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Structural studies of heterogeneous amyloid species of lysozymes and de novo protein albebetin and their cytotoxicity
Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
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 [en]
amyloid, cytotoxicity, atomic force microscopy
National Category
Biophysics
Identifiers
URN: urn:nbn:se:umu:diva-1085OAI: oai:DiVA.org:umu-1085DiVA: diva2:140152
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
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: 2017-12-14Bibliographically approved
2. Fibrillation of Carrier Protein Albebetin and Its Biologically Active Constructs. Multiple Oligomeric Intermediates and Pathways
Open this publication in new window or tab >>Fibrillation of Carrier Protein Albebetin and Its Biologically Active Constructs. Multiple Oligomeric Intermediates and Pathways
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2004 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Biochemistry, Vol. 43, no 30, 9610-9619 p.Article in journal (Refereed) Published
Abstract [en]

We showed that the genetically engineered carrier-protein albebetin and its biologically active constructs with interferon-2 octapeptide LKEKKYSP or differentiation factor hexapeptide TGENHR are inherently highly amyloidogenic at physiological pH. The kinetics of fibrillation were monitored by thioflavine-T (ThT) binding and the morphological changes by atomic force microscopy. Fibrillation proceeds via multiple pathways and includes a hierarchy of amyloid structures ranging from oligomers to protofilaments and fibrils. Comparative height and volume microscopic measurements allowed us to identify two distinct types of oligomeric intermediates: pivotal oligomers ca. 1.2 nm in height comprised of 10-12 monomers and on-pathway amyloid-competent oligomers ca. 2 nm in height constituted of 26-30 molecules. The former assemble into chains and rings with "bead-on-string morphology", in which a "bead" corresponds to an individual oligomer. Once formed, the rings and chains remain in solution simultaneously with fibrils. The latter give rise to protofilaments and fibrils, and their formation is concomitant with an increasing level of ThT binding. The amyloid nature of filamentous structures was confirmed by a pronounced ThT and Congo red binding and -sheet-rich far-UV circular dichroism. We suggest that transformation of the pivotal oligomers into the amyloid-prone ones is a limiting stage in amyloid assembly. Peptides, either fused to albebetin or added into solution, and an increased ionic strength promote fibrillation of albebetin (net charge of -12) by counterbalancing critical electrostatic repulsions. This finding demonstrates that the fibrillation of newly designed polypeptide-based products can produce multimeric amyloid species with a potentially "new" functionality, raising questions about their safety.

Identifiers
urn:nbn:se:umu:diva-14787 (URN)10.1021/bi0494121 (DOI)15274615 (PubMedID)
Available from: 2007-12-16 Created: 2007-12-16 Last updated: 2017-12-14Bibliographically approved
3. Cytotoxicity of albebetin oligomers depends on cross-beta-sheet formation
Open this publication in new window or tab >>Cytotoxicity of albebetin oligomers depends on cross-beta-sheet formation
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2006 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 580, no 10, 2451-2457 p.Article in journal (Refereed) Published
Abstract [en]

Prefibrillar cytotoxicity was suggested as a common amyloid characteristic. We showed two types of albebetin prefibrillar oligomers are formed during incubation at pH 7.3. Initial round-shaped oligomers consist of 10–15 molecules determined by atomic force microscopy, do not bind thioflavin-T and do not affect viability of granular neurons and SH-SY5Y cells. They are converted into ca. 30–40-mers possessing cross-β-sheet and reducing viability of neuronal cells. Neither monomers nor fibrils possess cytotoxicity. We suggest that oligomeric size is important for stabilising cross-β-sheet core critical for cytotoxicity. As albebetin was used as a carrier-protein for drug delivery, examination of amyloidogenicity is required prior polypeptide biomedical applications.

Keyword
Amyloid; Oligomers; Cytotoxicity; Atomic force microscopy
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-6632 (URN)10.1016/j.febslet.2006.03.074 (DOI)16638570 (PubMedID)
Available from: 2007-12-16 Created: 2007-12-16 Last updated: 2017-12-14Bibliographically approved
4. Lysozyme amyloid oligomers and fibrils induce cellular death via different apoptotic/necrotic pathways
Open this publication in new window or tab >>Lysozyme amyloid oligomers and fibrils induce cellular death via different apoptotic/necrotic pathways
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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.

Keyword
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:nbn:se:umu:diva-6630 (URN)10.1016/j.jmb.2006.10.101 (DOI)17134716 (PubMedID)
Available from: 2007-12-16 Created: 2007-12-16 Last updated: 2017-12-14Bibliographically approved

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