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Does the cytotoxic effect of transient amyloid oligomers from common equine lysozyme in vitro imply innate amyloid toxicity?
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Lundgren)
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
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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.

Place, publisher, year, edition, pages
2005. Vol. 280, no 8, 6269-6275 p.
Keyword [en]
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: urn:nbn:se:umu:diva-16484DOI: 10.1074/jbc.M407273200PubMedID: 15576361OAI: oai:DiVA.org:umu-16484DiVA: diva2:156157
Available from: 2007-12-16 Created: 2007-12-16 Last updated: 2017-12-14Bibliographically approved
In thesis
1. 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
2. Mechanisms involved in amyloid induced cytotoxicity
Open this publication in new window or tab >>Mechanisms involved in amyloid induced cytotoxicity
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Amyloidoses comprise a group of diseases where normal or mutated protein precipitates into amyloid fibrils. The deposition of fibrils causes dysfunction of organs and toxicity to nervous tissue. Up to date, 24 different proteins and peptides are known to be able to form amyloid fibrils. The most well known are Amyloid beta peptide and Prione protein causing Alzheimer’s disease and Creutzfeld Jacob’s disease respectively.

The aims of this thesis were to investigate the structural properties of cytotoxic amyloid and examine the mechanisms involved. The model protein mostly used in the studies was the plasma protein transthyretin (TTR). Familial Amyloidotic Polyneuropathy (FAP) is a hereditary, autosomal-dominant neurodegenerative disease caused by point mutations in the TTR gene. One of the most common variants of FAP is a mutation in position 30 where alanine is exchanged for methonine. This gives rise to “Skellefteåsjukan” in Sweden.

TTR is secreted into the plasma as a tetramer. Point mutations destabilize the tetramer leading to disassembled monomers, which undergo partial denaturation as an initiation step to aggregation and amyloid fibril formation. In vivo amyloidogenesis takes a long time and does not occur until late in adult life. Most of the clinical TTR mutations do not form amyloid in vitro under physiological conditions. We have created amyloidogenic TTR mutants that are prone to aggregate and form fibrils under physiological conditions. This provides us with a model system on the cellular level for studies of the mechanisms of amyloid associated cytotoxicity as we can control the aggregation process and capture defined stages in the TTR amyloidogenic pathway.

We used Atomic Force Microscopy (AFM) to follow the morphology of aggregates during fibril formation. Initially, amorphous aggregates were formed that subsequently matured into fibrillar structures, denoted protofilaments. This observation was interpreted as an optimisation of ß-strand registers. In addition we identified a correlation between the presence of early-formed aggregates of TTR and cytotoxicity. The toxic response was mediated via an apoptotic mechanism.

We were not able to more carefully determine the structure and size of the toxic TTR species. To address this problem we turned to another amyloidogenic protein, equine lysozyme (EL). Intermediate samples corresponding to the aggregation and growth phase of amyloid fibrils of EL were collected. These samples were subjected to cytotoxicity assays as well as monomeric starting material and mature amyloid fibrillar species. The results clearly showed that the soluble oligomers were cytotoxic in contrast to the monomers and fibrils. Our data indicate that the toxic properties of the oligomers are size dependent.

In this thesis we asked the question whether all mutated forms of TTR can be expressed and secreted or if there is a selection against the most aggressive mutations in vivo? We transfected hematopoetic K562 cells with wild type or mutant TTR, with or without the N-terminal signal peptide, responsible for secretion, to generate both extra- and intracellular TTR. We show that the post-translational quality control of the cells does not allow intracellular mutant TTR outside the secretory pathway, possibly due to the cytotoxic effects, while translocated to the secretory pathway made it escape the quality control permitting secretion and amyloid formation outside the cells.

We have further analyzed the cytotoxic mechanisms induced by TTR oligomers with a focus on intracellular apoptotic signalling pathways. We show that TTR oligomers bind to the surface of the target cells but are not taken up, that is in contrast to mature fibrils that do not bind them at all. The apoptotic response occurred in a caspase-independent and a free radical dependent way.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi, 2005. 49 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 972
Keyword
amyloid, transthyretin, cytotoxicity, apoptosis, caspases, aggregation
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-541 (URN)91-7305-897-1 (ISBN)
Public defence
2005-05-27, Major Groove, By 6L Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Available from: 2005-05-18 Created: 2005-05-18 Last updated: 2009-11-17Bibliographically approved

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Malisauskas, MantasOstman, JohanZamotin, VladimirLundgren, ErikMorozova-Roche, Ludmilla
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