Does the cytotoxic effect of transient amyloid oligomers from common equine lysozyme in vitro imply innate amyloid toxicity?
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.
Publication Date: 2004-12-01 PubMed ID: 15576361DOI: 10.1074/jbc.M407273200Google Scholar: Lookup
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- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article investigates the inherent toxicity of transient amyloid oligomers formed by equine lysozyme. The researchers found that the cytotoxicity is linked with oligomer size, potentially classifying soluble amyloid oligomers as primary therapeutic targets and markers for amyloid diseases.
Aim and methods of the research
- The research aimed to explore the cytotoxic effects of transient amyloid oligomers produced from an equine lysozyme, a commonly used protein in scientific research. It addressed the elusive aspect of which protein aggregates in amyloid diseases are responsible for causing cell death through specific molecular pathways.
- In the study, the researchers exposed equine lysozyme to different pH conditions and temperatures, and subsequently evaluated its ability to form soluble amyloid oligomers and protofilaments. The structures were also stained with traditional amyloid identifiers, thioflavin-T, and Congo red, to visually confirm their amyloid status.
- The researchers employed atomic force microscopy to further scrutinize the morphology of these structures and used these microscopic measurements to evaluate their molecular volumes.
Findings and interpretation
- The research found that monomeric lysozyme and protofilaments were non-cytotoxic. However, amyloid oligomers of varying sizes were found to induce cell death in different cell types, including primary neuronal cells, primary fibroblasts, and the neuroblastoma IMR-32 cell line.
- Cytotoxicity was measured using diverse techniques including ethidium bromide staining, MTT reduction, and TUNEL assays. Results from these methods highlighted that primary cultures were more susceptible to the toxic effects of soluble amyloid oligomers compared to the neuroblastoma cell line.
- Interestingly, the cytotoxic effect showed a correlation with oligomer size, where larger oligomers, including the 20-mer, found in samples incubated at a pH of 4.5, were considerably more toxic compared to smaller oligomers found at pH 2.0.
- Nevertheless, when the soluble amyloid oligomers formed into ring structures, the researchers did not observe correlative toxicity. This observation indicates that the cytotoxicity is not linked to the quantity of rings in the sample.
Concluding remarks and implications
- The study concludes that the cytotoxicity of transient oligomeric species formed from equine lysozyme might represent a natural characteristic of protein amyloid aggregation. This finding alludes to the possibility of targeting soluble amyloid oligomers therapeutically and using them as markers in diagnosing amyloid diseases.
- While the research provides evidence for the inherent toxicity in amyloid oligomers, more research is needed to further delve into the relationship between the structure and cytotoxic properties of these amyloid formation phases, including ring structures.
Cite This Article
APA
Malisauskas M, Ostman J, Darinskas A, Zamotin V, Liutkevicius E, Lundgren E, Morozova-Roche LA.
(2004).
Does the cytotoxic effect of transient amyloid oligomers from common equine lysozyme in vitro imply innate amyloid toxicity?
J Biol Chem, 280(8), 6269-6275.
https://doi.org/10.1074/jbc.M407273200 Publication
Researcher Affiliations
- Department of Medical Biochemistry and Biophysics, Umeå University, Umeå 90187, Sweden.
MeSH Terms
- Amyloid / metabolism
- Amyloidosis / etiology
- Amyloidosis / 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
Citations
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