.gov (United States)

    Patil, Sharadrao M; Mehta, Andrew; Jha, Suman; Alexandrescu, Andrei T

    2011-04-12

    Total internal reflection fluorescence microscopy has been used to visualize the fibrillization of amylin, a hormone which in aggregated forms plays a role in type 2 diabetes pathology. Data were obtained at acidic pH where fibrillization is hindered by the charging of histidine 18 and at slightly basic pH where the loss of charge on the histidine promotes aggregation. The experiments show three types of aggregate growth processes. In the earliest steps globular seeds are formed with some expanding radially during the course of the reaction. The dimensions of the globular seeds as well as their staining with the amyloid-specific dye thioflavin T indicate that they are plaques of short fibrils. The next species observed are fibrils that invariably grow from large globular seeds or smaller punctate granules. Fibril elongation appears to be unidirectional, although in some cases multiple fibrils radiate from a single seed or granule. After fibrils are formed, some show an increase in fluorescence intensity that we attribute to the growth of new fibrils alongside those previously formed. All three aggregation processes are suggestive of secondary (heterogeneous) nucleation mechanisms in which nucleation occurs on preformed fibrils. Consistently, electron micrographs show changes in fibril morphology well after fibrils are first formed, and the growth processes observed by fluorescence microscopy occur after the corresponding solution reactions have reached an initial apparent plateau. Taken together, the results highlight the importance of secondary nucleation in the fibrillization of amylin, as this could provide a pathway to continue fibril growth once an initial population of fibrils is established.

  16. Coconut (Cocos nucifera) Ethanolic Leaf Extract Reduces Amyloid-β (1-42) Aggregation and Paralysis Prevalence in Transgenic Caenorhabditis elegans Independently of Free Radical Scavenging and Acetylcholinesterase Inhibition.

    Science.gov (United States)

    Manalo, Rafael Vincent; Silvestre, Maries Ann; Barbosa, Aza Lea Anne; Medina, Paul Mark

    2017-04-21

    Virgin coconut oil (VCO) has been the subject of several studies which have aimed to alleviate Alzheimer's disease (AD) pathology, focusing on in vitro antioxidant and acetylcholinesterase (AChE) inhibitory activities. Here, we studied an underutilized and lesser-valued part of the coconut tree, specifically the leaves, using in vitro and in vivo approaches. Coconut leaf extract (CLE) was screened for antioxidant and AChE inhibitory properties in vitro and therapeutic effects in two strains of transgenic Caenorhabditis elegans expressing amyloid-β 1-42 (Aβ 1-42 ) in muscle cells. CLE demonstrated free radical scavenging activity with an EC 50 that is 79-fold less compared to ascorbic acid, and an AChE inhibitory activity that is 131-fold less compared to Rivastigmine. Surprisingly, in spite of its low antioxidant activity and AChE inhibition, CLE reduced Aβ deposits by 30.31% in CL2006 in a dose-independent manner, and reduced the percentage of paralyzed nematodes at the lowest concentration of CLE (159.38 μg/mL), compared to dH₂O/vehicle (control). Phytochemical analysis detected glycosides, anthocyanins, and hydrolyzable tannins in CLE, some of which are known to be anti-amyloidogenic. Taken together, these findings suggest that CLE metabolites alternatively decrease AB 1-42 aggregation and paralysis prevalence independently of free radical scavenging and AChE inhibition, and this warrants further investigation on the bioactive compounds of CLE.

  17. Proinsulin C-peptide interferes with insulin fibril formation

    International Nuclear Information System (INIS)

    Landreh, Michael; Stukenborg, Jan-Bernd; Willander, Hanna; Söder, Olle; Johansson, Jan; Jörnvall, Hans

    2012-01-01

    Highlights: ► Insulin and C-peptide can interact under insulin fibril forming conditions. ► C-peptide is incorporated into insulin aggregates and alters aggregation lag time. ► C-peptide changes insulin fibril morphology and affects backbone accessibility. ► C-peptide may be a regulator of fibril formation by β-cell granule proteins. -- Abstract: Insulin aggregation can prevent rapid insulin uptake and cause localized amyloidosis in the treatment of type-1 diabetes. In this study, we investigated the effect of C-peptide, the 31-residue peptide cleaved from proinsulin, on insulin fibrillation at optimal conditions for fibrillation. This is at low pH and high concentration, when the fibrils formed are regular and extended. We report that C-peptide then modulates the insulin aggregation lag time and profoundly changes the fibril appearance, to rounded clumps of short fibrils, which, however, still are Thioflavine T-positive. Electrospray ionization mass spectrometry also indicates that C-peptide interacts with aggregating insulin and is incorporated into the aggregates. Hydrogen/deuterium exchange mass spectrometry further reveals reduced backbone accessibility in insulin aggregates formed in the presence of C-peptide. Combined, these effects are similar to those of C-peptide on islet amyloid polypeptide fibrillation and suggest that C-peptide has a general ability to interact with amyloidogenic proteins from pancreatic β-cell granules. Considering the concentrations, these peptide interactions should be relevant also during physiological secretion, and even so at special sites post-secretory or under insulin treatment conditions in vivo.

  18. Electrochemistry of Alzheimer disease amyloid beta peptides.

    Science.gov (United States)

    Chiorcea-Paquim, Ana-Maria; Enache, Teodor Adrian; Oliveira-Brett, Ana Maria

    2018-02-13

    Alzheimer's disease (AD) is a widespread form of dementia that is estimated to affect 44.4 million people worldwide. AD pathology is closely related to the accumulation of amyloid beta (Aβ) peptides in fibrils and plagues, the small oligomeric intermediate species formed during the Aβ peptides aggregation presenting the highest neurotoxicity. This review discusses the recent advances on the Aβ peptides electrochemical characterisation. The Aβ peptides oxidation at a glassy carbon electrode occurs in one or two steps, depending on the amino acid sequence, length and content. The first electron transfer reaction corresponds to the tyrosine Tyr10 amino acid residue oxidation, and the second to all three histidine (His6, His13 and His14) and one methionine (Met35) amino acid residues. The Aβ peptides aggregation and amyloid fibril formation is electrochemically detected via the electroactive amino acids oxidation peak currents decrease that occurs in a time dependent manner. The Aβ peptides redox behaviour is correlated with changes in the adsorption morphology from initially random coiled structures, corresponding to the Aβ peptide monomers in random coil or in α-helix conformations, to aggregates and protofibrils and two types of fibrils, corresponding to the Aβ peptides in a β-sheet configuration, observed by atomic force microscopy. Electrochemical studies of Aβ peptides aggregation, mediated by the interaction with metal ions, in particular zinc, copper, and iron, and different methodologies concerning the detection of Aβ peptide biomarkers of AD in biological fluids, using electrochemical biosensors, are also discussed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. Efficacy and safety of rivaroxaban compared with warfarin among elderly patients with nonvalvular atrial fibrillation in the Rivaroxaban Once Daily, Oral, Direct Factor Xa Inhibition Compared With Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF).

    Science.gov (United States)

    Halperin, Jonathan L; Hankey, Graeme J; Wojdyla, Daniel M; Piccini, Jonathan P; Lokhnygina, Yuliya; Patel, Manesh R; Breithardt, Günter; Singer, Daniel E; Becker, Richard C; Hacke, Werner; Paolini, John F; Nessel, Christopher C; Mahaffey, Kenneth W; Califf, Robert M; Fox, Keith A A

    2014-07-08

    Nonvalvular atrial fibrillation is common in elderly patients, who face an elevated risk of stroke but difficulty sustaining warfarin treatment. The oral factor Xa inhibitor rivaroxaban was noninferior to warfarin in the Rivaroxaban Once Daily, Oral, Direct Factor Xa Inhibition Compared With Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF). This prespecified secondary analysis compares outcomes in older and younger patients. There were 6229 patients (44%) aged ≥75 years with atrial fibrillation and ≥2 stroke risk factors randomized to warfarin (target international normalized ratio=2.0-3.0) or rivaroxaban (20 mg daily; 15 mg if creatinine clearance <50 mL/min), double blind. The primary end point was stroke and systemic embolism by intention to treat. Over 10 866 patient-years, older participants had more primary events (2.57% versus 2.05%/100 patient-years; P=0.0068) and major bleeding (4.63% versus 2.74%/100 patient-years; P<0.0001). Stroke/systemic embolism rates were consistent among older (2.29% rivaroxaban versus 2.85% warfarin per 100 patient-years; hazard ratio=0.80; 95% confidence interval, 0.63-1.02) and younger patients (2.00% versus 2.10%/100 patient-years; hazard ratio=0.95; 95% confidence interval, 0.76-1.19; interaction P=0.313), as were major bleeding rates (≥75 years: 4.86% rivaroxaban versus 4.40% warfarin per 100 patient-years; hazard ratio=1.11; 95% confidence interval, 0.92-1.34; <75 years: 2.69% versus 2.79%/100 patient-years; hazard ratio=0.96; 95% confidence interval, 0.78-1.19; interaction P=0.336). Hemorrhagic stroke rates were similar in both age groups; there was no interaction between age and rivaroxaban response. Elderly patients had higher stroke and major bleeding rates than younger patients, but the efficacy and safety of rivaroxaban relative to warfarin did not differ with age, supporting rivaroxaban as an alternative for the elderly. © 2014 American Heart Association, Inc.

  20. A Novel Liposomal Nanoparticle for the Imaging of Amyloid Plaque by Magnetic Resonance Imaging.

    Science.gov (United States)

    Tanifum, Eric A; Ghaghada, Ketan; Vollert, Craig; Head, Elizabeth; Eriksen, Jason L; Annapragada, Ananth

    2016-01-01

    Amyloid binding molecules with greater hydrophilicity than existing ligands were synthesized. The lead candidate ET6-21 bound amyloid fibrils, and amyloid deposits in dog brain and human brain tissue ex vivo. The ligand was used to prepare novel amyloid-targeted liposomal nanoparticles. The preparation was tested in the Tg2576 and TetO/APP mouse models of amyloid deposition. Gd chelates and Indocyanine green were included in the particles for visualization by MRI and near-infrared microscopy. Upon intravenous injection, the particles successfully traversed the blood-brain barrier in these mice, and bound to the plaques. Magnetic resonance imaging (T1-MRI) conducted 4 days after injection demonstrated elevated signal in the brains of mice with amyloid plaques present. No signal was observed in amyloid-negative mice, or in amyloid-positive mice injected with an untargeted version of the same agent. The MRI results were confirmed by immunohistochemical and fluorescent microscopic examination of mouse brain sections, showing colocalization of the fluorescent tags and amyloid deposits.

  1. How does domain replacement affect fibril formation of the rabbit/human prion proteins.

    Directory of Open Access Journals (Sweden)

    Xu Yan

    Full Text Available It is known that in vivo human prion protein (PrP have the tendency to form fibril deposits and are associated with infectious fatal prion diseases, while the rabbit PrP does not readily form fibrils and is unlikely to cause prion diseases. Although we have previously demonstrated that amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and macromolecular crowding has different effects on fibril formation of the rabbit/human PrPs, we do not know which domains of PrPs cause such differences. In this study, we have constructed two PrP chimeras, rabbit chimera and human chimera, and investigated how domain replacement affects fibril formation of the rabbit/human PrPs.As revealed by thioflavin T binding assays and Sarkosyl-soluble SDS-PAGE, the presence of a strong crowding agent dramatically promotes fibril formation of both chimeras. As evidenced by circular dichroism, Fourier transform infrared spectroscopy, and proteinase K digestion assays, amyloid fibrils formed by human chimera have secondary structures and proteinase K-resistant features similar to those formed by the human PrP. However, amyloid fibrils formed by rabbit chimera have proteinase K-resistant features and secondary structures in crowded physiological environments different from those formed by the rabbit PrP, and secondary structures in dilute solutions similar to the rabbit PrP. The results from transmission electron microscopy show that macromolecular crowding caused human chimera but not rabbit chimera to form short fibrils and non-fibrillar particles.We demonstrate for the first time that the domains beyond PrP-H2H3 (β-strand 1, α-helix 1, and β-strand 2 have a remarkable effect on fibrillization of the rabbit PrP but almost no effect on the human PrP. Our findings can help to explain why amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and why macromolecular crowding has different

  2. Nanostructural Differentiation and Toxicity of Amyloid-β25-35 Aggregates Ensue from Distinct Secondary Conformation.

    Science.gov (United States)

    Song, Yongxiu; Li, Ping; Liu, Lei; Bortolini, Christian; Dong, Mingdong

    2018-01-15

    Amyloid nanostructures are originated from protein misfolding and aberrant aggregation, which is associated with the pathogenesis of many types of degenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease. The secondary conformation of peptides is of a fundamental importance for aggregation and toxicity of amyloid peptides. In this work, Aβ25-35, a fragment of amyloid β(1-42) (Aβ42), was selected to investigate the correlation between secondary structures and toxicity of amyloid fibrils. Furthermore, each aggregation assemblies show different cell membrane disruption and cytotoxicity. The structural analysis of amyloid aggregates originated from different secondary structure motifs is helpful to understand the mechanism of peptides/cell interactions in the pathogenesis of amyloid diseases.

  3. Amyloid-beta (Aβ₁₋₄₂)-induced paralysis in Caenorhabditis elegans is inhibited by the polyphenol quercetin through activation of protein degradation pathways.

    Science.gov (United States)

    Regitz, Charlotte; Dußling, Lisa Marie; Wenzel, Uwe

    2014-10-01

    Dietary polyphenols are suggested to play a role in the prevention of Alzheimer's disease, of which accumulation of aggregated beta amyloid (Aβ) is a key histopathological hallmark. We used the transgenic Caenorhabditis elegans strain CL2006, which expresses human Aβ₁₋₄₂ under control of a muscle-specific promoter and responds to Aβ₁₋₄₂ aggregation with paralysis, to test effects of the polyphenol quercetin on the phenotype. Quercetin dose-dependently decreased the amount of aggregated proteins in solution and also paralysis in CL2006. The knockdown of key components of unfolded protein response in mitochondria or the endoplasmic reticulum by RNA-interference (RNAi) enhanced paralysis in CL2006 but did not prevent the paralysis reducing activities of quercetin. RNAi for essential members of proteasomal protein degradation or macroautophagy also significantly increased paralysis but prevented quercetin from being effective. Quercetin increased proteasomal activity and, moreover, enhanced the flow of proteins through the macroautophagy pathway as reflected by reduced lysosome staining. The proteostasis network, including unfolded protein response, defines the aggregation of Aβ₁₋₄₂ and the associated paralysis phenotype in a nematode model for Alzheimer's disease. The polyphenol quercetin, by specifically activating macroautophagy and proteasomal degradation pathways, proved able to prevent Aβ₁₋₄₂ agregation and paralysis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Amyloid-beta oligomer detection by ELISA in cerebrospinal fluid and brain tissue

    NARCIS (Netherlands)

    Bruggink, K.A.; Jongbloed, W.; Biemans, E.A.L.M.; Veerhuis, R.; Claassen, J.A.H.R.; Kuiperij, H.B.; Verbeek, M.M.

    2013-01-01

    Amyloid-β (Aβ) deposits are important pathological hallmarks of Alzheimer's disease (AD). Aβ aggregates into fibrils; however, the intermediate oligomers are believed to be the most neurotoxic species and, therefore, are of great interest as potential biomarkers. Here, we have developed an

  5. Investigation on the Molecular Interactions Stabilizing the Structure of α-synuclein Fibril: An In silico Study.

    Science.gov (United States)

    Sanjeev, Airy; Mattaparthi, Venkata S K

    2017-01-01

    Amyloid fibrils represent stable form of many misfolded proteins associated with numerous diseases like Parkinson's Disease (PD), Type II diabetes and Alzheimer's disease (AD). α-synuclein protein is the principal constituent of Lewy bodies that are considered to be pathological hallmark of PD. Recently, a high resolution structure of α-synuclein protein that stacks together forming fibrils in brains of PD patients were identified. What structural features drive pathology of PD can now be possibly answered from the fibril structure of protein. To understand the molecular interactions those are responsible for the stability of the α- synuclein fibril structure. To study the molecular interactions stabilizing the α-synuclein fibril, we have used a high resolution amyloid fibril structure (PDB ID 2N0A). The molecular interactions in fibril structure were studied using PDBSum server. We then looked into the destabilization of α-synuclein fibril by disrupting the salt-bridge holding the strands and probable methods to decompose fibril into structurally distinct units using Top-domain web-server. The effect of salt-bridges on the stability of the fibril structure was studied by mutating one of the residues involved in the formation of salt-bridge using molecular dynamics simulation. Our results indicate a finite salt-bridge (E46-K80) is crucial for stability of protofibril. Besides, we observed hydrogen bonds and non-bonded contacts involved in fibril stabilization. We noticed α-synuclein dimer predominantly exists in conformations distinct from fibril. We characterized the salient molecular interactions in α-synuclein fibril and these findings may be useful to design potential inhibitors for the treatment of PD. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

    Science.gov (United States)

    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. Trifluoroethanol modulates amyloid formation by the all α-helical URN1 FF domain.

    Science.gov (United States)

    Marinelli, Patrizia; Castillo, Virginia; Ventura, Salvador

    2013-08-30

    Amyloid fibril formation is implicated in different human diseases. The transition between native α-helices and nonnative intermolecular β-sheets has been suggested to be a trigger of fibrillation in different conformational diseases. The FF domain of the URN1 splicing factor (URN1-FF) is a small all-α protein that populates a molten globule (MG) at low pH. Despite the fact that this conformation maintains most of the domain native secondary structure, it progressively converts into β-sheet enriched and highly ordered amyloid fibrils. In this study, we investigated if 2,2,2-trifluoroethanol (TFE) induced conformational changes that affect URN1-FF amyloid formation. Despite TFE having been shown to induce or increase the aggregation of both globular and disordered proteins at moderate concentrations, we demonstrate here that in the case of URN1-FF it reinforces its intrinsic α-helical structure, which competes the formation of aggregated assemblies. In addition, we show that TFE induces conformational diversity in URN1-FF fibrils, in such a way that the fibrils formed in the presence and absence of the cosolvent represent different polymorphs. It is suggested that the effect of TFE on both the soluble and aggregated states of URN1-FF depends on its ability to facilitate hydrogen bonding.

