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- Malisauskas, Mantas, et al.
(författare)
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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
- 2003
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Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 330:4, s. 879-890
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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- Malisauskas, Mantas, et al.
(författare)
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Does the cytotoxic effect of transient amyloid oligomers from common equine lysozyme in vitro imply innate amyloid toxicity?
- 2005
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 280:8, s. 6269-6275
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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- Malisauskas, Mantas, et al.
(författare)
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Lability landscape and protease resistance of human insulin amyloid : a new insight into its molecular properties
- 2010
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 396:1, s. 60-74
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid formation is a universal behavior of proteins central to many important human pathologies and industrial processes. The extreme stability of amyloids towards chemical and proteolytic degradation is an acquired property compared to the precursor proteins and is a major prerequisite for their accumulation. Here we report a study on the lability of human insulin amyloid as a function of pH and amyloid ageing. Using a range of methods such as AFM, thioflavin-T fluorescence, circular dichroism and gas phase electrophoretic mobility macromolecule analysis we probed the propensity of human insulin amyloid to propagate or dissociate in a wide span of pHs and ageing in a low concentration regime. We generated a three-dimensional amyloid lability landscape in coordinates of pH and amyloid ageing, which displays three distinctive features: (i) a maximum propensity to grow near pH 3.8 and an age corresponding the inflection point of the growth phase; (ii) an abrupt cut-off between growth and disaggregation at pH 8-10; (iii) isoclines shifted towards older age during the amyloid growth phase at pH 4-9, reflecting the greater stability of aged amyloid. Thus, lability of amyloid strongly depends on the ionization state of insulin and on the structure and maturity of amyloid fibrils. The stability of insulin amyloid towards protease K was assessed by using real-time AFM and thioflavin-T fluorescence. We estimated that amyloid fibrils can be digested both from the free ends and within the length of the fibril with a rate of ca. 4 nm/min. Our results highlight that amyloid structures, depending on solution conditions, can be less stable than commonly perceived. These results have wide implications for understanding the propagation of amyloids via a seeding mechanism as well as for understanding their natural clearance and dissociation under solution conditions unfavorable for amyloid formation in biological systems and industrial applications.
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- Malisauskas, Mantas, 1978-
(författare)
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The amyloid : structure, properties and application
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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- Malisauskas, Mantas, et al.
(författare)
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Ultrathin silver nanowires produced by amyloid biotemplating
- 2008
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Ingår i: Biotechnology progress (Print). - : Wiley. - 8756-7938 .- 1520-6033. ; 24:5, s. 1166-1170
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Tidskriftsartikel (refereegranskat)abstract
- By using a self-assembled amyloid from lysozyme as biotemplate we produced an ultrathin silver wire of 1 nm diameter and up to 2 μm in length, which is at the limit attainable in nanobiotechnological manufacturing. We showed that 2,2,2-trifluoroethanol produces a dual effect: it reduces ionic silver to colloidal nanoparticles with a regular size, depending on the length of incubation, and induces fibrillar assembly into the amyloid scaffold, forming the hollow channel filled with silver.
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- Morozova-Roche, Ludmilla, et al.
(författare)
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Fibrillation of Carrier Protein Albebetin and Its Biologically Active Constructs. Multiple Oligomeric Intermediates and Pathways
- 2004
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:30, s. 9610-9619
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 14. |
- Pansieri, Jonathan, et al.
(författare)
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Templating S100A9 amyloids on Aβ fibrillar surfaces revealed by charge detection mass spectrometry, microscopy, kinetic and microfluidic analyses
- 2020
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Ingår i: Chemical Science. - : Royal Society of Chemistry. - 2041-6520 .- 2041-6539. ; 11:27, s. 7031-7039
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Tidskriftsartikel (refereegranskat)abstract
- The mechanism of amyloid co-aggregation and its nucleation process are not fully understood in spite of extensive studies. Deciphering the interactions between proinflammatory S100A9 protein and Aβ42 peptide in Alzheimer's disease is fundamental since inflammation plays a central role in the disease onset. Here we use innovative charge detection mass spectrometry (CDMS) together with biophysical techniques to provide mechanistic insight into the co-aggregation process and differentiate amyloid complexes at a single particle level. Combination of mass and charge distributions of amyloids together with reconstruction of the differences between them and detailed microscopy reveals that co-aggregation involves templating of S100A9 fibrils on the surface of Aβ42 amyloids. Kinetic analysis further corroborates that the surfaces available for the Aβ42 secondary nucleation are diminished due to the coating by S100A9 amyloids, while the binding of S100A9 to Aβ42 fibrils is validated by a microfluidic assay. We demonstrate that synergy between CDMS, microscopy, kinetic and microfluidic analyses opens new directions in interdisciplinary research.
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