Biochemical study of the dicer-related helicases in C. elegans for anti-nematode drug discovery

2018 Fiscal Year Final Research Report

• Project/Area Number: 16K08229
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Biological pharmacy
• Research Institution: Toyohashi University of Technology
• Principal Investigator: Eki Toshihiko 豊橋技術科学大学, 工学(系)研究科(研究院), 教授 (40192512)
• Research Collaborator: HIROSE yuu ; KOBAYASHI taishi ; MURAKAMI takuro ; KIMURA kosei ; SHIRAKI hayato ; SUGINO misato ; IIDA seiya
• Project Period (FY): 2016-04-01 – 2019-03-31
• Keywords: 線虫 / ダイサー関連ヘリカーゼ / タンパク質間相互作用 / 抗線虫薬 / 核酸依存性ATPase / RNA干渉

Outline of Final Research Achievements

This study has aimed to clarify the biochemical properties of two RNAi factors DRH-3 (Dicer Related Helicase-3) and its paralog DRH-1 by purifying two DRH proteins. The fluorescence-based ATPase assay system was prepared and the double-stranded RNA-dependent ATPase activity of the purified DRH-3 was successfully found in this assay. I used this assay system to try the screening for potential DRH-3 inhibitors.
I prepared 14 GST-tagged mutated E1 proteins, four RNAi-related proteins and His- or His-SUMO-tagged DRH-3 to examine the protein-protein interaction between E1 (a novel protein with two Tudor domains) and DRH-3 by the pull-down method. The results suggested the Tudor domain/methylated residue-independent interaction between E1 and DRH-3 as well as the potential role of the N-terminal region of DRH-3 in the binding to E1.

Free Research Field

分子生物学

Academic Significance and Societal Importance of the Research Achievements

本研究成果として、線虫の繁殖に必須なDRH-3が二本鎖RNA依存性ATPase活性を持つことを明らかにした。未だ生化学的解析のなされていないDRH-1の精製法を確立したことや、DRH-3とE1との相互作用解析からTudorドメインやDRH-3のN末領域に関して新規な知見が得られたことは、線虫DRHの機能を分子レベルで解明するための重要な手がかりとなり、RNA干渉研究における学術的意義がある。本研究を基にDRH-3のATPase活性阻害剤を探索することで、神経系を標的とする既存薬とは作用機序の異なる新規抗線虫薬を開発できる可能性があり、今後、社会的意義のある成果に結びつくことが期待される。