  8. HP-β-cyclodextrin as an inhibitor of amyloid-β aggregation and toxicity.

    Science.gov (United States)

    Ren, Baiping; Jiang, Binbo; Hu, Rundong; Zhang, Mingzhen; Chen, Hong; Ma, Jie; Sun, Yan; Jia, Lingyun; Zheng, Jie

    2016-07-27

    Amyloid deposits of misfolded amyloid-β protein (Aβ) on neuronal cells are a pathological hallmark of Alzheimer's disease (AD). Prevention of the abnormal Aβ aggregation has been considered as a promising therapeutic strategy for AD treatment. To prevent reinventing the wheel, we proposed to search the existing drug database for other diseases to identify potential Aβ inhibitors. Herein, we reported the inhibitory activity of HP-β-cyclodextrin (HP-β-CD), a well-known sugar used in drug delivery, genetic vector, environmental protection and treatment of Niemann-Pick disease type C1 (NPC1), against Aβ1-42 aggregation and Aβ-induced toxicity, with the aim of adding a new function as a sugar-based Aβ inhibitor. Experimental data showed that HP-β-CD molecules were not only nontoxic to cells, but also greatly inhibitedfibrillization and reduced Aβ-induced toxicity in a concentration-dependent manner. At an optimal molar ratio of Aβ : HP-β-CD = 1 : 2, HP-β-CD enabled the reduction of 60% of Aβ fibrils and increased the cell viability to 92%. Such concentration-dependent inhibitor capacity of HP-β-CD was likely attributed to several combined effects, including the enhancement of Aβ-HP-β-CD interactions, prevention of structural transition of Aβ peptides towards β-sheet structures, and reduction of self-aggregation of HP-β-CD. In parallel, molecular simulations further revealed the atomic details of HP-β-CD interacting with the Aβ oligomer, showing that HP-β-CD had a high tendency to interact with hydrophobic residues of Aβ in two β-strands and the N-terminal tail. More importantly, we identified that the inner hydrophobic cavity of HP-β-CD was a key active site for Aβ inhibition. Once the inner cavity of HP-β-CD was blocked by a small hydrophobic molecule of ferulic acid, HP-β-CD completely lost its inhibition capacity against Aβ. Given the already established pharmaceutical functions of HP-β-CD in drug delivery, our findings

  9. Inhibitory Activities of Antioxidant Flavonoids from Tamarix gallica on Amyloid Aggregation Related to Alzheimer's and Type 2 Diabetes Diseases.

    Science.gov (United States)

    Ben Hmidene, Asma; Hanaki, Mizuho; Murakami, Kazuma; Irie, Kazuhiro; Isoda, Hiroko; Shigemori, Hideyuki

    2017-01-01

    The prevention of amyloid aggregation is promising for the treatment of age-related diseases such as Alzheimer's (AD) and type 2 diabetes (T2D). Ten antioxidant flavonoids isolated from the medicinal halophyte Tamarix gallica were tested for their amyloid aggregation inhibition potential. Glucuronosylated flavonoids show relatively strong inhibitory activity of Amyloid β (Aβ) and human islet amyloid polypeptide (hIAPP) aggregation compared to their aglycone analogs. Structure-activity relationship of the flavonoids suggests that the catechol moiety is important for amyloid aggregation inhibition, while the methylation of the carboxyl group in the glucuronide moiety and of the hydroxyl group in the aglycone flavonoids decreased it.

  10. Amylin under examination. Fibrillogenic polypeptide of pancreatic amyloid

    Directory of Open Access Journals (Sweden)

    Małgorzata Marszałek

    2015-06-01

    Full Text Available In patients or animals affected by 2 type diabetes mellitus (diabetes mellitus type 2, DM2, non-insulin-dependent diabetes mellitus, NIDDM or pancreatic tumor disease e.g., insulinoma, some pathological deposits, called amyloid, are observed among cells of islets of Langerhans. Among other constituents, pancreatic deposits consist of an insoluble, fibrillar form of peptide neurohormone termed amylin, produced by pancreatic beta cells. It is thought that formation of fibrillar deposits of misfolded and aggregated peptide is highly toxic to beta cells and leads to cell dysfunction, cell loss, pancreas destruction and progress of the disease. This relatively small, 37-amino acid peptide constitutes a serious scientific, research and to some extent a medical problem. This article presents amylin as a fibrillating molecule which participates in formation of amyloid deposits in human and animal pancreas, Langerhans islets as a microenvironment of pancreatic amyloid formation, occurrence of amylin and amyloid in animals and humans, and physico-chemical requirements to meet to name amylin deposit as amyloid.

  11. Heterogeneous Seeding of a Prion Structure by a Generic Amyloid Form of the Fungal Prion-forming Domain HET-s(218-289)

    Energy Technology Data Exchange (ETDEWEB)

    Wan, William; Bian, Wen; McDonald, Michele; Kijac, Aleksandra; Wemmer, David E.; Stubbs, Gerald [UCB; (Vanderbilt); (LBNL)

    2013-11-13

    The fungal prion-forming domain HET-s(218–289) forms infectious amyloid fibrils at physiological pH that were shown by solid-state NMR to be assemblies of a two-rung β-solenoid structure. Under acidic conditions, HET-s(218–289) has been shown to form amyloid fibrils that have very low infectivity in vivo, but structural information about these fibrils has been very limited. We show by x-ray fiber diffraction that the HET-s(218–289) fibrils formed under acidic conditions have a stacked β-sheet architecture commonly found in short amyloidogenic peptides and denatured protein aggregates. At physiological pH, stacked β-sheet fibrils nucleate the formation of the infectious β-solenoid prions in a process of heterogeneous seeding, but do so with kinetic profiles distinct from those of spontaneous or homogeneous (seeded with infectious β-solenoid fibrils) fibrillization. Several serial passages of stacked β-sheet-seeded solutions lead to fibrillization kinetics similar to homogeneously seeded solutions. Our results directly show that structural mutation can occur between substantially different amyloid architectures, lending credence to the suggestion that the processes of strain adaptation and crossing species barriers are facilitated by structural mutation.

  12. [A review for recent advances in AA amyloid research and therapeutic approach to AA amyloidosis complicating rheumatoid arthritis].

    Science.gov (United States)

    Tamura, Hiroaki; Hasegawa, Kiminori

    2009-02-01

    AA amyloidosis is a life threatening clinical complication of chronic inflammatory diseases such as rheumatoid arthritis. It has been demonstrated biochemically that amyloidosis resulted from abnormal folding of proteins, which are deposited as insoluble fibrils in extracellular tissue, leading to the disruption of their normal function. In this regard, amyloidosis has been recognized as a conformation disorder. Interestingly, genetic polymorphisms of amyloid precursor protein (SAA) have been reported to associate with increased risk for AA amyloidosis. Also recent biochemical research revealed that SAA is synthesized under the influence of the proinflammatory cytokines, such as IL-6, TNF-alpha, IL-1. Additionally, it was suggested that amyloid deposits in extracellular tissue could reflect to the serum level of SAA in the reversible fashion, leading to the hypothesis that the control of the SAA synthesis could be beneficial to the treatment of amyloidosis. In this context, anti-cytokine therapies may be most effective. Especially the inhibition of IL-6 is critical to suppression of SAA production, so treatment with a humanized monoclonal antibody against human IL-6 receptor may not only ameliorate RA disease activity but also pave the way for the treatment of AA amyloidosis.

  13. Doliroside A from Dolichos falcata Klein suppressing amyloid β-protein 42 fibrillogenesis: An insight at molecular level.

    Directory of Open Access Journals (Sweden)

    Dongpu Li

    Full Text Available A bioactive chemical constituent, doliroside A, from Chinese traditional herbal medicine Dolichos falcata Klein was isolated, purified and identified by 60% ethanol extraction, thin layer chromatography (TLC, high performance liquid chromatography (HPLC and nuclear magnetic resonance (NMR spectroscopy. Molecular interaction mechanism between doliroside and amyloid β42 protein was evaluated by thioflavin T fluorescence (ThT, circular dichroism (CD, atomic force microscope (AFM, and differential scanning calorimeter (DSC from the aspects of kinetics, secondary structure, morphology, and thermodynamics, respectively. Results show that the purity of doliroside A is 99.9% by HPLC, and its chemical structure is identified by 1H- and 13C-NMR. Doliroside A is observed to be concentration-dependent inhibiting the fibrillation of Aβ42 with the IC50 value of 26.57 ± 1.6 μM. CD and DSC results imply that doliroside A can bind to the nuclei and oligomers of Aβ42 to form a stable complex and suppress Aβ42 fibrillation. AFM images show that doliroside A, after bound to the nuclei and oligomers, redirect Aβ42 into off-pathway, amorphous oligomers. These findings not only provide a full insight into the molecular interaction mechanisms between Aβ42 and doliroside A, but also facilitate the development of new native anti-AD drug of doliroside A compound.

  14. Vulnerability to ventricular fibrillation

    Science.gov (United States)

    Janse, Michiel J.

    1998-03-01

    One of the factors that favors the development of ventricular fibrillation is an increase in the dispersion of refractoriness. Experiments will be described in which an increase in dispersion in the recovery of excitability was determined during brief episodes of enhanced sympathetic nerve activity, known to increase the risk of fibrillation. Whereas in the normal heart ventricular fibrillation can be induced by a strong electrical shock, a premature stimulus of moderate intensity only induces fibrillation in the presence of regional ischemia, which greatly increases the dispersion of refractoriness. One factor that is of importance for the transition of reentrant ventricular tachycardia to ventricular fibrillation during acute regional ischemia is the subendocardial Purkinje system. After selective destruction of the Purkinje network by lugol, reentrant tachycardias still develop in the ischemic region, but they do not degenerate into fibrillation. Finally, attempts were made to determine the minimal mass of thin ventricular myocardium required to sustain fibrillation induced by burst pacing. This was done by freezing of subendocardial and midmural layers. The rim of surviving epicardial muscle had to be larger than 20 g. Extracellular electrograms during fibrillation in both the intact and the "frozen" left ventricle were indistinguishable, but activation patterns were markedly different. In the intact ventricle epicardial activation was compatible with multiple wavelet reentry, in the "frozen" heart a single, or at most two wandering reentrant waves were seen.

  15. YY-1224, a terpene trilactone-strengthened Ginkgo biloba, attenuates neurodegenerative changes induced by β-amyloid (1-42) or double transgenic overexpression of APP and PS1 via inhibition of cyclooxygenase-2.

    Science.gov (United States)

    Li, Zheng-Yi; Chung, Yoon Hee; Shin, Eun-Joo; Dang, Duy-Khanh; Jeong, Ji Hoon; Ko, Sung Kwon; Nah, Seung-Yeol; Baik, Tae Gon; Jhoo, Jin Hyeong; Ong, Wei-Yi; Nabeshima, Toshitaka; Kim, Hyoung-Chun

    2017-04-27

    Ginkgo biloba has been reported to possess free radical-scavenging antioxidant activity and anti-inflammatory properties. In our pilot study, YY-1224, a terpene trilactone-strengthened extract of G. biloba, showed anti-inflammatory, neurotrophic, and antioxidant effects. We investigated the pharmacological potential of YY-1224 in β-amyloid (Aβ) (1-42)-induced memory impairment using cyclooxygenase-2 (COX-2) knockout (-/-) and APPswe/PS1dE9 transgenic (APP/PS1 Tg) mice. Repeated treatment with YY-1224 significantly attenuated Aβ (1-42)-induced memory impairment in COX-2 (+/+) mice, but not in COX-2 (-/-) mice. YY-1224 significantly attenuated Aβ (1-42)-induced upregulation of platelet-activating factor (PAF) receptor gene expression, reactive oxygen species, and pro-inflammatory factors. In addition, YY-1224 significantly inhibited Aβ (1-42)-induced downregulation of PAF-acetylhydrolase-1 (PAF-AH-1) and peroxisome proliferator-activated receptor γ (PPARγ) gene expression. These changes were more pronounced in COX-2 (+/+) mice than in COX-2 (-/-) mice. YY-1224 significantly attenuated learning impairment, Aβ deposition, and pro-inflammatory microglial activation in APP/PS1 Tg mice, whereas it significantly enhanced PAF-AH and PPARγ expression. A preferential COX-2 inhibitor, meloxicam, did not affect the pharmacological activity by YY-1224, suggesting that the COX-2 gene is a critical mediator of the neuroprotective effects of YY-1224. The protective activity of YY-1224 appeared to be more efficacious than a standard G. biloba extract (Gb) against Aβ insult. Our results suggest that the protective effects of YY-1224 against Aβ toxicity may be associated with its PAF antagonistic- and PPARγ agonistic-potential as well as inhibition of the Aβ-mediated pro-inflammatory switch of microglia phenotypes through suppression of COX-2 expression.

  16. Longitudinal study of experimental induction of AA amyloidosis in mice seeded with homologous and heterologous AA fibrils.

    Science.gov (United States)

    Muhammad, Naeem; Murakami, Tomoaki; Inoshima, Yasuo; Ishiguro, Naotaka

    2016-09-01

    To investigate pathogenesis and kinetics of experimentally induced murine AA amyloidosis seeded with homologous (murine) and heterologous (bovine) AA fibrils. Experimental AA amyloidosis was induced by administration of inflammatory stimulus and preformed AA fibrils to a total of 111 female C57/Black mice. In this longitudinal study, heterologous (bovine) as well as homologous (murine) AA fibrils were injected intraperitoneally to mice in various combinations. Re-stimulation was done at 120 or 300 days post first inoculation. To analyze the intensity of amyloid depositions in mice organs, immunohistochemical techniques and image J software were used. Assessment of cytokines level in sera was done using a Mouse Th1/Th2/Th17 Cytokine CBA Kit. Incidence and severity of AA amyloidosis were quite low in mice inoculated with heterologous bovine AA fibrils than homologous murine one. Homologous AA fibrils administration at first and second inoculation caused maximum amount of amyloid depositions and severe systemic form of amyloidosis. Increase in the level of pro-inflammatory cytokine IL-6 was observed after first inoculation, while second inoculation caused a further increase in the level of anti-inflammatory cytokine IL-10. AA amyloidosis can be induced by heterologous as well as homologous AA fibrils. Severity of AA amyloidosis induced with homologous AA fibrils is higher compared to heterologous AA fibrils.

  17. Ischaemic cardiac outcomes in patients with atrial fibrillation treated with vitamin K antagonism or factor Xa inhibition: results from the ROCKET AF trial

    Science.gov (United States)

    Mahaffey, Kenneth W.; Stevens, Susanna R.; White, Harvey D.; Nessel, Christopher C.; Goodman, Shaun G.; Piccini, Jonathan P.; Patel, Manesh R.; Becker, Richard C.; Halperin, Jonathan L.; Hacke, Werner; Singer, Daniel E.; Hankey, Graeme J.; Califf, Robert M.; Fox, Keith A.A.; Breithardt, Günter

    2014-01-01

    Aims We investigated the prevalence of prior myocardial infarction (MI) and incidence of ischaemic cardiovascular (CV) events among atrial fibrillation (AF) patients. Methods and results In ROCKET AF, 14 264 patients with nonvalvular AF were randomized to rivaroxaban or warfarin. The key efficacy outcome for these analyses was CV death, MI, and unstable angina (UA). This pre-specified analysis was performed on patients while on treatment. Rates are per 100 patient-years. Overall, 2468 (17%) patients had prior MI at enrollment. Compared with patients without prior MI, these patients were more likely to be male (75 vs. 57%), on aspirin at baseline (47 vs. 34%), have prior congestive heart failure (78 vs. 59%), diabetes (47 vs. 39%), hypertension (94 vs. 90%), higher mean CHADS2 score (3.64 vs. 3.43), and fewer prior strokes or transient ischaemic attacks (46 vs. 54%). CV death, MI, or UA rates tended to be lower in patients assigned rivaroxaban compared with warfarin [2.70 vs. 3.15; hazard ratio (HR) 0.86, 95% confidence interval (CI) 0.73–1.00; P = 0.0509]. CV death, MI, or UA rates were higher in those with prior MI compared with no prior MI (6.68 vs. 2.19; HR 3.04, 95% CI 2.59–3.56) with consistent results for CV death, MI, or UA for rivaroxaban compared with warfarin in prior MI compared with no prior MI (P interaction = 0.10). Conclusion Prior MI was common and associated with substantial risk for subsequent cardiac events. Patients with prior MI assigned rivaroxaban compared with warfarin had a non-significant 14% reduction of ischaemic cardiac events. PMID:24132190

  18. Ischaemic cardiac outcomes in patients with atrial fibrillation treated with vitamin K antagonism or factor Xa inhibition: results from the ROCKET AF trial.

    Science.gov (United States)

    Mahaffey, Kenneth W; Stevens, Susanna R; White, Harvey D; Nessel, Christopher C; Goodman, Shaun G; Piccini, Jonathan P; Patel, Manesh R; Becker, Richard C; Halperin, Jonathan L; Hacke, Werner; Singer, Daniel E; Hankey, Graeme J; Califf, Robert M; Fox, Keith A A; Breithardt, Günter

    2014-01-01

    We investigated the prevalence of prior myocardial infarction (MI) and incidence of ischaemic cardiovascular (CV) events among atrial fibrillation (AF) patients. In ROCKET AF, 14 264 patients with nonvalvular AF were randomized to rivaroxaban or warfarin. The key efficacy outcome for these analyses was CV death, MI, and unstable angina (UA). This pre-specified analysis was performed on patients while on treatment. Rates are per 100 patient-years. Overall, 2468 (17%) patients had prior MI at enrollment. Compared with patients without prior MI, these patients were more likely to be male (75 vs. 57%), on aspirin at baseline (47 vs. 34%), have prior congestive heart failure (78 vs. 59%), diabetes (47 vs. 39%), hypertension (94 vs. 90%), higher mean CHADS2 score (3.64 vs. 3.43), and fewer prior strokes or transient ischaemic attacks (46 vs. 54%). CV death, MI, or UA rates tended to be lower in patients assigned rivaroxaban compared with warfarin [2.70 vs. 3.15; hazard ratio (HR) 0.86, 95% confidence interval (CI) 0.73-1.00; P = 0.0509]. CV death, MI, or UA rates were higher in those with prior MI compared with no prior MI (6.68 vs. 2.19; HR 3.04, 95% CI 2.59-3.56) with consistent results for CV death, MI, or UA for rivaroxaban compared with warfarin in prior MI compared with no prior MI (P interaction = 0.10). Prior MI was common and associated with substantial risk for subsequent cardiac events. Patients with prior MI assigned rivaroxaban compared with warfarin had a non-significant 14% reduction of ischaemic cardiac events.

