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The amyloid: structure, properties and application
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein aggregation, leading to the formation and depositions of amyloids, is a cause for a number of diseases such as Alzheimer’s and Creutzfeld-Jacob’s disease, systemic amyloidoses, type II diabetes and others . More than 20 proteins are associated with protein misfolding diseases and even a larger number of proteins can self-assemble into amyloid in vitro. Relating structural and functional properties of amyloid is of particular interest, as this will lead to the identification of the main factors and mechanisms involved in the process of protein misfolding and aggregation; consequently, this will provide a basis for developing new strategies to treat protein misfolding diseases. The aim of the thesis is to investigate structural aspects of amyloid formation and relate that to the functional properties of amyloid. The first paper describes the amyloid formation of equine lysozyme (EL). We have demonstrated that EL enters an amyloid forming pathways under conditions where the molten globule state is populated. We have found that the morphology of the amyloids depend on the calcium-binding to lysozyme, specifically the holo-protein assembles into short, linear protofilaments, while the apo-EL forms ring-shaped structures. The morphology of EL amyloid significantly differs from the amyloid fibrils of human and hen lysozymes. We have suggested that the stable alpha-helical core of EL, which remains structured in the molten globule intermediate, may obstruct the formation of fibrilar interface and therefore leads to assembly of short, curly fibrils and rings.In the second paper, we describe the cytotoxicity of EL amyloids. We have analysed the amyloid intermediates on the pathway towards amyloid fibrils. The sizes of amyloid oligomers were determined by atomic force microscopy (AFM) and the formation of cross-beta sheet was shown by thioflavin T (ThT) binding. The toxicity studies show that the oligomers formed during amyloid growth phase are toxic to a range of cell lines and cultures and the toxicity is size-dependant.The last manuscript describes a novel method for manufacturing of silver nanowires by the biotemplating using amyloid fibrils. The amyloid assembled from an abundant and cheap hen egg white lysozyme was used as a scaffold for casting ultrathin silver nanowires. We have manufactured nanowires with a diameter of 1.0-2.5 nm and up to 2 micrometers in length. Up to date, it is the thinnest silver nanowires produced by using biotemplating and at least one order of magnitude thinner than nanowires manufactured by chemical synthesis.

Place, publisher, year, edition, pages
Umeå: Medicinsk kemi och biofysik , 2007. , 48 p.
Series
Umeå University medical dissertations, ISSN 0346-6612
Keyword [en]
amyloid, oligomers, AFM, cytotoxicity, biotemplating, lysozyme
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:umu:diva-1164ISBN: 978-91-7264-343-7 (print)OAI: oai:DiVA.org:umu-1164DiVA: diva2:140431
Public defence
2007-09-12, KB3A9, KBC huset, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2007-06-01 Created: 2007-06-01 Last updated: 2012-03-23Bibliographically approved
List of papers
1. Amyloid protofilaments from the calcium-binding protein equine lysozyme: formation of ring and linear structures depends on pH and metal ion concentration
Open this publication in new window or tab >>Amyloid protofilaments from the calcium-binding protein equine lysozyme: formation of ring and linear structures depends on pH and metal ion concentration
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2003 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 330, no 4, 879-890 p.Article in journal (Refereed) Published
Abstract [en]

The calcium-binding equine lysozyme has been found to undergo conversion into amyloid fibrils during incubation in solution at acidic pH. At pH 4.5 and 57 °C, where equine lysozyme forms a partially unfolded molten globule state, the protein forms protofilaments with a width of ca. 2 nm. In the absence of Ca2+ the protofilaments are present as annular structures with a diameter of 40–50 nm. In the presence of 10 mM CaCl2 the protofilaments of equine lysozyme are straight or curved; they can assemble into thicker threads, but they do not appear to undergo circularisation. At pH 2.0, where the protein is more destabilised compared to pH 4.5, fibril formation occurs at 37 °C and 57 °C. At pH 2.0, both ring-shaped and linear protofilaments are formed, in which periodic repeats of ca 35 nm can be distinguished clearly. The rings constitute about 10% of all fibrillar species under these conditions and they are characterised by a larger diameter of 70–80 nm. All the structures bind Congo red and thioflavine T in a manner similar to fibrils associated with a variety of amyloid diseases. At pH 2.0, fibril formation is accompanied by some acidic hydrolysis, producing specific fragmentation of the protein, leading to the accumulation of two peptides in particular, consisting of residues 1–80 and 54–125. At the initial stages of incubation, however, full-length equine lysozyme represents the dominant species within the fibrils. We propose that the ring-shaped structures observed here, and in the case of disease-associated proteins such as -synuclein, could be a second generic type of amyloid structure in addition to the more common linear fibrils.

