2023 Fiscal Year Research-status Report
高毒性型αシヌクレイン凝集構造の獲得機序解明と構造スイッチ治療の開発
| Project/Area Number |
22K15643
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| Research Institution | Osaka University |
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Principal Investigator |
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| Project Period (FY) |
2022-04-01 – 2025-03-31
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| Keywords | alpha synuclein / amyloid fibrils / fibril morphology / amyloidosis |
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| Outline of Annual Research Achievements |
1) Expression of recombinant alpha synuclein and amyloid fibril formation. There is a constant production of recombinant alpha synuclein to keep generating amyloid fibrils with them.
2) The visualization of mature amyloid fibrils has been performed using transmission electron microscopy. It has been possible to detect the presence of a reproducible morphology when amyloid fibrils are treated with a variety of compounds similar to the polyphenol compound A.
3) Molecular mechanisms of morphology switching. We have designed a strategy that involves a partial digestion of amyloid fibrils with proteases in order to obtain fragments that are planned to be analyzed by mass spectrometry. This tool is expected to corroborate the chemical reaction.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
1. At this point, the method for protein purification has totally been established, including controls of the protein purity and its performance at forming amyloid fibrils.
2. The morphology of the fibrils treated with the compound A and its derivatives is consistent from reaction to reaction, independently of the compound employed. This strongly suggests a common chemical event that takes place in all the reactions evaluated.
3. We have reconsidered the strategy to obtain chemical evidences that corroborate the same chemical reaction is taking place with all the compounds tested. Currently we have adjusted the original plan in order to integrate spectroscopic tools, more specifically mass spectrometry and 1D nuclear magnetic resonance, to provide a general mechanism of action. Eventually, we think the results can be employed to designed an optimized structure that shows an improved performance when reacting with amyloid fibrils.
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| Strategy for Future Research Activity |
Since the current status of the project, after adjusting the chemical strategy, 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. Essentially through nuclear magnetic resonance and mass spectrometry. One of the difficulties found was the technical limitation of the size of the switched morphologies to be analyzed by mass spectrometry and NMR. Now we have designed a pretreatment with proteases that more specifically cleave the reacted section of the fibrils and facilitate the spectroscopic analyses.
2) With the spectroscopic evidences, we plan to use the results to design an improved compound with better characteristics that the currently available in our materials. Two options will be explored: 1) similar compounds that show structural similarities; 2) evaluate a chemical synthesis of an optimized compound
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| Causes of Carryover |
Some of the resources had to be transferred to the following fiscal year to purchase periodically materials like buffers and purification compounds
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