  19. {beta} - amyloid imaging probes

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Jae Min [Seoul National University College of Medicine, Seoul (Korea, Republic of)

    2007-04-15

    Imaging distribution of {beta} - amyloid plaques in Alzheimer's disease is very important for early and accurate diagnosis. Early trial of the {beta} -amyloid plaques includes using radiolabeled peptides which can be only applied for peripheral {beta} - amyloid plaques due to limited penetration through the blood brain barrier (BBB). Congo red or Chrysamine G derivatives were labeled with Tc-99m for imaging {beta} - amyloid plaques of Alzheimer patient's brain without success due to problem with BBB penetration. Thioflavin T derivatives gave breakthrough for {beta} - amyloid imaging in vivo, and a benzothiazole derivative [C-11]6-OH-BTA-1 brought a great success. Many other benzothiazole, benzoxazole, benzofuran, imidazopyridine, and styrylbenzene derivatives have been labeled with F-18 and I-123 to improve the imaging quality. However, [C-11]6-OH-BTA-1 still remains as the best. However, short half-life of C-11 is a limitation of wide distribution of this agent. So, it is still required to develop an Tc-99m, F-18 or I-123 labeled agent for {beta} - amyloid imaging agent.

  20. Key aromatic/hydrophobic amino acids controlling a cross-amyloid peptide interaction versus amyloid self-assembly.

    Science.gov (United States)

    Bakou, Maria; Hille, Kathleen; Kracklauer, Michael; Spanopoulou, Anna; Frost, Christina V; Malideli, Eleni; Yan, Li-Mei; Caporale, Andrea; Zacharias, Martin; Kapurniotu, Aphrodite

    2017-09-01

    The interaction of the intrinsically disordered polypeptide islet amyloid polypeptide (IAPP), which is associated with type 2 diabetes (T2D), with the Alzheimer's disease amyloid-β (Aβ) peptide modulates their self-assembly into amyloid fibrils and may link the pathogeneses of these two cell-degenerative diseases. However, the molecular determinants of this interaction remain elusive. Using a systematic alanine scan approach, fluorescence spectroscopy, and other biophysical methods, including heterocomplex pulldown assays, far-UV CD spectroscopy, the thioflavin T binding assay, transmission EM, and molecular dynamics simulations, here we identified single aromatic/hydrophobic residues within the amyloid core IAPP region as hot spots or key residues of its cross-interaction with Aβ40(42) peptide. Importantly, we also find that none of these residues in isolation plays a key role in IAPP self-assembly, whereas simultaneous substitution of four aromatic/hydrophobic residues with Ala dramatically impairs both IAPP self-assembly and hetero-assembly with Aβ40(42). Furthermore, our experiments yielded several novel IAPP analogs, whose sequences are highly similar to that of IAPP but have distinct amyloid self- or cross-interaction potentials. The identified similarities and major differences controlling IAPP cross-peptide interaction with Aβ40(42) versus its amyloid self-assembly offer a molecular basis for understanding the underlying mechanisms. We propose that these insights will aid in designing intervention strategies and novel IAPP analogs for the management of type 2 diabetes, Alzheimer's disease, or other diseases related to IAPP dysfunction or cross-amyloid interactions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Amyloid β Oligomeric Species Present in the Lag Phase of Amyloid Formation.

    Directory of Open Access Journals (Sweden)

    Martin Wolff

    Full Text Available Alzheimer's disease (AD-associated amyloid β peptide (Aβ is one of the main actors in AD pathogenesis. Aβ is characterized by its high tendency to self-associate, leading to the generation of oligomers and amyloid fibrils. The elucidation of pathways and intermediates is crucial for the understanding of protein assembly mechanisms in general and in conjunction with neurodegenerative diseases, e.g., for the identification of new therapeutic targets. Our study focused on Aβ42 and its oligomeric assemblies in the lag phase of amyloid formation, as studied by sedimentation velocity (SV centrifugation. The assembly state of Aβ during the lag phase, the time required by an Aβ solution to reach the exponential growth phase of aggregation, was characterized by a dominant monomer fraction below 1 S and a population of oligomeric species between 4 and 16 S. From the oligomer population, two major species close to a 12-mer and an 18-mer with a globular shape were identified. The recurrence of these two species at different initial concentrations and experimental conditions as the smallest assemblies present in solution supports the existence of distinct, energetically favored assemblies in solution. The sizes of the two species suggest an Aβ42 aggregation pathway that is based on a basic hexameric building block. The study demonstrates the potential of SV analysis for the evaluation of protein aggregation pathways.

  2. Aggregation properties of a short peptide that mediates amyloid fibril ...

    Indian Academy of Sciences (India)

    Mandelkow E 2005 Tau aggregation is driven by a transition from random coil to beta sheet structure. Biochim. Biophys. Acta 1739 158–166 von Bergen M, Barghorn S, Li L, Marx A, Biernat J, Mandelkow. EM and Mandelkow E 2001 Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical.

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

    DEFF Research Database (Denmark)

    Pigolotti, Simone; Lizana, Ludvig; Otzen, Daniel

    2013-01-01

    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 t...

  4. Digoxin use in patients with atrial fibrillation and adverse cardiovascular outcomes: a retrospective analysis of the Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF).

    Science.gov (United States)

    Washam, Jeffrey B; Stevens, Susanna R; Lokhnygina, Yuliya; Halperin, Jonathan L; Breithardt, Günter; Singer, Daniel E; Mahaffey, Kenneth W; Hankey, Graeme J; Berkowitz, Scott D; Nessel, Christopher C; Fox, Keith A A; Califf, Robert M; Piccini, Jonathan P; Patel, Manesh R

    2015-06-13

    Digoxin is a widely used drug for ventricular rate control in patients with atrial fibrillation (AF), despite a scarcity of randomised trial data. We studied the use and outcomes of digoxin in patients in the Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF). For this retrospective analysis, we included and classified patients from ROCKET AF on the basis of digoxin use at baseline and during the study. Patients in ROCKET AF were recruited from 45 countries and had AF and risk factors putting them at moderate-to-high risk of stroke, with or without heart failure. We used Cox proportional hazards regression models adjusted for baseline characteristics and drugs to investigate the association of digoxin with all-cause mortality, vascular death, and sudden death. ROCKET AF was registered with ClinicalTrials.gov, number NCT00403767. In 14,171 randomly assigned patients, digoxin was used at baseline in 5239 (37%). Patients given digoxin were more likely to be female (42% vs 38%) and have a history of heart failure (73% vs 56%), diabetes (43% vs 38%), and persistent AF (88% vs 77%; p<0·0001 for each comparison). After adjustment, digoxin was associated with increased all-cause mortality (5·41 vs 4·30 events per 100 patients-years; hazard ratio 1·17; 95% CI 1·04-1·32; p=0·0093), vascular death (3·55 vs 2·69 per 100 patient-years; 1·19; 1·03-1·39, p=0·0201), and sudden death (1·68 vs 1·12 events per 100 patient-years; 1·36; 1·08-1·70, p=0·0076). Digoxin treatment was associated with a significant increase in all-cause mortality, vascular death, and sudden death in patients with AF. This association was independent of other measured prognostic factors, and although residual confounding could account for these results, these data show the possibility of digoxin having these effects. A randomised trial of digoxin in treatment of AF patients

  5. Quenched hydrogen-deuterium exchange NMR of a disease-relevant Aβ(1-42) amyloid polymorph.

    Science.gov (United States)

    Wälti, Marielle Aulikki; Orts, Julien; Riek, Roland

    2017-01-01

    Alzheimer's disease is associated with the aggregation into amyloid fibrils of Aβ(1-42) and Aβ(1-40) peptides. Interestingly, these fibrils often do not obtain one single structure but rather show different morphologies, so-called polymorphs. Here, we compare quenched hydrogen-deuterium (H/D) exchange of a disease-relevant Aβ(1-42) fibril for which the 3D structure has been determined by solid-state NMR with H/D exchange previously determined on another structural polymorph. This comparison reveals secondary structural differences between the two polymorphs suggesting that the two polymorphisms can be classified as segmental polymorphs.

  6. Albumin fibrillization induces apoptosis via integrin/FAK/Akt pathway

    Directory of Open Access Journals (Sweden)

    Liang Chi-Ming

    2009-01-01

    Full Text Available Abstract Background Numerous proteins can be converted to amyloid-like fibrils to increase cytotoxicity and induce apoptosis, but the methods generally require a high concentration of protein, vigorous shaking, or fibril seed. As well, the detailed mechanism of the cytotoxic effects is not well characterized. In this study, we have developed a novel process to convert native proteins into the fibrillar form. We used globular bovine serum albumin (BSA as a model protein to verify the properties of the fibrillar protein, investigated its cellular effects and studied the signaling cascade induced by the fibrillar protein. Results We induced BSA, a non-cytotoxic globular protein, to become fibril by a novel process involving Superdex-200 column chromatography in the presence of anionic or zwittergenic detergent(s. The column pore size was more important than column matrix composite in fibril formation. The fibrillar BSA induced apoptosis in BHK-21 cell as well as breast cancer cell line T47D. Pre-treating cells with anti-integrin antibodies blocked the apoptotic effect. Fibrillar BSA, but not globular BSA, bound to integrin, dephosphorylated focal adhesion kinase (FAK, Akt and glycogen synthase kinase-3β (GSK-3β. Conclusion We report on a novel process for converting globular proteins into fibrillar form to cause apoptosis by modulating the integrin/FAK/Akt/GSK-3β/caspase-3 signaling pathway. Our findings may be useful for understanding the pathogenesis of amyloid-like fibrils and applicable for the development of better therapeutic agents that target the underlying mechanism(s of the etiologic agents.

  7. Factors associated with major bleeding events: insights from the ROCKET AF trial (rivaroxaban once-daily oral direct factor Xa inhibition compared with vitamin K antagonism for prevention of stroke and embolism trial in atrial fibrillation).

    Science.gov (United States)

    Goodman, Shaun G; Wojdyla, Daniel M; Piccini, Jonathan P; White, Harvey D; Paolini, John F; Nessel, Christopher C; Berkowitz, Scott D; Mahaffey, Kenneth W; Patel, Manesh R; Sherwood, Matthew W; Becker, Richard C; Halperin, Jonathan L; Hacke, Werner; Singer, Daniel E; Hankey, Graeme J; Breithardt, Gunter; Fox, Keith A A; Califf, Robert M

    2014-03-11

    This study sought to report additional safety results from the ROCKET AF (Rivaroxaban Once-daily oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation). The ROCKET AF trial demonstrated similar risks of stroke/systemic embolism and major/nonmajor clinically relevant bleeding (principal safety endpoint) with rivaroxaban and warfarin. The risk of the principal safety and component bleeding endpoints with rivaroxaban versus warfarin were compared, and factors associated with major bleeding were examined in a multivariable model. The principal safety endpoint was similar in the rivaroxaban and warfarin groups (14.9 vs. 14.5 events/100 patient-years; hazard ratio: 1.03; 95% confidence interval: 0.96 to 1.11). Major bleeding risk increased with age, but there were no differences between treatments in each age category (<65, 65 to 74, ≥75 years; pinteraction = 0.59). Compared with those without (n = 13,455), patients with a major bleed (n = 781) were more likely to be older, current/prior smokers, have prior gastrointestinal (GI) bleeding, mild anemia, and a lower calculated creatinine clearance and less likely to be female or have a prior stroke/transient ischemic attack. Increasing age, baseline diastolic blood pressure (DBP) ≥90 mm Hg, history of chronic obstructive pulmonary disease or GI bleeding, prior acetylsalicylic acid use, and anemia were independently associated with major bleeding risk; female sex and DBP <90 mm Hg were associated with a decreased risk. Rivaroxaban and warfarin had similar risk for major/nonmajor clinically relevant bleeding. Age, sex, DBP, prior GI bleeding, prior acetylsalicylic acid use, and anemia were associated with the risk of major bleeding. (An Efficacy and Safety Study of Rivaroxaban With Warfarin for the Prevention of Stroke and Non-Central Nervous System Systemic Embolism in Patients With Non-Valvular Atrial Fibrillation: NCT00403767

  8. Affinity of (nat/68)Ga-Labelled Curcumin and Curcuminoid Complexes for β-Amyloid Plaques: Towards the Development of New Metal-Curcumin Based Radiotracers.

    Science.gov (United States)

    Rubagotti, Sara; Croci, Stefania; Ferrari, Erika; Iori, Michele; Capponi, Pier C; Lorenzini, Luca; Calzà, Laura; Versari, Annibale; Asti, Mattia

    2016-09-06

    Curcumin derivatives labelled with fluorine-18 or technetium-99m have recently shown their potential as diagnostic tools for Alzheimer's disease. Nevertheless, no study by exploiting the labelling with gallium-68 has been performed so far, in spite of its suitable properties (positron emitter, generator produced radionuclide). Herein, an evaluation of the affinity for synthetic β-amyloid fibrils and for amyloid plaques of three (nat/68)Ga-labelled curcumin analogues, namely curcumin curcumin (CUR), bis-dehydroxy-curcumin (bDHC) and diacetyl-curcumin (DAC), was performed. Affinity and specificity were tested in vitro on amyloid synthetic fibrils by using gallium-68 labelled compounds. Post-mortem brain cryosections from Tg2576 mice were used for the ex vivo visualization of amyloid plaques. The affinity of (68)Ga(CUR)₂⁺, (68)Ga(DAC)₂⁺, and (68)Ga(bDHC)₂⁺ for synthetic β-amyloid fibrils was moderate and their uptake could be observed in vitro. On the other hand, amyloid plaques could not be visualized on brain sections of Tg2576 mice after injection, probably due to the low stability of the complexes in vivo and of a hampered passage through the blood-brain barrier. Like curcumin, all (nat/68)Ga-curcuminoid complexes maintain a high affinity for β-amyloid plaques. However, structural modifications are still needed to improve their applicability as radiotracers in vivo.

  9. Photoinduced fibrils formation of chicken egg white lysozyme under native conditions.

    Science.gov (United States)

    Xie, Jin-Bing; Cao, Yi; Pan, Hai; Qin, Meng; Yan, Zhi-Qiang; Xiong, Xiang; Wang, Wei

    2012-11-01

    Recent findings showed that transiently accessing structurally native-like yet energetically higher conformational states is sufficient to trigger the formation of protein fibrils. Typically, these conformational states are made available through changing solvent conditions or introducing mutations. Here we show a novel way to initialize fibril formation for Chicken egg white lysozyme (CEWL) under native conditions via controlled UV illumination. Through a cassette of tryptophan-based photochemistry, the two terminal disulfide bonds in CEWL can be selectively reduced. The reduced CEWL is then converted to conformational states with the C-terminal fragment floppy upon thermal fluctuation. These states serve as precursors for the fibrillar aggregation. Intriguingly, the CEWL fibrils are stabilized by intermolecular disulfide bonds instead of noncovalent β-sheet structures, distinct from the amyloid-like lysozyme fibrils reported before. Based on the experimental evidences and all-atom molecular dynamics simulation, we proposed a "runaway domain-swapping" model for the structure of the CEWL fibrils, in which each CEWL molecule swaps the C-terminal fragment into the complementary position of the adjacent molecule along the fibrils. We anticipate that this fibrillation mechanism can be extended to many other disulfide-containing proteins. Our study stands for the first example of formation of protein fibrils under native conditions upon UV illumination and poses the potential danger of low UV dose to organisms at the protein level. Copyright © 2012 Wiley Periodicals, Inc.

  10. A Kinetic Aggregation Assay Enabling Selective and Sensitive Aβ Amyloid Quantification in Cells and Tissues

    Science.gov (United States)

    Du, Deguo; Murray, Amber N.; Cohen, Ehud; Kim, Hyun-Eui; Simkovsky, Ryan; Dillin, Andrew; Kelly, Jeffery W.

    2011-01-01

    The process of amyloid-β (Aβ) fibril formation is genetically and pathologically linked to Alzheimer's disease (AD). Thus, a selective and sensitive method for the quantification of Aβ amyloid fibrils in complex biological samples enables a variety of hypotheses to be tested. Herein we report the basis for a quantitative in vitro kinetic aggregation assay that detects seeding-competent Aβ aggregates in mammalian cell culture media, in Caenorhabditis elegans lysate and in mouse brain homogenate. Sonicated, proteinase K treated Aβ-fibril-containing tissue homogenates or cell culture media were added to an initially monomeric Aβ1–40 reporter peptide to seed an in vitro nucleated aggregation reaction. The reduction in the half time (t50) of the amyloid growth phase is proportional to the quantity of seeding-competent Aβ aggregates present in the biological sample. An ion exchange resin amyloid isolation strategy from complex biological samples is demonstrated as an alternative to improve the sensitivity and linearity of the kinetic aggregation assay. PMID:21268584

  11. Nanoscopic and Photonic Ultrastructural Characterization of Two Distinct Insulin Amyloid States

    Directory of Open Access Journals (Sweden)

    Mikael Lindgren

    2012-02-01

    Full Text Available Two different conformational isoforms or amyloid strains of insulin with different cytotoxic capacity have been described previously. Herein these filamentous and fibrillar amyloid states of insulin were investigated using biophysical and spectroscopic techniques in combination with luminescent conjugated oligothiophenes (LCO. This new class of fluorescent probes has a well defined molecular structure with a distinct number of thiophene units that can adopt different dihedral angles depending on its binding site to an amyloid structure. Based on data from surface charge, hydrophobicity, fluorescence spectroscopy and imaging, along with atomic force microscopy (AFM, we deduce the ultrastructure and fluorescent properties of LCO stained insulin fibrils and filaments. Combined total internal reflection fluorescence microscopy (TIRFM and AFM revealed rigid linear fibrous assemblies of fibrils whereas filaments showed a short curvilinear morphology which assemble into cloudy deposits. All studied LCOs bound to the filaments afforded more blue-shifted excitation and emission spectra in contrast to those corresponding to the fibril indicating a different LCO binding site, which was also supported by less efficient hydrophobic probe binding. Taken together, the multi-tool approach used here indicates the power of ultrastructure identification applying AFM together with LCO fluorescence interrogation, including TIRFM, to resolve structural differences between amyloid states.

