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Fibrillation of Carrier Protein Albebetin and Its Biologically Active Constructs. Multiple Oligomeric Intermediates and 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 Science and Technology, Department of Chemistry.
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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
Description
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.

Place, publisher, year, edition, pages
2004. Vol. 43, no 30, 9610-9619 p.
Identifiers
URN: urn:nbn:se:umu:diva-14787DOI: 10.1021/bi0494121PubMedID: 15274615OAI: oai:DiVA.org:umu-14787DiVA: diva2:154459
Available from: 2007-12-16 Created: 2007-12-16 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)
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Available from: 2007-04-17 Created: 2007-04-17 Last updated: 2010-01-18Bibliographically approved

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