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Lysozyme amyloid oligomers and fibrils induce cellular death via different apoptotic/necrotic pathways
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 Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
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2007 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 365, no 5, 1337-1349 p.Article in journal (Refereed) Published
Abstract [en]

Among the newly discovered amyloid properties, its cytotoxicity plays a key role. Lysozyme is a ubiquitous protein involved in systemic amyloidoses in vivo and forming amyloid under destabilising conditions in vitro. We characterized both oligomers and fibrils of hen lysozyme by atomic force microscopy and demonstrated their dose (5–50 μM) and time-dependent (6–48 h) effect on neuroblastoma SH-SY5Y cell viability. We revealed that fibrils induce a decrease of cell viability after 6 h due to membrane damage shown by inhibition of WST-1 reduction, early lactate dehydrogenase release, and propidium iodide intake; by contrast, oligomers activate caspases after 6 h but cause the cell viability to decline only after 48 h, as shown by fluorescent-labelled annexin V binding to externalized phosphatidylserine, propidium iodide DNA staining, lactate dehydrogenase release, and by typical apoptotic shrinking of cells. We conclude that oligomers induce apoptosis-like cell death, while the fibrils lead to necrosis-like death. As polymorphism is a common property of an amyloid, we demonstrated that it is not a single uniform species but rather a continuum of cross-β-sheet-containing amyloids that are cytotoxic. An abundance of lysozyme highlights a universal feature of this phenomenon, indicating that amyloid toxicity should be assessed in all clinical applications involving proteinaceous materials.

Place, publisher, year, edition, pages
2007. Vol. 365, no 5, 1337-1349 p.
Keyword [en]
amyloid cytotoxicity; oligomers; fibrils; apoptosis; necrosis
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-6630DOI: 10.1016/j.jmb.2006.10.101PubMedID: 17134716OAI: oai:DiVA.org:umu-6630DiVA: diva2:146300
Available from: 2007-12-16 Created: 2007-12-16 Last updated: 2012-05-25Bibliographically 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. Amyloids here, amyloids there…What’s wrong with them?
Open this publication in new window or tab >>Amyloids here, amyloids there…What’s wrong with them?
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Amyloid formation is inherent property of proteins which under certain circumstances can become a pathologic feature of a group of diseases called amyloidosis. There are about 30 known human amyloidosis and more than 27 identified proteins involved in these pathologies.  Besides these proteins, there are a growing number of proteins non-related to diseases shown to form amyloid-like structures in vitro, which make them excellent tools for studying amyloid formation mechanisms, physicochemical properties of different amyloid species and the nature of their influence on tissues and cells.  It is important to understand the mechanisms by which amyloids interact with different types of cells, as the leading hypothesis in amyloid field suggests that amyloids and especially their intermediate states are the main harmful, toxic species causing tissue and cell degeneration.

Using de-novo synthesized protein albebetin as a model of amyloidogenic protein, we demonstrated that it forms amyloid-like structures under physiological conditions (pH 7 and 37°C). During aggregation it forms 2 different types of intermediate oligomers — cross-b sheet containing and lacking β-sheet oligomers. Only the former induces cellular toxicity in a dose dependent manner. Further aggregation leads to the formation of fully mature amyloid-like fibrils, which are not toxic to the cells during studied period of incubation.

Another model protein in our studies was hen egg white lysozyme, which readily forms amyloid under denaturing conditions (pH 2,2 and 57°C). In contrast to albebetin and many other proteins reported in the literature, we showed that both oligomers and mature fibrils from hen lysozyme affect cell viability. Targeting different mechanisms involved in cellular death, we revealed that oligomers induce slow and apoptotic-like cell death, while mature fibrils cause rapid and mainly necrotic-like cellular death.   

One of the important aspects of amyloid studies is to develop measures for inhibiting or re-directing the process of amyloid formation to abolish or neutralize toxic amyloid species. Among the agents having inhibitory or modulatory properties small, phenol containing molecules are widely studied. We investigated the effect of the novel nootropic drug noopept on amyloid formation process of α-synuclein, as this drug is a small dipeptide containing a phenol ring. We showed that noopept is able to modulate amyloid formation process by accelerating it to rapid conversion of α-synuclein into fully mature fibrils, thus eliminating the stage of population of toxic oligomeric species.  Using wide range of cytotoxicity assays we showed that amyloid-like fibrils formed in the presence of noopept have no cytotoxic properties.  As this medicine is becoming popular and freely available in some countries as a cognitive enhancer, neuroprotective and nootropic agent, further detailed investigations and clinical trials are needed to assess the safety and benefit of noopept in particular for the patients with amyloid related neurodegenerative diseases (such as Parkinson’s or Alzheimer’s diseases).    

While in vitro models are useful to study some specific aspects of protein aggregation, their properties and effects on cell viability, it is very difficult or practically impossible to create an absolutely accurate model of in vivo situation. Therefore, it is important to turn to in vivo/ex vivo studies to relate the knowledge accumulated from in vitro studies to the real situation in the body.

Using human brain hippocampus tissues from individuals with Alzheimer’s disease, we found that besides well-known and widely accepted main pathological hallmark — Ab peptide deposition, S100A9 and S100A8 pro-inflammatory calcium-binding proteins are also localized in the plaques and in surrounding tissues and very explicitly co-localized with Ab. Moreover, we found the presence of S100A9 within the neuronal cells, which has not been reported before and can be an important clue for understanding the mechanisms of neurodegeneration. In vitro cytotoxicity studies showed that S100A9 protein can efficiently induce cytotoxicity when added exogenously to the neuronal cell culture. These findings suggest that S100A8 and S100A9 proteins play an important role in Alzheimer’s pathology, and potentially can be candidates for the amyloid plaque formation and neurodegeneration. Whether they are associated with inflammatory processes underlying the early onset of disease or produced and accumulated as a consequence of A-beta induced pathology remain to be clarified.

We found that Alzheimer’s disease is not the only pathology associated with A-beta and S100A9 deposition in a form of plaques. Immunohistochemical studies of an aortic valve surgically removed from a patient with aortic stenosis revealed plaque-like structures positively stained with A-beta and S100A9 proteins. These areas are also positively stained with fibril-specific antibodies as well as with Congo red, which also shows very distinct apple-green birefringence under the polarized light. Besides, there is intracellular localization and co-localization of both proteins in interstitial cells throughout the whole fibrous tissue of the valve. The presented case report is the first finding suggesting inflammatory protein S100A9 as well as A-beta peptide as potential candidates for amyloid formation in aortic stenosis valves.  We suggest that there is a specific interaction between A-beta and S100A9 during amyloid formation, which can be involved in amyloid-associated pathology in various tissues and organs in the body, which can potentially be caused by inflammatory processes, particularly by its chronic, long lasting forms.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2012. 67 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1511
Keyword
Amyloids, oligomers, fibrils, cytotoxicity, Alzheimer's disease, Aortic stenosis, S100A9
National Category
Cell and Molecular Biology Other Basic Medicine
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-55621 (URN)978-91-7459-447-8 (ISBN)
Public defence
2012-06-15, KB3B1, KBC, Umeå University, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2012-05-25 Created: 2012-05-24 Last updated: 2012-05-25Bibliographically approved

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Gharibyan, AnnaMorozova-Roche, Ludmilla

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