  12. Overexpression of heparanase lowers the amyloid burden in amyloid-β precursor protein transgenic mice.

    Science.gov (United States)

    Jendresen, Charlotte B; Cui, Hao; Zhang, Xiao; Vlodavsky, Israel; Nilsson, Lars N G; Li, Jin-Ping

    2015-02-20

    Heparan sulfate (HS) and HS proteoglycans (HSPGs) colocalize with amyloid-β (Aβ) deposits in Alzheimer disease brain and in Aβ precursor protein (AβPP) transgenic mouse models. Heparanase is an endoglycosidase that specifically degrades the unbranched glycosaminoglycan side chains of HSPGs. The aim of this study was to test the hypothesis that HS and HSPGs are active participators of Aβ pathogenesis in vivo. We therefore generated a double-transgenic mouse model overexpressing both human heparanase and human AβPP harboring the Swedish mutation (tgHpa*Swe). Overexpression of heparanase did not affect AβPP processing because the steady-state levels of Aβ1-40, Aβ1-42, and soluble AβPP β were the same in 2- to 3-month-old double-transgenic tgHpa*Swe and single-transgenic tgSwe mice. In contrast, the Congo red-positive amyloid burden was significantly lower in 15-month-old tgHpa*Swe brain than in tgSwe brain. Likewise, the Aβ burden, measured by Aβx-40 and Aβx-42 immunohistochemistry, was reduced significantly in tgHpa*Swe brain. The intensity of HS-stained plaques correlated with the Aβx-42 burden and was reduced in tgHpa*Swe mice. Moreover, the HS-like molecule heparin facilitated Aβ1-42-aggregation in an in vitro Thioflavin T assay. The findings suggest that HSPGs contribute to amyloid deposition in tgSwe mice by increasing Aβ fibril formation because heparanase-induced fragmentation of HS led to a reduced amyloid burden. Therefore, drugs interfering with Aβ-HSPG interactions might be a potential strategy for Alzheimer disease treatment. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. What Is Atrial Fibrillation?

    Science.gov (United States)

    ANSWERS by heart Cardiovascular Conditions What Is Atrial Fibrillation? Your heart has a natural pacemaker, called the “sinus node,” that makes electrical signals. These signals cause the heart to contract and pump ...

  14. Amyloid Beta Mediates Memory Formation

    Science.gov (United States)

    Garcia-Osta, Ana; Alberini, Cristina M.

    2009-01-01

    The amyloid precursor protein (APP) undergoes sequential cleavages to generate various polypeptides, including the amyloid [beta] (1-42) peptide (A[beta][1-42]), which is believed to play a major role in amyloid plaque formation in Alzheimer's disease (AD). Here we provide evidence that, in contrast with its pathological role when accumulated,…

  15. Solution structures of {beta}-amyloid{sub 10-35} and {beta}-amyloid{sub 10-35} PEG3000 aggregates.

    Energy Technology Data Exchange (ETDEWEB)

    Benzinger, T. L. S.; Burkoth, T. S.; Gordon, D.; Lynn, D. G.; Meredith, S. C.; Morgan, D. M.; Seifert, S.; Thiyagarajan, P.; Urban, V.

    1999-07-02

    Small angle neutron and x-ray scattering (SANS/SAXS) studies were conducted on the structure of the aggregates formed from both the truncated model peptide {beta}-Amyloid(10-35) (A{beta}{sub 10-35}) and a block copolymer {beta}-Amyloid (10-35)-PEG3000 (A{beta}{sub 10-35}-PEG) in D{sub 2}O at pHs from 3.0 to 7.0. These studies indicate that A{beta}{sub 10-35} aggregates into rod-like particles (fibril) and their radii are strongly dependent on the Pm of the solution. The fibril-fibril association in A{beta}{sub 10-35} solutions is less of pH < 5.6, but becomes larger at higher pH. A{beta}{sub 10-35}-PEG also assembles into rod-like particles whose radius is larger by about 30 {angstrom} than that for A{beta}{sub 10-35} fibril at pH 4.2, while it is about 23 {angstrom} larger at higher pH. Contrast matching SAXS/SANS experiments that eliminate the coherent scattering from PEG reveal that PEG moiety is located at the periphery of the fibril. Also, the mass per unit length of the peptide portion is similar for both A{beta}{sub 10-35} and A{beta}{sub 10-35}-PEG fibrils at pH 5.6. The mass per unit length of the rods from SANS provides key information on the packing of A{beta}{sub 10-35} peptides in the fibril.

  16. BETASCAN: probable beta-amyloids identified by pairwise probabilistic analysis.

    Directory of Open Access Journals (Sweden)

    Allen W Bryan

    2009-03-01

    Full Text Available Amyloids and prion proteins are clinically and biologically important beta-structures, whose supersecondary structures are difficult to determine by standard experimental or computational means. In addition, significant conformational heterogeneity is known or suspected to exist in many amyloid fibrils. Recent work has indicated the utility of pairwise probabilistic statistics in beta-structure prediction. We develop here a new strategy for beta-structure prediction, emphasizing the determination of beta-strands and pairs of beta-strands as fundamental units of beta-structure. Our program, BETASCAN, calculates likelihood scores for potential beta-strands and strand-pairs based on correlations observed in parallel beta-sheets. The program then determines the strands and pairs with the greatest local likelihood for all of the sequence's potential beta-structures. BETASCAN suggests multiple alternate folding patterns and assigns relative a priori probabilities based solely on amino acid sequence, probability tables, and pre-chosen parameters. The algorithm compares favorably with the results of previous algorithms (BETAPRO, PASTA, SALSA, TANGO, and Zyggregator in beta-structure prediction and amyloid propensity prediction. Accurate prediction is demonstrated for experimentally determined amyloid beta-structures, for a set of known beta-aggregates, and for the parallel beta-strands of beta-helices, amyloid-like globular proteins. BETASCAN is able both to detect beta-strands with higher sensitivity and to detect the edges of beta-strands in a richly beta-like sequence. For two proteins (Abeta and Het-s, there exist multiple sets of experimental data implying contradictory structures; BETASCAN is able to detect each competing structure as a potential structure variant. The ability to correlate multiple alternate beta-structures to experiment opens the possibility of computational investigation of prion strains and structural heterogeneity of amyloid

  17. Curcumin Promotes A-beta Fibrillation and Reduces Neurotoxicity in Transgenic Drosophila

    Science.gov (United States)

    Caesar, Ina; Jonson, Maria; Nilsson, K. Peter R.; Thor, Stefan; Hammarström, Per

    2012-01-01

    The pathology of Alzheimer's disease (AD) is characterized by the presence of extracellular deposits of misfolded and aggregated amyloid-β (Aβ) peptide and intraneuronal accumulation of tangles comprised of hyperphosphorylated Tau protein. For several years, the natural compound curcumin has been proposed to be a candidate for enhanced clearance of toxic Aβ amyloid. In this study we have studied the potency of feeding curcumin as a drug candidate to alleviate Aβ toxicity in transgenic Drosophila. The longevity as well as the locomotor activity of five different AD model genotypes, measured relative to a control line, showed up to 75% improved lifespan and activity for curcumin fed flies. In contrast to the majority of studies of curcumin effects on amyloid we did not observe any decrease in the amount of Aβ deposition following curcumin treatment. Conformation-dependent spectra from p-FTAA, a luminescent conjugated oligothiophene bound to Aβ deposits in different Drosophila genotypes over time, indicated accelerated pre-fibrillar to fibril conversion of Aβ1–42 in curcumin treated flies. This finding was supported by in vitro fibrillation assays of recombinant Aβ1–42. Our study shows that curcumin promotes amyloid fibril conversion by reducing the pre-fibrillar/oligomeric species of Aβ, resulting in a reduced neurotoxicity in Drosophila. PMID:22348084

  18. Characterization of fibrillation process of α-synuclein at the initial stage

    International Nuclear Information System (INIS)

    Tashiro, Mitsuru; Kojima, Masaki; Kihara, Hiroshi; Kasai, Kouki; Kamiyoshihara, Tomoaki; Ueda, Kenji; Shimotakahara, Sakurako

    2008-01-01

    α-Synuclein is the major component of the filamentous Lewy bodies and Lewy-related neurites, neuropathological hallmarks of Parkinson's disease. Although numerous studies on α-synuclein fibrillation have been reported, the molecular mechanisms of aggregation and fibrillation at the initial stage are still unclear. In the present study, structural properties and propensities to form fibrils of α-synuclein at the initial stage were investigated using 2D 1 H- 15 N NMR spectroscopy, electron microscope, and small angle X-ray scattering (SAXS). Observation of the 2D 1 H- 15 N HSQC spectra indicated significant attenuation of many cross peak intensities in the regions of KTKEGV-type repeats and the non-Aβ component of Alzheimer's disease amyloid (NAC), suggesting that these regions contributed fibril formation. Oligomerization comprising heptamer was successfully monitored at the initial stage using the time-dependent SAXS measurements

  19. Detection of AA-type amyloid protein in labial salivary glands.

    Science.gov (United States)

    Sacsaquispe, Sonia-Julia; Antúnez-de Mayolo, Eleazar-Antonio; Vicetti, Rodolfo; Delgado, Wilson-Alejandro

    2011-03-01

    Among the diverse forms of amyloidosis, secondary type is the most frequent one. Diagnosis of amyloid deposition is based on the identification of the fibrillary protein amyloid by means of Congo Red (CR) or crystal violet (CV) stains, but these techniques do not differentiate between the different types of amyloid fibrils. The aim of this study was to identify by immunofluorescence (IF) AA amyloid a pathological fibrillar low-molecular-weight protein formed by cleavage of serum amyloid A (SAA) protein in labial salivary gland (LSG) biopsies from patients with secondary amyloidosis. 98 LSG were studied, 65 were from patients with secondary amyloidosis and 33 from subjects with chronic inflammatory diseases without evidence of this anomaly. All sections were stained with hematoxylin and eosin (H &E), CV, CR and IF using anti-AA antibodies. Positive and negative controls were used for all techniques. CV and CR demonstrated that the amyloid substance was found mainly distributed periductally (93.8%), followed by periacinar and perivascular locations (p <0.001); however, the IF demonstrated that amyloid AA substance predominates in the periacinar area (73.8%), followed by periductal and perivascular locations (p <0.001). IF has a sensitivity of 83%, 100% of specificity, 100% of predictive positive value and 75% of predictive negative value. The results of this study confirm the efficacy of the LSG biopsy as a highly reliable method for diagnosis of secondary amyloidosis.

  20. Insights into the variability of nucleated amyloid polymerization by a minimalistic model of stochastic protein assembly

    Energy Technology Data Exchange (ETDEWEB)

    Eugène, Sarah, E-mail: Sarah.Eugene@inria.fr; Doumic, Marie, E-mail: Philippe.Robert@inria.fr, E-mail: Marie.Doumic@inria.fr [INRIA de Paris, 2 Rue Simone Iff, CS 42112, 75589 Paris Cedex 12 (France); Sorbonne Universités, UPMC Université Pierre et Marie Curie, UMR 7598, Laboratoire Jacques-Louis Lions, F-75005 Paris (France); Xue, Wei-Feng, E-mail: W.F.Xue@kent.ac.uk [School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ (United Kingdom); Robert, Philippe, E-mail: Philippe.Robert@inria.fr [INRIA de Paris, 2 Rue Simone Iff, CS 42112, 75589 Paris Cedex 12 (France)

    2016-05-07

    Self-assembly of proteins into amyloid aggregates is an important biological phenomenon associated with human diseases such as Alzheimer’s disease. Amyloid fibrils also have potential applications in nano-engineering of biomaterials. The kinetics of amyloid assembly show an exponential growth phase preceded by a lag phase, variable in duration as seen in bulk experiments and experiments that mimic the small volumes of cells. Here, to investigate the origins and the properties of the observed variability in the lag phase of amyloid assembly currently not accounted for by deterministic nucleation dependent mechanisms, we formulate a new stochastic minimal model that is capable of describing the characteristics of amyloid growth curves despite its simplicity. We then solve the stochastic differential equations of our model and give mathematical proof of a central limit theorem for the sample growth trajectories of the nucleated aggregation process. These results give an asymptotic description for our simple model, from which closed form analytical results capable of describing and predicting the variability of nucleated amyloid assembly were derived. We also demonstrate the application of our results to inform experiments in a conceptually friendly and clear fashion. Our model offers a new perspective and paves the way for a new and efficient approach on extracting vital information regarding the key initial events of amyloid formation.

  1. Prevention of the β-amyloid peptide-induced inflammatory process by inhibition of double-stranded RNA-dependent protein kinase in primary murine mixed co-cultures

    Directory of Open Access Journals (Sweden)

    Terro F

    2011-06-01

    Full Text Available Abstract Background Inflammation may be involved in the pathogenesis of Alzheimer's disease (AD. There has been little success with anti-inflammatory drugs in AD, while the promise of anti-inflammatory treatment is more evident in experimental models. A new anti-inflammatory strategy requires a better understanding of molecular mechanisms. Among the plethora of signaling pathways activated by β-amyloid (Aβ peptides, the nuclear factor-kappa B (NF-κB pathway could be an interesting target. In virus-infected cells, double-stranded RNA-dependent protein kinase (PKR controls the NF-κB signaling pathway. It is well-known that PKR is activated in AD. This led us to study the effect of a specific inhibitor of PKR on the Aβ42-induced inflammatory response in primary mixed murine co-cultures, allowing interactions between neurons, astrocytes and microglia. Methods Primary mixed murine co-cultures were prepared in three steps: a primary culture of astrocytes and microglia for 14 days, then a primary culture of neurons and astrocytes which were cultured with microglia purified from the first culture. Before exposure to Aβ neurotoxicity (72 h, co-cultures were treated with compound C16, a specific inhibitor of PKR. Levels of tumor necrosis factor-α (TNFα, interleukin (IL-1β, and IL-6 were assessed by ELISA. Levels of PT451-PKR and activation of IκB, NF-κB and caspase-3 were assessed by western blotting. Apoptosis was also followed using annexin V-FITC immunostaining kit. Subcellular distribution of PT451-PKR was assessed by confocal immunofluorescence and morphological structure of cells by scanning electron microscopy. Data were analysed using one-way ANOVA followed by a Newman-Keuls' post hoc test Results In these co-cultures, PKR inhibition prevented Aβ42-induced activation of IκB and NF-κB, strongly decreased production and release of tumor necrosis factor (TNFα and interleukin (IL-1β, and limited apoptosis. Conclusion In spite of the

  2. The N-terminal helix controls the transition between the soluble and amyloid states of an FF domain.

    Science.gov (United States)

    Castillo, Virginia; Chiti, Fabrizio; Ventura, Salvador

    2013-01-01

    Protein aggregation is linked to the onset of an increasing number of human nonneuropathic (either localized or systemic) and neurodegenerative disorders. In particular, misfolding of native α-helical structures and their self-assembly into nonnative intermolecular β-sheets has been proposed to trigger amyloid fibril formation in Alzheimer's and Parkinson's diseases. Here, we use a battery of biophysical techniques to elucidate the conformational conversion of native α-helices into amyloid fibrils using an all-α FF domain as a model system. We show that under mild denaturing conditions at low pH this FF domain self-assembles into amyloid fibrils. Theoretical and experimental dissection of the secondary structure elements in this domain indicates that the helix 1 at the N-terminus has both the highest α-helical and amyloid propensities, controlling the transition between soluble and aggregated states of the protein. The data illustrates the overlap between the propensity to form native α-helices and amyloid structures in protein segments. The results presented contribute to explain why proteins cannot avoid the presence of aggregation-prone regions and indeed use stable α-helices as a strategy to neutralize such potentially deleterious stretches.

  3. Nucleation and growth of elastin-like peptide fibril multilayers: an in situ atomic force microscopy study.

    Science.gov (United States)

    Yang, Guocheng; Wong, Michael K; Lin, Lauren E; Yip, Christopher M

    2011-12-09

    Controlling how molecules assemble into complex supramolecular architectures requires careful consideration of the subtle inter- and intra-molecular interactions that control their association. This is particularly crucial in the context of assembly at interfaces, where both surface chemistry and structure can play a role in directing structure formation. We report here the results of a study into the self-assembly of the elastin-like peptide EP I on structurally modified highly ordered pyrolytic graphite, including the role of spatial confinement on fibril nucleation and the growth of oriented fibril multilayers. In situ atomic force microscopy performed in fluid and at elevated temperature provided direct evidence of frustrated fibril nuclei and oriented growth of independent fibril domains. These results portend the application of this in situ strategy for studies of the nucleation and growth mechanisms of other fibril- and amyloid-forming proteins.

  4. Calcium-dependent and -independent binding of the pentraxin serum amyloid P component to glycosaminoglycans and amyloid proteins

    DEFF Research Database (Denmark)

    Danielsen, B; Sørensen, I J; Nybo, Mads

    1997-01-01

    and beta2M) by ELISA. An increase in the dose-dependent binding of SAP to heparan sulfate, AA-protein and beta2M was observed as the pH decreased from 8.0 to 5.0. Furthermore, a lower, but significant Ca2(+)-independent binding of SAP to heparan sulfate, dermatan sulfate, AA protein and the amyloid...... precursor protein beta2M was observed. This binding was also enhanced at slightly acid pH, most pronounced at pH 5.0. The results of this study indicate that SAP can exhibit both Ca2(+)-dependent and -independent binding to ligands involved in amyloid fibril formation and that the binding is enhanced under...

  5. Unfolding, aggregation, and seeded amyloid formation of lysine-58-cleaved beta(2)-microglobulin

    DEFF Research Database (Denmark)

    Heegaard, N.H.H.; Jørgensen, T.J.D.; Rozlosnik, N.

    2005-01-01

    . Using amide hydrogen/deuterium exchange monitored by mass spectrometry, we show that Delta K58-beta(2)m has increased unfolding rates compared to wt-beta(2)m and that unfolding is highly temperature dependent. The unfolding rate is I order of magnitude faster in Delta K58-beta(2)M than in wt-beta(2)m...... in wt-beta(2)m shows extensive amyloid fibrillation in Delta K58-beta(2)m samples. The results highlight the instability and amyloidogenicity under near physiological conditions of a slightly modified beta(2)m variant generated by limited proteolysis and illustrate stages of amyloid formation from early...

