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Amyloid protofilaments from the calcium-binding protein equine lysozyme: formation of ring and linear structures depends on pH and metal ion concentration
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
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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.

Place, publisher, year, edition, pages
2003. Vol. 330, no 4, 879-890 p.
Keyword [en]
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: urn:nbn:se:umu:diva-9922DOI: 10.1016/S0022-2836(03)00551-5PubMedID: 12850154OAI: oai:DiVA.org:umu-9922DiVA: diva2:149593
Available from: 2008-05-23 Created: 2008-05-23 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Structural studies of heterogeneous amyloid species of lysozymes and de novo protein albebetin and their cytotoxicity
Open this publication in new window or tab >>Structural studies of heterogeneous amyloid species of lysozymes and de novo protein albebetin and their cytotoxicity
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A number of diseases are linked to protein folding problems which lead to the deposition of insoluble protein plaques in the brain or other organs. These diseases include prion diseases such as Creutzfeld-Jakob disease, Alzheimer's disease, Parkinson's disease and type II (non-insulin dependent) diabetes. The protein plaques are found to consist of amyloid fibrils - cross-beta-sheet polymers with the beta-strands arranged perpendicular to the long axis of the fibre. Studies of ex vivo fibrils and fibrils produced in vitro showed that amyloid structures possess similar tinctorial and morphological properties. These suggest that the ability to form amyloid fibrils is an inherent property of polypeptide chains.

The aims of this thesis were to investigate the structural properties of cytotoxic amyloid and examine the involved mechanisms. The model proteins used in the studies were the equine and hen lysozymes and de novo designed protein albebetin.

Lysozymes are naturally ubiquitous proteins. Equine lysozyme belongs to an extended family of structurally related lysozymes and α-lactalbumins and can be considered as an evolutional bridge between them. Hen lysozyme is one of the most characterized protein and its amyloidogenic properties were described earlier. De novo protein albebetin and its constructs are designed to perform the function of grafted polypeptide sequence.

Fibrils of equine lysozyme are formed at acidic pH and elevated temperatures where a partially folded molten globule state is populated. We have shown that lysozyme assembles into annular and linear protofilaments in a calcium-dependent manner.

We showed that albebetin and its constructs are inherently highly amyloidogenic under physiological conditions. Fibrillation proceeds via multiple pathways and includes a hierarchy of amyloid structures ranging from oligomers to protofilaments and fibrils, among which two distinct types of oligomeric intermediates were characterized. Pivotal oligomers comprise of 10-12 monomers and on-pathway amyloid-prone oligomers constitute of 26-30 molecules. We suggest that transformation of the pivotal oligomers into the amyloid-prone ones is a limiting stage in albebetin fibrillation. Cytotoxic studies of albebetin amyloid species have revealed that initial, pivotal oligomers do not effect on cell viability while amyloid-prone ones induce cell death. We suggest that oligomeric size is important for the stabilizing cross-beta-sheet core which is crucial for cell toxicity.

Cytotoxic studies of both oligomers and fibrils of hen lysozyme have revealed that both species induce cell death. The amyloid sample containing cross-β-sheet oligomers induces an apoptosis-like cell death. The oligomers without cross-β-sheet appeared to be non-toxic, indicating that the stabilization of this structural pattern is critical for the induced toxicity. In contrast, the fibrils induce more rapid, necrosis-like death.

These studies gained insights into a structure–function relationship of different forms of amyloid and general pathways of cell death. This is an important step in understanding the mechanisms of amyloid-associated degeneration and defining specific therapeutic targets.

Place, publisher, year, edition, pages
Umeå: Medicinsk kemi och biofysik, 2007. 69 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1110
Keyword
amyloid, cytotoxicity, atomic force microscopy
National Category
Biophysics
Identifiers
urn:nbn:se:umu:diva-1085 (URN)
Public defence
2007-05-08, KB3A9, KBC huset, Umeå university, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2007-04-17 Created: 2007-04-17 Last updated: 2010-01-18Bibliographically approved
2. The amyloid: structure, properties and application
Open this publication in new window or tab >>The amyloid: structure, properties and application
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
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:nbn:se:umu:diva-1164 (URN)978-91-7264-343-7 (ISBN)
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

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Malisauskas, MantasZamotin, VladimirJass, JanaMorozova-Roche, Ludmilla
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