| 研究課題/領域番号 |
22K15643
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| 研究機関 | 大阪大学 |
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研究代表者 |
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| 研究期間 (年度) |
2022-04-01 – 2025-03-31
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| キーワード | alpha synuclein / amyloid fibrils / fibril morphology |
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| 研究実績の概要 |
1) Expression of recombinant alpha synuclein and amyloid fibril formation. i) Employing a well established E. coli expression system, recombinant alpha synuclein has been purified until reaching a purity of > 95 %, as determined by SDS-PAGE and mass spectrometry. ii) Monitoring the formation of amyloid fibrils through Thioflavin T intensity fluorescence, it has been possible to optimize the conditions that generate fibrils with reproducible physicochemical and morphological characteristics.
2) The visualization of mature amyloid fibrils has been performed using transmission electron microscopy. The protocol of negative staining has been optimized with phosphotungstic acid solutions. The visualization settings in the microscope were also adjusted to obtain high quality images.
3) The morphology switching of fibrils was evaluated using the polyphenol compound A. The conditions of morphology switching were also optimized in order to generate reproducible results. In consequence, we were able to modulate the morphology of the fibrils after treatment with the compound A, as corroborated with electron microscopy.
4) Molecular mechanisms of morphology switching. We started exploring the chemical processes that take place during the fibril morphology change. We have obtained evidence of possible chemical reactions, although this part will be develop in deeper detail during the current fiscal year.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
1. The purification of recombinant protein could be successfully performed, then there was no delay to generate amyloid fibrils that satisfy the minimal requirements of quality. In addition, the fibril morphologies were consistently uniform from batch to batch of purification, day to day of experiment, and examiner to examiner that followed the same standardized protocol.
2. As done with the protein purification, the standardization of the protocol of microscopy visualization allowed to obtain images with the highest resolution and quality that let us characterize properly the morphological properties of a fibril and perform statistical analyses of the parameters of the fibrils.
3. The evaluation of the morphological characteristics of the fibrils and their modulation represent the most important objective achieved during the last first year. It was crucial to corroborate that the fibrils could be regulated in shape, size, and quantity without producing depolymerization of the fibrils. The current compound A satisfy the formerly mentioned criteria.
4. We started the chemical characterization of the reaction that took place and allowed the regulation of fibrils morphology. We are still exploring the mechanism of the reaction, however, we think the fundamental step has already been identified. Currently, this step is under evaluation to strengthen the experimental evidences and proceed to evaluate similar compounds in order to optimize the structure of our candidate compounds.
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| 今後の研究の推進方策 |
Since the current status of the project seems to be in track with the originally planned objectives, we are considering to continue with the next stage of the experimental schedule:
1) Complete the chemical characterization of the reaction that takes place between the fibrils and the compounds. We are planning to perform spectroscopic analyses, essentially nuclear magnetic resonance and mass spectrometry. These experiments would allow us to detect the molecular intermediate of the reaction and get a clearer image of the steps involved during the morphological switch.
2) Compare compounds with similar structure. The core structure required for the morphological switching has been identified, then we plan to evaluate similar compounds that contain the fundamental core structure in order to optimize the chemical reaction; and even propose the synthesis of a new chemical compound that permits a more efficient reaction but that does not acquire a higher cytotoxicity.
3) Evaluate the morphological change and its effect in cytotoxicity. One of the most important objective of this project is to demonstrate that the fibrils whose morphology has been regulated with our candidate compounds reduce the toxicity of the fibrils. Employing a cell system and a caenorhabditis elegans model, we plan to corroborate the effect of fibrils treated and not treated with our candidate compounds. These in vivo systems have already been established in the team for other purposes.
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| 次年度使用額が生じた理由 |
Due to the need of continue with the research plan and keep it on track, it was required to adjust the amount of money to purchase materials and reagents during the previous fiscal year.
However, we consider the approved budget will sufficiently allow the completion of the project on time.
The designed experimental schedule has been planned taking into account that the total amount of expenses will match properly the total amount of the approved budget.
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