  6. Effect of the surface charge of artificial model membranes on the aggregation of amyloid β-peptide.

    Science.gov (United States)

    Sabaté, Raimon; Espargaró, Alba; Barbosa-Barros, Lucyanna; Ventura, Salvador; Estelrich, Joan

    2012-08-01

    The neurotoxicity effect of the β-amyloid (Aβ) peptide, the primary constituent of senile plaques in Alzheimer's disease, occurs through interactions with neuronal membranes. Here, we attempt to clarify the mechanisms and consequences of the interaction of Aβ with lipid membranes. We have used liposomes as a model of biological membrane, and have devoted particular attention to the bilayer charge effect. Our results show that insertion and surface association of peptide with membrane, increased in a membrane charge-dependent manner, lead to a reduction of Aβ soluble species, lag time elongation and an increase in the inter-molecular β-sheet ratio of amyloid fibrils. In addition, our findings suggest that the fine balance between peptide insertion and surface association modulates Aβ aggregation, influencing the amyloid fibrils concentration as well as their morphology. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  7. Amide proton solvent protection in amylin fibrils probed by quenched hydrogen exchange NMR.

    Directory of Open Access Journals (Sweden)

    Andrei T Alexandrescu

    Full Text Available Amylin is an endocrine hormone that accumulates in amyloid plaques in patients with advanced type 2 diabetes. The amyloid plaques have been implicated in the destruction of pancreatic β-cells, which synthesize amylin and insulin. To better characterize the secondary structure of amylin in amyloid fibrils we assigned the NMR spectrum of the unfolded state in 95% DMSO and used a quenched hydrogen-deuterium exchange technique to look at amide proton solvent protection in the fibrils. In this technique, partially exchanged fibrils are dissolved in 95% DMSO and information about amide proton occupancy in the fibrils is determined from DMSO-denatured monomers. Hydrogen exchange lifetimes at pH 7.6 and 37°C vary between ∼5 h for the unstructured N-terminus to 600 h for amide protons in the two β-strands that form inter-molecular hydrogen bonds between amylin monomers along the length of the fibril. Based on the protection data we conclude that residues A8-H18 and I26-Y37 comprise the two β-strands in amylin fibrils. There is variation in protection within the β-strands, particularly for strand β1 where only residues F15-H18 are strongly protected. Differences in protection appear to be due to restrictions on backbone dynamics imposed by the packing of two-layers of C2-symmetry-related β-hairpins in the protofilament structure, with strand β1 positioned on the surface and β2 in the interior.

  8. Silymarin effect on amyloid-β plaque accumulation and gene expression of APP in an Alzheimer’s disease rat model

    OpenAIRE

    Yaghmaei, Parichehreh; Azarfar, Katia; Dezfulian, Mehrooz; Ebrahim-Habibi, Azadeh

    2014-01-01

    Background The deposition of amyloid peptides is associated with Alzheimer’s disease (AD). These amyloid peptides are derived from the amyloid protein precursor (APP). Silymarin, a standardized extract of milk thistle, which is currently used in liver diseases, may be effective in the inhibition of amyloid formation. However, its effect has not been assessed on APP expression. Results In this study, first, the effect of silymarin was examined on the passive avoidance learning in a rat model o...

  9. Identification of key amino acid residues modulating intracellular and in vitro microcin E492 amyloid formation

    Directory of Open Access Journals (Sweden)

    Paulina eAguilera

    2016-01-01

    Full Text Available Microcin E492 (MccE492 is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril’s morphology and formation kinetics in vitro have been well characterized, however it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in E. coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophillic probes, 2-4´-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54-63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59, which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54-63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although

  10. [Signaling indicator of ventricular fibrillation].

    Science.gov (United States)

    Agizim, G M; Pasichnik, T V; Sherman, A M

    1976-01-01

    The purpose of the work was to design an appliance for reliable detection of ventricular fibrillation. Frequency-amplitude spectrum of electrocardiograms taken in dogs with normal rhythm and fibrillation, as well as the impulse ratio in normalcy, tachycardia and in fibrillation were investigated. An appliance for automatic separation of ventricular fibrillation by referring to pulse ratio is described. The designed device is shown to have a high degree of discrimination and noise-immunity.

  11. Positron emission tomography (PET) utilizing Pittsburgh compound B (PIB) for detection of amyloid heart deposits in hereditary transthyretin amyloidosis (ATTR).

    Science.gov (United States)

    Pilebro, Björn; Arvidsson, Sandra; Lindqvist, Per; Sundström, Torbjörn; Westermark, Per; Antoni, Gunnar; Suhr, Ole; Sörensen, Jens

    2018-02-01

    DPD scintigraphy has been advocated for imaging cardiac amyloid in ATTR amyloidosis. PET utilizing 11 C-Pittsburgh compound B (PIB) is the gold standard for imaging brain amyloid in Alzheimer's disease. PIB was recently shown to identify cardiac amyloidosis in both AL and ATTR amyloidosis. In the ATTR population, two types of amyloid fibrils exist, one containing fragmented and full-length TTR (type A) and the other only full-length TTR (type B). The aim of this study was to further evaluate PIB-PET in patients with hereditary ATTR amyloidosis. Ten patients with biopsy-proven V30M ATTR amyloidosis and discrete or no signs of cardiac involvement were included. Patients were grouped according to TTR-fragmentation. All underwent DPD scintigraphy, echocardiography, and PIB-PET. A left ventricular PIB-retention index (PIB-RI) was established and compared to five normal volunteers. PIB-RI was increased in all patients (P PIB-PET, in contrast to DPD scintigraphy, has the potential to specifically identify cardiac amyloid depositions irrespective of amyloid fibril composition. The heart appears to be a target organ for amyloid deposition in ATTR amyloidosis.

  12. A Food and Drug Administration-approved asthma therapeutic agent impacts amyloid β in the brain in a transgenic model of Alzheimer disease.

    Science.gov (United States)

    Hori, Yukiko; Takeda, Shuko; Cho, Hansang; Wegmann, Susanne; Shoup, Timothy M; Takahashi, Kazue; Irimia, Daniel; Elmaleh, David R; Hyman, Bradley T; Hudry, Eloise

    2015-01-23

    Interfering with the assembly of Amyloid β (Aβ) peptides from monomer to oligomeric species and fibrils or promoting their clearance from the brain are targets of anti-Aβ-directed therapies in Alzheimer disease. Here we demonstrate that cromolyn sodium (disodium cromoglycate), a Food and Drug Administration-approved drug already in use for the treatment of asthma, efficiently inhibits the aggregation of Aβ monomers into higher-order oligomers and fibrils in vitro without affecting Aβ production. In vivo, the levels of soluble Aβ are decreased by over 50% after only 1 week of daily intraperitoneally administered cromolyn sodium. Additional in vivo microdialysis studies also show that this compound decreases the half-life of soluble Aβ in the brain. These data suggest a clear effect of a peripherally administered, Food and Drug Administration-approved medication on Aβ economy, supporting further investigation of the potential long-term efficacy of cromolyn sodium in Alzheimer disease. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Atrial Fibrillation (AF or AFib)

    Science.gov (United States)

    ... Peripheral Artery Disease Venous Thromboembolism Aortic Aneurysm More Atrial Fibrillation (AF or AFib) Updated:Aug 14,2017 What ... to the Terms and Conditions and Privacy Policy Atrial Fibrillation • Introduction • What is Atrial Fibrillation? • Why AFib Matters ...

  14. Efficient purification of arsenic-contaminated water using amyloid-carbon hybrid membranes.

    Science.gov (United States)

    Bolisetty, Sreenath; Reinhold, Noemi; Zeder, Christophe; Orozco, Monica N; Mezzenga, Raffaele

    2017-05-23

    We show the purification of arsenic-contaminated water using amyloid fibril-based membranes, which adsorb both the arsenate (+5) and arsenite (+3) oxidation forms at efficiencies of ∼99%. Binding isotherms indicate that amyloid fibrils possess multiple binding residues capable of strongly adsorbing arsenic ions via metal-ligand interactions, delaying the saturation of the membrane. We also show that these membranes can be reused for several cycles without any efficiency drop, and validate our technology in purifying real contaminated ground water by removing arsenic with an efficiency as high as 99.6%. These results make this technology promising for inexpensive, efficient and low-energy removal of arsenic from contaminated water.

  15. Atrial Fibrillation and Hyperthyroidism

    Directory of Open Access Journals (Sweden)

    Jayaprasad N

    2005-10-01

    Full Text Available Atrial fibrillation occurs in 10 – 15% of patients with hyperthyroidism. Low serum thyrotropin concentration is an independent risk factor for atrial fibrillation. Thyroid hormone contributes to arrythmogenic activity by altering the electrophysiological characteristics of atrial myocytes by shortening the action potential duration, enhancing automaticity and triggered activity in the pulmonary vein cardio myocytes. Hyperthyroidism results in excess mortality from increased incidence of circulatory diseases and dysrhythmias. Incidence of cerebral embolism is more in hyperthyroid patients with atrial fibrillation, especially in the elderly and anti-coagulation is indicated in them. Treatment of hyperthyroidism results in conversion to sinus rhythm in up to two-third of patients. Beta-blockers reduce left ventricular hypertrophy and atrial and ventricular arrhythmias in patients with hyperthyroidism. Treatment of sub clinical hyperthyroidism is controversial. Optimizing dose of thyroxine treatment in those with replacement therapy and beta-blockers is useful in exogenous subclinical hyperthyroidism.

  16. Concentration dependent switch in the kinetic pathway of lysozyme fibrillation: Spectroscopic and microscopic analysis

    Science.gov (United States)

    Kiran Kumar, E.; Prasad, Deepak Kumar; Prakash Prabhu, N.

    2017-08-01

    Formation of amyloid fibrils is found to be a general tendency of many proteins. Investigating the kinetic mechanisms and structural features of the intermediates and the final fibrillar state is essential to understand their role in amyloid diseases. Lysozyme, a notable model protein for amyloidogenic studies, readily formed fibrils in vitro at neutral pH in the presence of urea. It, however, showed two different kinetic pathways under varying urea concentrations when probed with thioflavin T (ThT) fluorescence. In 2 M urea, lysozyme followed a nucleation-dependent fibril formation pathway which was not altered by varying the protein concentration from 2 mg/ml to 8 mg/ml. In 4 M urea, the protein exhibited concentration dependent change in the mechanism. At lower protein concentrations, lysozyme formed fibrils without any detectable nuclei (nucleation-independent polymerization pathway). When the concentration of the protein was increased above 3 mg/ml, the protein followed nucleation-dependent polymerization pathway as observed in the case of 2 M urea condition. This was further verified using microscopic images of the fibrils. The kinetic parameters such as lag time, elongation rate, and fibrillation half-time, which were derived from ThT fluorescence changes, showed linear dependency against the initial protein concentration suggested that under the nucleation-dependent pathway conditions, the protein followed primary-nucleation mechanism without any significant secondary nucleation events. The results also suggested that the differences in the initial protein conformation might alter the mechanism of fibrillation; however, at the higher protein concentrations lysozyme shifted to nucleation-dependent pathway.

  17. Amyloid-linked cellular toxicity triggered by bacterial inclusion bodies

    International Nuclear Information System (INIS)

    Gonzalez-Montalban, Nuria; Villaverde, Antonio; Aris, Anna

    2007-01-01

    The aggregation of proteins in the form of amyloid fibrils and plaques is the characteristic feature of some pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. The mechanisms by which the aggregation processes result in cell damage are under intense investigation but recent data indicate that prefibrillar aggregates are the most proximate mediators of toxicity rather than mature fibrils. Since it has been shown that prefibrillar forms of the nondisease-related misfolded proteins are highly toxic to cultured mammalian cells we have studied the cytoxicity associated to bacterial inclusion bodies that have been recently described as protein deposits presenting amyloid-like structures. We have proved that bacterial inclusion bodies composed by a misfolding-prone β-galactosidase fusion protein are clearly toxic for mammalian cells but the β-galactosidase wild type enzyme forming more structured thermal aggregates does not impair cell viability, despite it also binds and enter into the cells. These results are in the line that the most cytotoxic aggregates are early prefibrilar assemblies but discard the hypothesis that the membrane destabilization is Key event to subsequent disruption of cellular processes, such as ion balance, oxidative state and the eventually cell death

  18. Constant region of a kappa III immunoglobulin light chain as a major AL-amyloid protein

    DEFF Research Database (Denmark)

    Engvig, J P; Olsen, K E; Gislefoss, R E

    1998-01-01

    AL-amyloidoses are generally described as a group of disorders in which N-terminal fragments of monoclonal immunoglobulin light chains are transferred into amyloid fibrils. We have, by amino acid sequence analyses and immunological methods, characterized the Bence-Jones protein and the correspond......AL-amyloidoses are generally described as a group of disorders in which N-terminal fragments of monoclonal immunoglobulin light chains are transferred into amyloid fibrils. We have, by amino acid sequence analyses and immunological methods, characterized the Bence-Jones protein...... and the corresponding AL protein as a kappa III immunoglobulin light chain from material of a patient with systemic AL-amyloidosis presenting as a local inguinal tumour. The two proteins showed some unique features. The major part of the AL amyloid fibril protein consisted of C-terminal fragments of the Bence......-Jones protein. Furthermore, both the Bence-Jones protein and the AL protein were glycosylated, with possibly a glycosylation in the constant part of the light chain....

  19. Modulation of atrial fibrillation

    NARCIS (Netherlands)

    Geuzebroek, G.S.C.

    2013-01-01

    In this thesis we investigate the results of various surgical procedures for atrial fibrillation which have been performed in the last 2 decades in the Sint Antonius Hospital, Nieuwegein, The Netherlands. In the 1990s the classical Maze III procedure was the main surgical technique for

  20. Orientation of aromatic residues in amyloid cores: Structural insights into prion fiber diversity

    KAUST Repository

    Reymer, Anna

    2014-11-17

    Structural conversion of one given protein sequence into different amyloid states, resulting in distinct phenotypes, is one of the most intriguing phenomena of protein biology. Despite great efforts the structural origin of prion diversity remains elusive, mainly because amyloids are insoluble yet noncrystalline and therefore not easily amenable to traditional structural-biology methods. We investigate two different phenotypic prion strains, weak and strong, of yeast translation termination factor Sup35 with respect to angular orientation of tyrosines using polarized light spectroscopy. By applying a combination of alignment methods the degree of fiber orientation can be assessed, which allows a relatively accurate determination of the aromatic ring angles. Surprisingly, the strains show identical average orientations of the tyrosines, which are evenly spread through the amyloid core. Small variations between the two strains are related to the local environment of a fraction of tyrosines outside the core, potentially reflecting differences in fibril packing.

  1. Histochemical Differential Diagnosis and Polarization Optical Analysis of Amyloid and Amyloidosis

    Directory of Open Access Journals (Sweden)

    M. Bély

    2006-01-01

    Full Text Available Amyloidosis is characterized by extracellular deposition of protein fibrils of chemically heterogeneous composition. Early recognition and identification of amyloid deposits allows an early start of therapy, which may entail a better prognosis. Congo red staining according to Romhányi (1971 is a highly specific and sensitive method for early microscopic recognition of amyloidosis. The main and most important types of amyloidosis may be distinguished by classic histochemical methods of performate pretreatment according to Romhányi (1979, or by KMnO4 oxidation according to Wright (1977 followed by Congo red staining and viewed under polarized light. Differences in the speed of breakdown (disintegration of amyloid deposits according to Bély and Apáthy allow a more precise distinction of various types of amyloid.

  2. Sugar microarray via click chemistry: molecular recognition with lectins and amyloid β (1–42

    Directory of Open Access Journals (Sweden)

    Erino Matsumoto, Takahiro Yamauchi, Tomohiro Fukuda and Yoshiko Miura

    2009-01-01

    Full Text Available Sugar microarrays were fabricated on various substrates via click chemistry. Acetylene-terminated substrates were prepared by forming self-assembled monolayers (SAMs on a gold substrate with alkyl-disulfide and on silicon, quartz and glass substrates with a silane-coupling reagent. The gold substrates were subjected to surface plasmon resonance measurements, and the quartz and glass substrates were subjected to spectroscopy measurements and optical microscopy observation. The saccharide-immobilized substrate on the gold substrate showed specific interaction with the corresponding lectin, and the saccharides showed inert surface properties to other proteins with a high signal-to-noise ratio. We also focused on the saccharide–protein interaction on protein amyloidosis of Alzheimer amyloid β. Amyloid β peptide showed conformation transition on the saccharide-immobilization substrate into a β-sheet, and fibril formation and amyloid aggregates were found on the specific saccharides.

  3. Variation of amino acid sequences of serum amyloid a (SAA) and immunohistochemical analysis of amyloid a (AA) in Japanese domestic cats.

    Science.gov (United States)

    Tei, Meina; Uchida, Kazuyuki; Chambers, James K; Watanabe, Ken-Ichi; Tamamoto, Takashi; Ohno, Koichi; Nakayama, Hiroyuki

    2018-02-02

    Amyloid A (AA) amyloidosis, a fatal systemic amyloid disease, occurs secondary to chronic inflammatory conditions in humans. Although persistently elevated serum amyloid A (SAA) levels are required for its pathogenesis, not all individuals with chronic inflammation necessarily develop AA amyloidosis. Furthermore, many diseases in cats are associated with the elevated production of SAA, whereas only a small number actually develop AA amyloidosis. We hypothesized that a genetic mutation in the SAA gene may strongly contribute to the pathogenesis of feline AA amyloidosis. In the present study, genomic DNA from four Japanese domestic cats (JDCs) with AA amyloidosis and from five without amyloidosis was analyzed using polymerase chain reaction (PCR) amplification and direct sequencing. We identified the novel variation combination of 45R-51A in the deduced amino acid sequences of four JDCs with amyloidosis and five without. However, there was no relationship between amino acid variations and the distribution of AA amyloid deposits, indicating that differences in SAA sequences do not contribute to the pathogenesis of AA amyloidosis. Immunohistochemical analysis using antisera against the three different parts of the feline SAA protein-i.e., the N-terminal, central, and C-terminal regions-revealed that feline AA contained the C-terminus, unlike human AA. These results indicate that the cleavage and degradation of the C-terminus are not essential for amyloid fibril formation in JDCs.