Keyword
amyloidosis, lysozyme, protofilament, circularisation, acidic hydrolysis
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-9922 (URN)10.1016/S0022-2836(03)00551-5 (DOI)12850154 (PubMedID)
Available from: 2008-05-23 Created: 2008-05-23 Last updated: 2012-03-23Bibliographically approved
2. Does the cytotoxic effect of transient amyloid oligomers from common equine lysozyme in vitro imply innate amyloid toxicity?
Open this publication in new window or tab >>Does the cytotoxic effect of transient amyloid oligomers from common equine lysozyme in vitro imply innate amyloid toxicity?
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2005 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 8, 6269-6275 p.Article in journal (Refereed) Published
Abstract [en]

In amyloid diseases, it is not evident which protein aggregates induce cell death via specific molecular mechanisms and which cause damage because of their mass accumulation and mechanical properties. We showed that equine lysozyme assembles into soluble amyloid oligomers and protofilaments at pH 2.0 and 4.5, 57 degrees C. They bind thioflavin-T and Congo red similar to common amyloid structures, and their morphology was monitored by atomic force microscopy. Molecular volume evaluation from microscopic measurements allowed us to identify distinct types of oligomers, ranging from tetramer to octamer and 20-mer. Monomeric lysozyme and protofilaments are not cytotoxic, whereas the oligomers induce cell death in primary neuronal cells, primary fibroblasts, and the neuroblastoma IMR-32 cell line. Cytotoxicity was accessed by ethidium bromide staining, MTT reduction, and TUNEL assays. Primary cultures were more susceptible to the toxic effect induced by soluble amyloid oligomers than the neuroblastoma cell line. The cytotoxicity correlates with the size of oligomers; the sample incubated at pH 4.5 and containing larger oligomers, including 20-mer, appears to be more cytotoxic than the lysozyme sample kept at pH 2.0, in which only tetramers and octamers were found. Soluble amyloid oligomers may assemble into rings; however, there was no correlation between the quantity of rings in the sample and its toxicity. The cytotoxicity of transient oligomeric species of the ubiquitous protein lysozyme indicates that this is an intrinsic feature of protein amyloid aggregation, and therefore soluble amyloid oligomers can be used as a primary therapeutic target and marker of amyloid disease.

Keyword
Amyloid/*metabolism, Amyloidosis/etiology/pathology, Animals, Cell Death, Cell Line; Tumor, Cells; Cultured, Dimerization, Fibroblasts/pathology, Horses, Hydrogen-Ion Concentration, Mice, Mice; Inbred BALB C, Microscopy; Atomic Force, Muramidase/*metabolism, Neuroblastoma/pathology, Neurons/*pathology
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-16484 (URN)10.1074/jbc.M407273200 (DOI)15576361 (PubMedID)
Available from: 2007-12-16 Created: 2007-12-16 Last updated: 2012-03-23Bibliographically approved
3. Ultrathin silver nanowires produced by amyloid biotemplating
Open this publication in new window or tab >>Ultrathin silver nanowires produced by amyloid biotemplating
2008 (English)In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 24, no 5, 1166-1170 p.Article in journal (Refereed) Published
Abstract [en]

By using a self-assembled amyloid from lysozyme as biotemplate we produced an ultrathin silver wire of 1 nm diameter and up to 2 μm in length, which is at the limit attainable in nanobiotechnological manufacturing. We showed that 2,2,2-trifluoroethanol produces a dual effect: it reduces ionic silver to colloidal nanoparticles with a regular size, depending on the length of incubation, and induces fibrillar assembly into the amyloid scaffold, forming the hollow channel filled with silver.

Keyword
amyloid;biopolymers;biotemplating;nanowires;silver;2, 2, 2-trifluoroethanol
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-2437 (URN)10.1002/btpr.49 (DOI)
Available from: 2007-06-01 Created: 2007-06-01 Last updated: 2012-03-23Bibliographically approved

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