  4. Sodium dodecyl sulphate (SDS) induced changes in propensity and kinetics of α-lactalbumin fibrillation.

    Science.gov (United States)

    Kumar, E Kiran; Qumar, Shamsul; Prabhu, N Prakash

    2015-11-01

    Understanding surfactants induced changes on protein folding, aggregation, and fibrillation has a lot of implications in their laboratory and industrial applications. The effect of an anionic surfactant, sodium dodecyl sulphate (SDS), on fibrillation of an acidic protein α-lactalbumin (α-LA) at neutral pH condition was investigated. SDS at lower concentrations increased the lag time by nearly two-fold whereas the fibril elongation rate was not significantly altered. At the concentrations above 0.2mM, SDS lengthened the lag time by many-fold (∼60), but fibril elongation was accelerated by 3-6 fold. At the concentrations above 2mM, SDS inhibited α-LA fibrillation and led it to the formation of amorphous aggregates. These results were compared with the effect of SDS on the fibrillation of lysozyme, a basic protein. Though fibril inhibition was observed on both the proteins at the micellar concentrations of SDS, there were differences in the effect on lag time and elongation rate at the lower concentrations of SDS. This suggests that the inhibition of protein fibrillation by SDS-micelles might be a common mechanism irrespective of the surface charges on protein. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Complement activation by the amyloid proteins A beta peptide and beta 2-microglobulin

    DEFF Research Database (Denmark)

    Nybo, Mads; Nielsen, E H; Svehag, S E

    1999-01-01

    Complement activation (CA) has been reported to play a role in the pathogenesis of Alzheimer's disease (AD). To investigate whether CA may contribute to amyloidogenesis in general, the CA potential of different amyloid fibril proteins was tested. CA induced by A beta preparations containing soluble...... protein, protofilaments and some fibrils or only fibrils in a solid phase system (ELISA) was modest with a slow kinetics compared to the positive delta IgG control. Soluble A beta induced no detectable CA in a liquid phase system (complement consumption assay) while fibrillar A beta caused CA at 200 mg....../ml and higher concentrations. Soluble beta 2-microglobulin (beta 2M) purified from peritoneal dialysates was found to be as potent a complement activator as A beta in both solid and liquid phase systems while beta 2M purified from urine exhibited lower activity, a difference which may be explained...

  6. HYPERTHYROIDISM AND ATRIAL FIBRILLATION

    Directory of Open Access Journals (Sweden)

    I. M. Marusenko

    2017-01-01

    Full Text Available Review on a problem of the development of atrial fibrillation in patients with thyrotoxicosis is presented. Thyrotoxicosis is one of the most frequent endocrine diseases, conceding only to a diabetes mellitus. The most frequent reasons of hyperthyroidism are Graves’ disease and functional thyroid autonomy. The authors give an analysis of data on the cardiac effects of thyrotoxicosis, features of heart remodeling under the influence of thyroid hyperfunction, prevalence of atrial fibrillation in thyrotoxicosis, depending on age, as well as the possibility of restoring sinus rhythm in the combination of these diseases. Particular attention is paid to the effect on the heart of subclinical thyrotoxicosis, which is defined as a dysfunction of the thyroid gland, characterized by low serum concentration of thyrotropin, normal values of free thyroxine and free triiodothyronine. Subclinical hyperthyroidism is also capable of causing heart remodeling and diastolic dysfunction.Prevalence of thyrotoxicosis in elderly people is higher in areas of iodine deficiency; it is relevant for our country due to the large territory of iodine deficiency. In elderly patients, the cardiac effects of thyrotoxicosis prevail in the clinical picture, that makes it difficult to diagnose endocrine disorders, and correction of thyrotoxicosis is critically important for the successful control of the heart rhythm. The article also discusses the problem of thyrotoxic cardiomyopathy, caused by the toxic effect of excess thyroid hormones: features of this heart disorder, factors affecting its formation, clinical significance and contribution to the development of rhythm disturbances. The greatest significance is the development of atrial fibrillation as a result of thyrotox-icosis in older patients who already have various cardiovascular diseases.Atrial fibrillation is the most frequent heart rhythm disorder in thyrotoxicosis. The main cause of arrhythmia in hyperthyroidism is the

  7. Contribution of Electrostatics in the Fibril Stability of a Model Ionic-Complementary Peptide.

    Science.gov (United States)

    Owczarz, Marta; Casalini, Tommaso; Motta, Anna C; Morbidelli, Massimo; Arosio, Paolo

    2015-12-14

    In this work we quantified the role of electrostatic interactions in the self-assembly of a model amphiphilic peptide (RADA 16-I) into fibrillar structures by a combination of size exclusion chromatography and molecular simulations. For the peptide under investigation, it is found that a net charge of +0.75 represents the ideal condition to promote the formation of regular amyloid fibrils. Lower net charges favor the formation of amorphous precipitates, while larger net charges destabilize the fibrillar aggregates and promote a reversible dissociation of monomers from the ends of the fibrils. By quantifying the dependence of the equilibrium constant of this reversible reaction on the pH value and the peptide net charge, we show that electrostatic interactions contribute largely to the free energy of fibril formation. The addition of both salt and a charged destabilizer (guanidinium hydrochloride) at moderate concentration (0.3-1 M) shifts the monomer-fibril equilibrium toward the fibrillar state. Whereas the first effect can be explained by charge screening of electrostatic repulsion only, the promotion of fibril formation in the presence of guanidinium hydrochloride is also attributed to modifications of the peptide conformation. The results of this work indicate that the global peptide net charge is a key property that correlates well with the fibril stability, although the peptide conformation and the surface charge distribution also contribute to the aggregation propensity.

  8. Thioredoxin-interacting protein promotes islet amyloid polypeptide expression through miR-124a and FoxA2.

    Science.gov (United States)

    Jing, Gu; Westwell-Roper, Clara; Chen, Junqin; Xu, Guanlan; Verchere, C Bruce; Shalev, Anath

    2014-04-25

    Thioredoxin-interacting protein (TXNIP) is up-regulated by glucose and diabetes and plays a critical role in glucotoxicity, inflammation, and beta-cell apoptosis, whereas we have found that TXNIP deficiency protects against diabetes. Interestingly, human islet amyloid polypeptide (IAPP) is also induced by glucose, aggregates into insoluble amyloid fibrils found in islets of most individuals with type 2 diabetes and promotes inflammation and beta-cell cytotoxicity. However, so far no connection between TXNIP and IAPP signaling had been reported. Using TXNIP gain and loss of function experiments, INS-1 beta-cells and beta-cell-specific Txnip knock-out mice, we now found that TXNIP regulates IAPP expression. Promoter analyses and chromatin-immunoprecipitation assays further demonstrated that TXNIP increases IAPP expression at the transcriptional level, and we discovered that TXNIP-induced FoxA2 (forkhead box A2) transcription factor expression was conferring this effect by promoting FoxA2 enrichment at the proximal FoxA2 site in the IAPP promoter. Moreover, we found that TXNIP down-regulates miR-124a expression, a microRNA known to directly target FoxA2. Indeed, miR-124a overexpression led to decreased FoxA2 expression and IAPP promoter occupancy and to a significant reduction in IAPP mRNA and protein expression and also effectively inhibited TXNIP-induced IAPP expression. Thus, our studies have identified a novel TXNIP/miR-124a/FoxA2/IAPP signaling cascade linking the critical beta-cell signaling pathways of TXNIP and IAPP and thereby provide new mechanistic insight into an important aspect of transcriptional regulation and beta-cell biology.

  9. Thioredoxin-interacting Protein Promotes Islet Amyloid Polypeptide Expression through miR-124a and FoxA2*

    Science.gov (United States)

    Jing, Gu; Westwell-Roper, Clara; Chen, Junqin; Xu, Guanlan; Verchere, C. Bruce; Shalev, Anath

    2014-01-01

    Thioredoxin-interacting protein (TXNIP) is up-regulated by glucose and diabetes and plays a critical role in glucotoxicity, inflammation, and beta-cell apoptosis, whereas we have found that TXNIP deficiency protects against diabetes. Interestingly, human islet amyloid polypeptide (IAPP) is also induced by glucose, aggregates into insoluble amyloid fibrils found in islets of most individuals with type 2 diabetes and promotes inflammation and beta-cell cytotoxicity. However, so far no connection between TXNIP and IAPP signaling had been reported. Using TXNIP gain and loss of function experiments, INS-1 beta-cells and beta-cell-specific Txnip knock-out mice, we now found that TXNIP regulates IAPP expression. Promoter analyses and chromatin-immunoprecipitation assays further demonstrated that TXNIP increases IAPP expression at the transcriptional level, and we discovered that TXNIP-induced FoxA2 (forkhead box A2) transcription factor expression was conferring this effect by promoting FoxA2 enrichment at the proximal FoxA2 site in the IAPP promoter. Moreover, we found that TXNIP down-regulates miR-124a expression, a microRNA known to directly target FoxA2. Indeed, miR-124a overexpression led to decreased FoxA2 expression and IAPP promoter occupancy and to a significant reduction in IAPP mRNA and protein expression and also effectively inhibited TXNIP-induced IAPP expression. Thus, our studies have identified a novel TXNIP/miR-124a/FoxA2/IAPP signaling cascade linking the critical beta-cell signaling pathways of TXNIP and IAPP and thereby provide new mechanistic insight into an important aspect of transcriptional regulation and beta-cell biology. PMID:24627476

  10. Magnetite-Amyloid-β deteriorates activity and functional organization in an in vitro model for Alzheimer’s disease

    Science.gov (United States)

    Teller, Sara; Tahirbegi, Islam Bogachan; Mir, Mònica; Samitier, Josep; Soriano, Jordi

    2015-11-01

    The understanding of the key mechanisms behind human brain deterioration in Alzheimer’ disease (AD) is a highly active field of research. The most widespread hypothesis considers a cascade of events initiated by amyloid-β peptide fibrils that ultimately lead to the formation of the lethal amyloid plaques. Recent studies have shown that other agents, in particular magnetite, can also play a pivotal role. To shed light on the action of magnetite and amyloid-β in the deterioration of neuronal circuits, we investigated their capacity to alter spontaneous activity patterns in cultured neuronal networks. Using a versatile experimental platform that allows the parallel monitoring of several cultures, the activity in controls was compared with the one in cultures dosed with magnetite, amyloid-β and magnetite-amyloid-β complex. A prominent degradation in spontaneous activity was observed solely when amyloid-β and magnetite acted together. Our work suggests that magnetite nanoparticles have a more prominent role in AD than previously thought, and may bring new insights in the understanding of the damaging action of magnetite-amyloid-β complex. Our experimental system also offers new interesting perspectives to explore key biochemical players in neurological disorders through a controlled, model system manner.

  11. Heterologous amyloid seeding: revisiting the role of acetylcholinesterase in Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Létitia Jean

    2007-07-01

    Full Text Available Neurodegenerative diseases associated with abnormal protein folding and ordered aggregation require an initial trigger which may be infectious, inherited, post-inflammatory or idiopathic. Proteolytic cleavage to generate vulnerable precursors, such as amyloid-beta peptide (Abeta production via beta and gamma secretases in Alzheimer's Disease (AD, is one such trigger, but the proteolytic removal of these fragments is also aetiologically important. The levels of Abeta in the central nervous system are regulated by several catabolic proteases, including insulysin (IDE and neprilysin (NEP. The known association of human acetylcholinesterase (hAChE with pathological aggregates in AD together with its ability to increase Abeta fibrilization prompted us to search for proteolytic triggers that could enhance this process. The hAChE C-terminal domain (T40, AChE(575-614 is an exposed amphiphilic alpha-helix involved in enzyme oligomerisation, but it also contains a conformational switch region (CSR with high propensity for conversion to non-native (hidden beta-strand, a property associated with amyloidogenicity. A synthetic peptide (AChE(586-599 encompassing the CSR region shares homology with Abeta and forms beta-sheet amyloid fibrils. We investigated the influence of IDE and NEP proteolysis on the formation and degradation of relevant hAChE beta-sheet species. By combining reverse-phase HPLC and mass spectrometry, we established that the enzyme digestion profiles on T40 versus AChE(586-599, or versus Abeta, differed. Moreover, IDE digestion of T40 triggered the conformational switch from alpha- to beta-structures, resulting in surfactant CSR species that self-assembled into amyloid fibril precursors (oligomers. Crucially, these CSR species significantly increased Abeta fibril formation both by seeding the energetically unfavorable formation of amyloid nuclei and by enhancing the rate of amyloid elongation. Hence, these results may offer an explanation

  12. Characterization of the response of primary cells relevant to dialysis-related amyloidosis to β2-microglobulin monomer and fibrils.

    Directory of Open Access Journals (Sweden)

    Morwenna Y Porter

    Full Text Available The formation of insoluble amyloid fibrils is associated with an array of devastating human diseases. Dialysis-related amyloidosis (DRA is a severe complication of hemodialysis that results in the progressive destruction of the bones and joints. Elevated concentrations of β(2-microglobulin (β(2m in the serum of subjects on hemodialysis promote the formation of amyloid fibrils in the osteoarticular tissues, but the cellular basis for the destruction of these tissues in DRA is poorly understood. In this study we performed a systematic analysis of the interaction of monomeric and fibrillar β(2m with primary human cells of the types present in the synovial joints of subjects with DRA. Building upon observations that macrophages infiltrate β(2m amyloid deposits in vivo we demonstrate that monocytes, the precursors of macrophages, cannot degrade β(2m fibrils, and that both monomeric β(2m and fibrillar β(2m are cytotoxic to these cells. β(2m fibrils also impair the formation of bone resorbing osteoclasts from monocytes and reduce the viability of osteoblasts, the cell type that produces bone. As a consequence, we predict that β(2m amyloid will disrupt the remodelling of the bone, which is critical for the maintenance of this tissue. Moreover, we show that β(2m fibrils reduce the viability of chondrocytes, rationalizing the loss of cartilage in DRA. Together, our observations demonstrate that β(2m cytotoxicity has multiple cellular targets in the osteoarticular tissues and is likely to be a key factor in the bone and joint destruction characteristic of DRA.

  13. Fracture mechanics of collagen fibrils

    DEFF Research Database (Denmark)

    Svensson, Rene B; Mulder, Hindrik; Kovanen, Vuokko

    2013-01-01

    technique to measure the mechanical behavior of individual collagen fibrils loaded to failure. Fibrils from human patellar tendons, rat-tail tendons (RTTs), NaBH₄ reduced RTTs, and tail tendons of Zucker diabetic fat rats were tested. We found a characteristic three-phase stress-strain behavior in the human...... and the plateau continued until failure. The importance of cross-link lability was investigated by NaBH₄ reduction of the rat-tail fibrils, which did not alter their behavior. These findings shed light on the function of cross-links at the fibril level, but further studies will be required to establish...

  14. Evidence for Intramolecular Antiparallel Beta-Sheet Structure in Alpha-Synuclein Fibrils from a Combination of Two-Dimensional Infrared Spectroscopy and Atomic Force Microscopy

    NARCIS (Netherlands)

    Roeters, Steven J; Iyer, Aditya; Pletikapić, Galja; Kogan, Vladimir; Subramaniam, Vinod; Woutersen, Sander

    2017-01-01

    The aggregation of the intrinsically disordered protein alpha-synuclein (αS) into amyloid fibrils is thought to play a central role in the pathology of Parkinson's disease. Using a combination of techniques (AFM, UV-CD, XRD, and amide-I 1D- and 2D-IR spectroscopy) we show that the structure of αS

  15. Synthesis, characterization, and preclinical validation of a PET radiopharmaceutical for interrogating Aβ (β-amyloid) plaques in Alzheimer's disease.

    Science.gov (United States)

    Sundaram, Guruswami Sm; Dhavale, Dhruva; Prior, Julie L; Sivapackiam, Jothilingam; Laforest, Richard; Kotzbauer, Paul; Sharma, Vijay

    2015-12-01

    PET radiopharmaceuticals capable of imaging β-amyloid (Aβ) plaque burden in the brain could offer highly valuable diagnostic tools for clinical studies of Alzheimer's disease. To further supplement existing armamentarium of FDA-approved agents as well as those under development, and to correlate multiphoton-imaging data reported earlier, herein, we describe preclinical validation of a PET tracer. A novel PET radiopharmaceutical ((18)F-7B) was synthesized and characterized. To assess its affinity for Aβ, binding assays with Aβ1-42 fibrils, Alzheimer's disease (AD) homogenates, and autoradiography studies and their IHC correlations were performed. For assessing its overall pharmacokinetic profiles in general and its ability to cross the blood-brain barrier (BBB) in particular, biodistribution studies in normal mice were performed. Finally, for evaluating potential for (18)F-7B to serve as a targeted Aβ probe, the microPET/CT imaging was performed in age-matched amyloid precursor protein/presenilin-1 (APP/PS1) mice and wild-type (WT) counterparts. The radiotracer (18)F-7B shows saturable binding to autopsy-confirmed AD homogenates (K d = 17.7 nM) and Aβ1-42 fibrils (K d = 61 nM). Preliminary autoradiography studies show binding of (18)F-7B to cortical Aβ plaques in autopsy-confirmed AD tissue sections, inhibition of that binding by unlabeled counterpart 7A-indicating specificity, and a good correlation of tracer binding with Aβ immunostaining. The agent indicates high initial penetration into brains (7.23 ± 0.47%ID/g; 5 min) of normal mice, thus indicating a 5-min/120-min brain uptake clearance ratio of 4.7, a benchmark value (>4) consistent with the ability of agents to traverse the BBB to enable PET brain imaging. Additionally, (18)F-7B demonstrates the presence of parental species in human serum. Preliminary microPET/CT imaging demonstrates significantly higher retention of (18)F-7B in brains of transgenic mice compared with their WT counterparts

  16. Assessment of the nature interactions of β-amyloid protein by a nanoprobe method.

    Science.gov (United States)

    Caballero, Leonardo; Mena, Juan; Morales-Alvarez, Aurora; Kogan, Marcelo J; Melo, Francisco

    2015-01-01

    We present a method based on atomic force microscopy (AFM) to assess the work of adhesion between the interfaces of gold AFM tips functionalized with three peptides derived from β-sheet breaker LPFFD [CLPFFD-NH2 (i0) and their isomers CDLPFF-NH2 (i1) and CLPDFF-NH2 (i2)], and the beta-amyloid protein (Aβ1-42). β-Amyloid protein was deposited onto a highly oriented graphite (HOPG) surface as protofibrils and fibrils. The presence of the residues Leu (L), Phe (F), and Phe (F), which are also present in the native sequence, confirm that the peptides are able to bind to the aggregates of Aβ1-42 fibrils and protofibrils. Force of adhesion data were directly obtained from the maximum force on retraction, and the work of adhesion was calculated from the Jhonson-Kendall-Roberts model (JKR-Model). Both the polar and dispersive contributions to the surface energy of the peptides i0, i1, and i2, as well as Aβ1-42 fibrils and protofibrils, were determined by means of measuring the contact angle and using the two-fluid method. The macroscopic energies of the functionalized gold surfaces do not differ significantly between isomers, which confirms the similar nature of the peptides i0, i1, and i2 but suggests that the macroscopic measurements are not able to distinguish specific sequences. The nanoprobe reveals a typical adhesion work value associated with the interaction of protofibrils with i0 and i2; this value is three times higher than that of i1. The difference is attributed to the hydrophobic nature of protofibrils, the predominant exposition of hydrophobic residues of the peptides i0 and i2, with respect to i1, and the degree of functionalization. i0 and i2 presented a slight adhesion with Aβ fibrils, which is associated with the exposed hydrophilic groups of these fibrils (onto HOPG) compared to the protofibrils. However, i1 showed interaction with both Aβ fibrils and protofibrils. For this, we propose an explanation based on the fact that the peptide i1 locates

  17. The amyloid architecture provides a scaffold for enzyme-like catalysts.

    Science.gov (United States)

    Al-Garawi, Z S; McIntosh, B A; Neill-Hall, D; Hatimy, A A; Sweet, S M; Bagley, M C; Serpell, L C

    2017-08-03

    Natural biological enzymes possess catalytic sites that are generally surrounded by a large three-dimensional scaffold. However, the proportion of the protein molecule that participates in the catalytic reaction is relatively small. The generation of artificial or miniature enzymes has long been a focus of research because enzyme mimetics can be produced with high activity at low cost. These enzymes aim to mimic the active sites without the additional architecture contributed by the protein chain. Previous work has shown that amyloidogenic peptides are able to self-assemble to create an active site that is capable of binding zinc and catalysing an esterase reaction. Here, we describe the structural characterisation of a set of designed peptides that form an amyloid-like architecture and reveal that their capability to mimic carbonic anhydrase and serve as enzyme-like catalysts is related to their ability to self-assemble. These amyloid fibril structures can bind the metal ion Zn 2+ via a three-dimensional arrangement of His residues created by the amyloid architecture. Our results suggest that the catalytic efficiency of amyloid-like assembly is not only zinc-dependent but also depends on an active centre created by the peptides which is, in turn, dependent on the ordered architecture. These fibrils have good esterase activity, and they may serve as good models for the evolution of modern-day enzymes. Furthermore, they may be useful in designing self-assembling fibrils for applications as metal ion catalysts. This study also demonstrates that the ligands surrounding the catalytic site affect the affinity of the zinc-binding site to bind the substrate contributing to the enzymatic activity of the assembled peptides.

  18. MRI evaluation of amyloid myopathy

    International Nuclear Information System (INIS)

    Metzler, J.P.; Fleckenstein, J.L.; Veterans Affairs Medical Center, Dallas, TX; White, C.L. III; Veterans Affairs Medical Center, Dallas, TX; Haller, R.G.; Greenlee, R.G. Jr.; Veterans Affairs Medical Center, Dallas, TX; Frenkel, E.P.; Veterans Affairs Medical Center, Dallas, TX

    1992-01-01

    Amyloid myopathy is a rare complication of primary amyloidosis. The magnetic resonance imaging (MRI) features of two patients with amyloid myopathy were studied. Slight prolongation of muscle T1 and T2 relaxation times was evident but the striking abnormality was marked reticulation of the subcutaneous fat. The clinical findings of indurated extremities far exceeds the minimal signal intenisty alteration seen in the muscles. The MR appearance of amyloid myopathy differs from that of other neuromuscular conditions in the minimal changes found in muscle, but the striking abnormality seen in subcutaneous fat makes it distinct from many neuromuscular conditions. (orig.)

  19. Experimental induction of chicken amyloid A amyloidosis in white layer chickens by inoculation with inactivated vaccines.

    Science.gov (United States)

    Habibi, Wazir Ahmad; Hirai, Takuya; Niazmand, Mohammad Hakim; Okumura, Naoko; Yamaguchi, Ryoji

    2017-10-01

    We investigated the amyloidogenic potential of inactivated vaccines and the localized production of serum amyloid A (SAA) at the injection site in white layer chickens. Hens in the treated group were injected intramuscularly three times with high doses of inactivated oil-emulsion Salmonella Enteritidis vaccine and multivalent viral and bacterial inactivated oil-emulsion vaccines at two-week intervals. Chickens in the control group did not receive any inoculum. In the treated group, emaciation and granulomas were present, while several chickens died between 4 and 6 weeks after the first injection. Hepatomegaly was seen at necropsy, and the liver parenchyma showed inconsistent discolouration with patchy green to yellowish-brown areas, or sometimes red-brown areas with haemorrhage. Amyloid deposition in the liver, spleen, duodenum, and at injection sites was demonstrated using haematoxylin and eosin staining, Congo red, and immunohistochemistry. The incidence of chicken amyloid A (AA) amyloidosis was 47% (28 of 60) in the treated group. In addition, RT-PCR was used to identify chicken SAA mRNA expression in the liver and at the injection sites. Furthermore, SAA mRNA was detected by in situ hybridization in fibroblasts at the injection sites, and also in hepatocytes. We believe that this is the first report of the experimental induction of systemic AA amyloidosis in white layer chickens following repeated inoculation with inactivated vaccines without the administration of amyloid fibrils or other amyloid-enhancing factors.

  20. Role of gut microbiota and nutrients in amyloid formation and pathogenesis of Alzheimer disease.

    Science.gov (United States)

    Pistollato, Francesca; Sumalla Cano, Sandra; Elio, Iñaki; Masias Vergara, Manuel; Giampieri, Francesca; Battino, Maurizio

    2016-10-01

    It has been hypothesized that alterations in the composition of the gut microbiota might be associated with the onset of certain human pathologies, such as Alzheimer disease, a neurodegenerative syndrome associated with cerebral accumulation of amyloidfibrils. It has been shown that bacteria populating the gut microbiota can release significant amounts of amyloids and lipopolysaccharides, which might play a role in the modulation of signaling pathways and the production of proinflammatory cytokines related to the pathogenesis of Alzheimer disease. Additionally, nutrients have been shown to affect the composition of the gut microbiota as well as the formation and aggregation of cerebral amyloid-β. This suggests that modulating the gut microbiome and amyloidogenesis through specific nutritional interventions might prove to be an effective strategy to prevent or reduce the risk of Alzheimer disease. This review examines the possible role of the gut in the dissemination of amyloids, the role of the gut microbiota in the regulation of the gut-brain axis, the potential amyloidogenic properties of gut bacteria, and the possible impact of nutrients on modulation of microbiota composition and amyloid formation in relation to the pathogenesis of Alzheimer disease. © The Author(s) 2016. Published by Oxford University Press on behalf of the International Life Sciences Institute. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. Dimethyl Sulfoxide Induced Destabilization and Disassembly of Various Structural Variants of Insulin Fibrils Monitored by Vibrational Circular Dichroism.

    Science.gov (United States)

    Zhang, Ge; Babenko, Viktoria; Dzwolak, Wojciech; Keiderling, Timothy A

    2015-12-15

    Dimethyl sulfoxide (DMSO) induced destabilization of insulin fibrils has been previously studied by Fourier transform infrared spectroscopy and interpreted in terms of secondary structural changes. The variation of this process for fibrils with different types of higher-order morphological structures remained unclear. Here, we utilize vibrational circular dichroism (VCD), which has been reported to provide a useful biophysical probe of the supramolecular chirality of amyloid fibrils, to characterize changes in the macroscopic chirality following DMSO-induced disassembly for two types of insulin fibrils formed under different conditions, at different reduced pH values with and without added salt and agitation. We confirm that very high concentrations of DMSO can disaggregate both types of insulin fibrils, which initially maintained a β-sheet conformation and eventually changed their secondary structure to a disordered form. The two types responded to varying concentrations of DMSO, and disaggregation followed different mechanisms. Interconversion of specific insulin fibril morphological types also occurred during the destabilization process as monitored by VCD. With transmission electron microscopy, we were able to correlate the changes in VCD sign patterns to alteration of morphology of the insulin fibrils.

  2. Oxidation reduces the fibrillation but not the neurotoxicity of the prion peptide PrP106-126

    DEFF Research Database (Denmark)

    Bergstrøm, Linda Alice; Chabry, J.; Bastholm, L.

    2007-01-01

    There is increasing evidence that soluble oligomers of misfolded protein may play a role in the pathogenesis of protein misfolding diseases including the transmissible spongiform encephalopathies (TSE) where the protein involved is the prion protein, PrP. The effect of oxidation on fibrillation...... tendency and neurotoxicity of different molecular variants of the prion peptide PrP106-126 was investigated. It was found that methionine oxidation significantly reduced amyloid fibril formation and proteinase K resistance, but it did not reduce (but rather increase slightly) the neurotoxicity...

  3. Atrial fibrillation and female sex.

    Science.gov (United States)

    Anselmino, Matteo; Battaglia, Alberto; Gallo, Cristina; Gili, Sebastiano; Matta, Mario; Castagno, Davide; Ferraris, Federico; Giustetto, Carla; Gaita, Fiorenzo

    2015-12-01

    Atrial fibrillation is the most common supraventricular arrhythmia. Its prevalence increases with age and preferentially affects male patients. Over 75 years of age, however, female patients being more prevalent, the absolute number of patients affected is similar between sexes. Despite this, few data are available in the literature concerning sex-related differences in atrial fibrillation patients. The present systematic review therefore considers comorbidities, referring symptoms, quality of life, pharmacological approaches and trans-catheter ablation in female rather than in male atrial fibrillation patients in search of parameters that may have an impact on the treatment outcome. In brief, female atrial fibrillation patients more commonly present comorbidities, leading to a higher prevalence of persistent atrial fibrillation; moreover, they refer to hospital care later and with a longer disease history. Atrial fibrillation symptoms relate to low quality of life in female patients; in fact, atrial fibrillation paroxysm usually presents higher heart rate, leading to preferentially adopt a rate rather than a rhythm-control strategy. Female atrial fibrillation patients present an increased risk of stroke, worsened by the lower oral anticoagulant prescription rate related to the concomitant higher haemorrhagic risk profile. Trans-catheter ablation is under-used in female patients and, on the contrary, they are more commonly affected by anti-arrhythmic drug side effects.

  4. Interaction of amyloid inhibitor proteins with amyloid beta peptides: insight from molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Payel Das

    Full Text Available Knowledge of the detailed mechanism by which proteins such as human αB- crystallin and human lysozyme inhibit amyloid beta (Aβ peptide aggregation is crucial for designing treatment for Alzheimer's disease. Thus, unconstrained, atomistic molecular dynamics simulations in explicit solvent have been performed to characterize the Aβ17-42 assembly in presence of the αB-crystallin core domain and of lysozyme. Simulations reveal that both inhibitor proteins compete with inter-peptide interaction by binding to the peptides during the early stage of aggregation, which is consistent with their inhibitory action reported in experiments. However, the Aβ binding dynamics appear different for each inhibitor. The binding between crystallin and the peptide monomer, dominated by electrostatics, is relatively weak and transient due to the heterogeneous amino acid distribution of the inhibitor surface. The crystallin-bound Aβ oligomers are relatively long-lived, as they form more extensive contact surface with the inhibitor protein. In contrast, a high local density of arginines from lysozyme allows strong binding with Aβ peptide monomers, resulting in stable complexes. Our findings not only illustrate, in atomic detail, how the amyloid inhibitory mechanism of human αB-crystallin, a natural chaperone, is different from that of human lysozyme, but also may aid de novo design of amyloid inhibitors.

  5. Regulation of presynaptic Ca2+, synaptic plasticity and contextual fear conditioning by a N-terminal β-amyloid fragment.

    Science.gov (United States)

    Lawrence, James L M; Tong, Mei; Alfulaij, Naghum; Sherrin, Tessi; Contarino, Mark; White, Michael M; Bellinger, Frederick P; Todorovic, Cedomir; Nichols, Robert A

    2014-10-22

    Soluble β-amyloid has been shown to regulate presynaptic Ca(2+) and synaptic plasticity. In particular, picomolar β-amyloid was found to have an agonist-like action on presynaptic nicotinic receptors and to augment long-term potentiation (LTP) in a manner dependent upon nicotinic receptors. Here, we report that a functional N-terminal domain exists within β-amyloid for its agonist-like activity. This sequence corresponds to a N-terminal fragment generated by the combined action of α- and β-secretases, and resident carboxypeptidase. The N-terminal β-amyloid fragment is present in the brains and CSF of healthy adults as well as in Alzheimer's patients. Unlike full-length β-amyloid, the N-terminal β-amyloid fragment is monomeric and nontoxic. In Ca(2+) imaging studies using a model reconstituted rodent neuroblastoma cell line and isolated mouse nerve terminals, the N-terminal β-amyloid fragment proved to be highly potent and more effective than full-length β-amyloid in its agonist-like action on nicotinic receptors. In addition, the N-terminal β-amyloid fragment augmented theta burst-induced post-tetanic potentiation and LTP in mouse hippocampal slices. The N-terminal fragment also rescued LTP inhibited by elevated levels of full-length β-amyloid. Contextual fear conditioning was also strongly augmented following bilateral injection of N-terminal β-amyloid fragment into the dorsal hippocampi of intact mice. The fragment-induced augmentation of fear conditioning was attenuated by coadministration of nicotinic antagonist. The activity of the N-terminal β-amyloid fragment appears to reside largely in a sequence surrounding a putative metal binding site, YEVHHQ. These findings suggest that the N-terminal β-amyloid fragment may serve as a potent and effective endogenous neuromodulator. Copyright © 2014 the authors 0270-6474/14/3414210-09$15.00/0.

  6. Evaluation of systemic amyloidosis by scintigraphy with 123I-labeled serum amyloid P component

    International Nuclear Information System (INIS)

    Hawkins, P.N.; Lavender, J.P.; Pepys, M.B.

    1990-01-01

    In systemic amyloidosis the distribution and progression of disease have been difficult to monitor, because they can be demonstrated only by biopsy. Serum amyloid P component (SAP) is a normal circulating plasma protein that is deposited on amyloid fibrils because of its specific binding affinity for them. We investigated whether labeled SAP could be used to locate amyloid deposits. Purified human SAP labeled with iodine-123 was given intravenously to 50 patients with biopsy-proved systemic amyloidosis--25 with the AL (primary) type and 25 with the AA (secondary) type--and to 26 control patients with disease and 10 healthy subjects. Whole-body images and regional views were obtained after 24 hours and read in a blinded fashion. In the patients with amyloidosis the 123I-SAP was localized rapidly and specifically in amyloid deposits. The scintigraphic images obtained were characteristic and appeared to identify the extent of amyloid deposition in all 50 patients. There was no uptake of the 123I-SAP by the control patients and the healthy subjects. In all patients with AA amyloidosis the spleen was affected, whereas the scans showed uptake in the heart, skin, carpal region, and bone marrow only in patients with the AL type. Positive images were seen in six patients in whom biopsies had been negative or unsuccessful; in all six, amyloid was subsequently found on biopsy or at autopsy. Progressive amyloid deposition was observed in 9 of 11 patients studied serially. Scintigraphy after the injection of 123I-SAP can be used for diagnosing, locating, and monitoring the extent of systemic amyloidosis

  7. Dementia and Atrial Fibrillation

    DEFF Research Database (Denmark)

    Pastori, Daniele; Miyazawa, Kazuo; Lip, Gregory Y H

    2018-01-01

    The risk of developing dementia is increased in patients with atrial fibrillation (AF), with the incidence of both conditions increasing with aging. Patients with dementia frequently do not receiving adequate thrombo-prophylaxis, because of the inability to monitor INR and/or to achieve...... in therapeutic range during VKAs therapy, the assessment of cognitive impairment may help identify those patients who may benefit from switching to NOACs. In conclusion, patients with AF and dementia benefit from anticoagulation and should not be denied receiving adequate stroke prevention. Cognitive function...

  8. Cerebrospinal Fluid Amyloid β40 Is Decreased in Cerebral Amyloid Angiopathy

    Science.gov (United States)

    Verbeek, Marcel M.; Kremer, Berry P. H.; Rikkert, Marcel Olde; van Domburg, Peter H. M. F.; Skehan, Maureen E.; Greenberg, Steven M.

    2013-01-01

    Cerebral amyloid angiopathy is caused by deposition of the amyloid β protein in the cerebral vasculature. In analogy to previous observations in Alzheimer disease, we hypothesized that analysis of amyloid β40 and β42 proteins in the cerebrospinal fluid might serve as a molecular biomarker. We observed strongly decreased cerebrospinal fluid amyloid β40 (p < 0.01 vs controls or Alzheimer disease) and amyloid β42 concentrations (p < 0.001 vs controls and p < 0.05 vs Alzheimer disease) in cerebral amyloid angiopathy patients. The combination of amyloid β42 and total tau discriminated cerebral amyloid angiopathy from controls, with an area under the receiver operator curve of 0.98. Our data are consistent with neuropathological evidence that amyloid β40 as well as amyloid β42 protein are selectively trapped in the cerebral vasculature from interstitial fluid drainage pathways that otherwise transport amyloid β proteins toward the cerebrospinal fluid. PMID:19743453

  9. Loss of metal ions, disulfide reduction and mutations related to familial ALS promote formation of amyloid-like aggregates from superoxide dismutase.

    Directory of Open Access Journals (Sweden)

    Zeynep A Oztug Durer

    Full Text Available Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1 are one of the causes of familial amyotrophic lateral sclerosis (FALS. Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1 formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1.

  10. Loss of metal ions, disulfide reduction and mutations related to familial ALS promote formation of amyloid-like aggregates from superoxide dismutase.

    Science.gov (United States)

    Oztug Durer, Zeynep A; Cohlberg, Jeffrey A; Dinh, Phong; Padua, Shelby; Ehrenclou, Krista; Downes, Sean; Tan, James K; Nakano, Yoko; Bowman, Christopher J; Hoskins, Jessica L; Kwon, Chuhee; Mason, Andrew Z; Rodriguez, Jorge A; Doucette, Peter A; Shaw, Bryan F; Valentine, Joan Selverstone

    2009-01-01

    Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (FALS). Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1.

  11. Interaction and inhibitory influence of the azo dye carmoisine on lysozyme amyloid fibrillogenesis.

    Science.gov (United States)

    Basu, Anirban; Suresh Kumar, Gopinatha

    2017-07-25

    The binding of the common food colorant carmoisine and its inhibitory effect on amyloid fibrillation in lysozyme have been investigated. Since humans are increasingly exposed to various food colorants like carmoisine, such studies are highly relevant. In the presence of lysozyme, the carmoisine absorption spectrum exhibited hypochromic changes. The intrinsic fluorescence of lysozyme was also quenched on interaction. Time-resolved fluorescence results suggested that the binding mechanism involved ground state complexation. The binding was predominantly dominated by non-polyelectrolytic forces. The molecular distance between the donor (lysozyme) and the acceptor (carmoisine), calculated from FRET theory, was found to be 3.37 nm, indicating that carmoisine binds close to Trp-62/63 residues in the β-domain of the protein. Information on alterations in the microenvironment surrounding the Trp-residues was also obtained from synchronous fluorescence data. Carmoisine binding induced significant loss in the alpha helical organization of lysozyme. The binding, nevertheless, did not influence the thermal stability of lysozyme significantly. The binding reaction was exothermic and driven by large negative enthalpy and small but favourable entropic contributions. Thioflavin T assay, far-UV circular dichroism studies and AFM imaging profiles testified that carmoisine had a significant inhibitory effect on amyloid fibrillogenesis in lysozyme. Carmoisine also had a definitive defibrillating effect on existing fibrils. The results may provide new insights for designing new small molecule inhibitors for amyloid related diseases.

  12. Towards a Pharmacophore for Amyloid

    Energy Technology Data Exchange (ETDEWEB)

    Landau, Meytal; Sawaya, Michael R.; Faull, Kym F.; Laganowsky, Arthur; Jiang, Lin; Sievers, Stuart A.; Liu, Jie; Barrio, Jorge R.; Eisenberg, David (UCLA)

    2011-09-16

    Diagnosing and treating Alzheimer's and other diseases associated with amyloid fibers remains a great challenge despite intensive research. To aid in this effort, we present atomic structures of fiber-forming segments of proteins involved in Alzheimer's disease in complex with small molecule binders, determined by X-ray microcrystallography. The fiber-like complexes consist of pairs of {beta}-sheets, with small molecules binding between the sheets, roughly parallel to the fiber axis. The structures suggest that apolar molecules drift along the fiber, consistent with the observation of nonspecific binding to a variety of amyloid proteins. In contrast, negatively charged orange-G binds specifically to lysine side chains of adjacent sheets. These structures provide molecular frameworks for the design of diagnostics and drugs for protein aggregation diseases. The devastating and incurable dementia known as Alzheimer's disease affects the thinking, memory, and behavior of dozens of millions of people worldwide. Although amyloid fibers and oligomers of two proteins, tau and amyloid-{beta}, have been identified in association with this disease, the development of diagnostics and therapeutics has proceeded to date in a near vacuum of information about their structures. Here we report the first atomic structures of small molecules bound to amyloid. These are of the dye orange-G, the natural compound curcumin, and the Alzheimer's diagnostic compound DDNP bound to amyloid-like segments of tau and amyloid-{beta}. The structures reveal the molecular framework of small-molecule binding, within cylindrical cavities running along the {beta}-spines of the fibers. Negatively charged orange-G wedges into a specific binding site between two sheets of the fiber, combining apolar binding with electrostatic interactions, whereas uncharged compounds slide along the cavity. We observed that different amyloid polymorphs bind different small molecules, revealing that a

  13. Curcumin Alters the Salt Bridge-containing Turn Region in Amyloid β(1–42) Aggregates*

    Science.gov (United States)

    Mithu, Venus Singh; Sarkar, Bidyut; Bhowmik, Debanjan; Das, Anand Kant; Chandrakesan, Muralidharan; Maiti, Sudipta; Madhu, Perunthiruthy K.

    2014-01-01

    Amyloid β (Aβ) fibrillar deposits in the brain are a hallmark of Alzheimer disease (AD). Curcumin, a common ingredient of Asian spices, is known to disrupt Aβ fibril formation and to reduce AD pathology in mouse models. Understanding the structural changes induced by curcumin can potentially lead to AD pharmaceutical agents with inherent bio-compatibility. Here, we use solid-state NMR spectroscopy to investigate the structural modifications of amyloid β(1–42) (Aβ42) aggregates induced by curcumin. We find that curcumin induces major structural changes in the Asp-23–Lys-28 salt bridge region and near the C terminus. Electron microscopy shows that the Aβ42 fibrils are disrupted by curcumin. Surprisingly, some of these alterations are similar to those reported for Zn2+ ions, another agent known to disrupt the fibrils and alter Aβ42 toxicity. Our results suggest the existence of a structurally related family of quasi-fibrillar conformers of Aβ42, which is stabilized both by curcumin and by Zn2+. PMID:24599958

  14. Curcumin alters the salt bridge-containing turn region in amyloid β(1-42) aggregates.

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    Mithu, Venus Singh; Sarkar, Bidyut; Bhowmik, Debanjan; Das, Anand Kant; Chandrakesan, Muralidharan; Maiti, Sudipta; Madhu, Perunthiruthy K

    2014-04-18

    Amyloid β (Aβ) fibrillar deposits in the brain are a hallmark of Alzheimer disease (AD). Curcumin, a common ingredient of Asian spices, is known to disrupt Aβ fibril formation and to reduce AD pathology in mouse models. Understanding the structural changes induced by curcumin can potentially lead to AD pharmaceutical agents with inherent bio-compatibility. Here, we use solid-state NMR spectroscopy to investigate the structural modifications of amyloid β(1-42) (Aβ42) aggregates induced by curcumin. We find that curcumin induces major structural changes in the Asp-23-Lys-28 salt bridge region and near the C terminus. Electron microscopy shows that the Aβ42 fibrils are disrupted by curcumin. Surprisingly, some of these alterations are similar to those reported for Zn(2+) ions, another agent known to disrupt the fibrils and alter Aβ42 toxicity. Our results suggest the existence of a structurally related family of quasi-fibrillar conformers of Aβ42, which is stabilized both by curcumin and by Zn(2+.)

  15. Pittsburgh Compound-B (PiB) binds amyloid β-protein protofibrils.

    Science.gov (United States)

    Yamin, Ghiam; Teplow, David B

    2017-01-01

    The neuropathology of Alzheimer's disease (AD) includes amyloid plaque formation by the amyloid β-protein (Aβ) and intracellular paired helical filament formation by tau protein. These neuropathogenetic features correlate with disease progression and have been revealed in brains of AD patients using positron emission tomography (PET). One of the most useful positron emission tomography imaging agents has been Pittsburgh Compound-B (PiB). However, since its introduction in 2002, substantial evidence has accumulated suggesting that Aβ oligomerization and protofibril formation, rather than fibril formation per se, may be the more important pathogenetic event in AD. Detecting protofibrils and oligomeric forms of Aβ thus may be of value. We report here the results of experiments to determine whether PiB binds to oligomers or protofibrils formed by Aβ40 and Aβ42. We observed strong binding to Aβ42 fibrils, significant binding to protofibrils, and weaker binding to Aβ42 oligomers. PiB also binds Aβ40 fibrils, but its binding to Aβ40 protofibrils and oligomers is substantially lower than for that observed for Aβ42. © 2016 International Society for Neurochemistry.

  16. Evaluation of Polyphenol Anthocyanin-Enriched Extracts of Blackberry, Black Raspberry, Blueberry, Cranberry, Red Raspberry, and Strawberry for Free Radical Scavenging, Reactive Carbonyl Species Trapping, Anti-Glycation, Anti-β-Amyloid Aggregation, and Microglial Neuroprotective Effects

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    Hang Ma

    2018-02-01

    Full Text Available Glycation is associated with several neurodegenerative disorders, including Alzheimer’s disease (AD, where it potentiates the aggregation and toxicity of proteins such as β-amyloid (Aβ. Published studies support the anti-glycation and neuroprotective effects of several polyphenol-rich fruits, including berries, which are rich in anthocyanins. Herein, blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts were evaluated for: (1 total phenolic and anthocyanins contents, (2 free radical (DPPH scavenging and reactive carbonyl species (methylglyoxal; MGO trapping, (3 anti-glycation (using BSA-fructose and BSA-MGO models, (4 anti-Aβ aggregation (using thermal- and MGO-induced fibrillation models, and, (5 murine microglia (BV-2 neuroprotective properties. Berry crude extracts (CE were fractionated to yield anthocyanins-free (ACF and anthocyanins-enriched (ACE extracts. The berry ACEs (at 100 μg/mL showed superior free radical scavenging, reactive carbonyl species trapping, and anti-glycation effects compared to their respective ACFs. The berry ACEs (at 100 μg/mL inhibited both thermal- and MGO-induced Aβ fibrillation. In addition, the berry ACEs (at 20 μg/mL reduced H2O2-induced reactive oxygen species production, and lipopolysaccharide-induced nitric oxide species in BV-2 microglia as well as decreased H2O2-induced cytotoxicity and caspase-3/7 activity in BV-2 microglia. The free radical scavenging, reactive carbonyl trapping, anti-glycation, anti-Aβ fibrillation, and microglial neuroprotective effects of these berry extracts warrant further in vivo studies to evaluate their potential neuroprotective effects against AD.

  17. Proteomic screening for amyloid proteins.

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    Anton A Nizhnikov

    Full Text Available Despite extensive study, progress in elucidation of biological functions of amyloids and their role in pathology is largely restrained due to the lack of universal and reliable biochemical methods for their discovery. All biochemical methods developed so far allowed only identification of glutamine/asparagine-rich amyloid-forming proteins or proteins comprising amyloids that form large deposits. In this article we present a proteomic approach which may enable identification of a broad range of amyloid-forming proteins independently of specific features of their sequences or levels of expression. This approach is based on the isolation of protein fractions enriched with amyloid aggregates via sedimentation by ultracentrifugation in the presence of strong ionic detergents, such as sarkosyl or SDS. Sedimented proteins are then separated either by 2D difference gel electrophoresis or by SDS-PAGE, if they are insoluble in the buffer used for 2D difference gel electrophoresis, after which they are identified by mass-spectrometry. We validated this approach by detection of known yeast prions and mammalian proteins with established capacity for amyloid formation and also revealed yeast proteins forming detergent-insoluble aggregates in the presence of human huntingtin with expanded polyglutamine domain. Notably, with one exception, all these proteins contained glutamine/asparagine-rich stretches suggesting that their aggregates arose due to polymerization cross-seeding by human huntingtin. Importantly, though the approach was developed in a yeast model, it can easily be applied to any organism thus representing an efficient and universal tool for screening for amyloid proteins.

  18. Curcumin Ameliorates the Reduction Effect of PGE2 on Fibrillar β-Amyloid Peptide (1-42)-Induced Microglial Phagocytosis through the Inhibition of EP2-PKA Signaling in N9 Microglial Cells

    Science.gov (United States)

    Yang, Ju; Shen, Ting-ting; Chen, Yi; Yang, Xue-Sen

    2016-01-01

    Inflammatory activation of microglia and β amyloid (Aβ) deposition are considered to work both independently and synergistically to contribute to the increased risk of Alzheimer’s disease (AD). Recent studies indicate that long-term use of phenolic compounds provides protection against AD, primarily due to their anti-inflammatory actions. We previously suggested that phenolic compound curcumin ameliorated phagocytosis possibly through its anti-inflammatory effects rather than direct regulation of phagocytic function in electromagnetic field-exposed N9 microglial cells (N9 cells). Here, we explored the prostaglandin-E2 (PGE2)-related signaling pathway that involved in curcumin-mediated phagocytosis in fibrillar β-amyloid peptide (1–42) (fAβ42)-stimulated N9 cells. Treatment with fAβ42 increased phagocytosis of fluorescent-labeled latex beads in N9 cells. This increase was attenuated in a dose-dependent manner by endogenous and exogenous PGE2, as well as a selective EP2 or protein kinase A (PKA) agonist, but not by an EP4 agonist. We also found that an antagonist of EP2, but not EP4, abolished the reduction effect of PGE2 on fAβ42-induced microglial phagocytosis. Additionally, the increased expression of endogenous PGE2, EP2, and cyclic adenosine monophosphate (AMP), and activation of vasodilator-stimulated phosphoprotein, cyclic AMP responsive element-binding protein, and PKA were depressed by curcumin administration. This reduction led to the amelioration of the phagocytic abilities of PGE2-stimulated N9 cells. Taken together, these data suggested that curcumin restored the attenuating effect of PGE2 on fAβ42-induced microglial phagocytosis via a signaling mechanism involving EP2 and PKA. Moreover, due to its immune modulatory effects, curcumin may be a promising pharmacological candidate for neurodegenerative diseases. PMID:26824354

  19. Peripheral treatment with enoxaparin exacerbates amyloid plaque pathology in Tg2576 mice.

    Science.gov (United States)

    Cui, Hao; King, Anna E; Jacobson, Glenn A; Small, David H

    2017-04-01

    Alzheimer's disease (AD) is a complex, progressive neurological disorder characterized by the formation of extracellular amyloid plaques composed of β-amyloid protein (Aβ), the key component in pathogenesis of AD. Peripheral administration of enoxaparin (ENO) reportedly reduces the level of Aβ and the amyloid plaques in the cortex of amyloid precursor protein (APP) transgenic mice. However, the exact mechanism of these effects is unclear. Our previous studies indicated that ENO can inhibit APP processing to Aβ in primary cortical cells from Tg2576 mice by downregulating BACE1 levels. This study examines whether ENO-induced reduction of amyloid load is due to the decreased APP processing to Aβ in Tg2576 mice. Surprisingly, our results indicated that ENO significantly increases the Aβ42/Aβ40 ratio in cortex and enhances the amyloid plaque load in both cortex and hippocampus, although overall APP processing was not influenced by ENO. Moreover, ENO stimulated the aggregation of both Aβ40 and Aβ42 in vitro. Although ENO has been reported to improve cognition in vivo and has potential as a therapeutic agent for AD, the results from our study suggest that ENO can exacerbate the amyloid pathology, and the strategy of using ENO for the treatment of AD may require further assessment. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  20. Disaggregation of amyloid plaque in brain of Alzheimer's disease transgenic mice with daily subcutaneous administration of a tetravalent bispecific antibody that targets the transferrin receptor and the Abeta amyloid peptide.

    Science.gov (United States)

    Sumbria, Rachita K; Hui, Eric Ka-Wai; Lu, Jeff Zhiqiang; Boado, Ruben J; Pardridge, William M

    2013-09-03

    Anti-amyloid antibodies (AAA) are under development as new therapeutics that disaggregate the amyloid plaque in brain in Alzheimer's disease (AD). However, the AAAs are large molecule drugs that do not cross the blood-brain barrier (BBB), in the absence of BBB disruption. In the present study, an AAA was re-engineered for receptor-mediated transport across the BBB via the endogenous BBB transferrin receptor (TfR). A single chain Fv (ScFv) antibody form of an AAA was fused to the carboxyl terminus of each heavy chain of a chimeric monoclonal antibody (mAb) against the mouse TfR, and this produced a tetravalent bispecific antibody designated the cTfRMAb-ScFv fusion protein. Unlike a conventional AAA, which has a plasma half-time of weeks, the cTfRMAb-ScFv fusion protein is cleared from plasma in mice with a mean residence time of about 3 h. Therefore, a novel protocol was developed for the treatment of one year old presenilin (PS)-1/amyloid precursor protein (APP) AD double transgenic PSAPP mice, which were administered daily subcutaneous (sc) injections of 5 mg/kg of the cTfRMAb-ScFv fusion protein for 12 consecutive weeks. At the end of the treatment, brain amyloid plaques were quantified with confocal microscopy using both Thioflavin-S staining and immunostaining with the 6E10 antibody against Abeta amyloid fibrils. Fusion protein treatment caused a 57% and 61% reduction in amyloid plaque in the cortex and hippocampus, respectively. No increase in plasma immunoreactive Abeta amyloid peptide, and no cerebral microhemorrhage, was observed. Chronic daily sc treatment of the mice with the fusion protein caused no immune reactions and only a low titer antidrug antibody response. In conclusion, re-engineering AAAs for receptor-mediated BBB transport allows for reduction in brain amyloid plaque without cerebral microhemorrhage following daily sc treatment for 12 weeks.

  1. Pharmacological Treatment for Atrial Fibrillation

    Directory of Open Access Journals (Sweden)

    Kaoru Sugi, MD PhD

    2005-01-01

    Full Text Available Pharmacological treatment for atrial fibrillation has a variety of purposes, such as pharmacological defibrillation, maintenance of sinus rhythm, heart rate control to prevent congestive heart failure and prevention of both cerebral infarction and atrial remodeling. Sodium channel blockers are superior to potassium channel blockers for atrial defibrillation, while both sodium and potassium channel blockers are effective in the maintenance of sinus rhythm. In general, digitalis or Ca antagonists are used to control heart rate during atrial fibrillation to prevent congestive heart failure, while amiodarone or bepridil also reduce heart rates during atrial fibrillation. Anticoagulant therapy with warfarin is recommended to prevent cerebral infarction and angiotensin converting enzyme antagonists or angiotensin II receptor blockers are also used to prevent atrial remodeling. One should select appropriate drugs for treatment of atrial fibrillation according to the patient's condition.

  2. A Free Energy Barrier Caused by the Refolding of an Oligomeric Intermediate Controls the Lag Time of Amyloid Formation by hIAPP.

    Science.gov (United States)

    Serrano, Arnaldo L; Lomont, Justin P; Tu, Ling-Hsien; Raleigh, Daniel P; Zanni, Martin T

    2017-11-22

    Transiently populated oligomers formed en route to amyloid fibrils may constitute the most toxic aggregates associated with many amyloid-associated diseases. Most nucleation theories used to describe amyloid aggregation predict low oligomer concentrations and do not take into account free energy costs that may be associated with structural rearrangements between the oligomer and fiber states. We have used isotope labeling and two-dimensional infrared spectroscopy to spectrally resolve an oligomeric intermediate during the aggregation of the human islet amyloid protein (hIAPP or amylin), the protein associated with type II diabetes. A structural rearrangement includes the F 23 G 24 A 25 I 26 L 27 region of hIAPP, which starts from a random coil structure, evolves into ordered β-sheet oligomers containing at least 5 strands, and then partially disorders in the fibril structure. The supercritical concentration is measured to be between 150 a