Identification of target substrates of uncharacterized F-box proteins.

• Project/Area Number: 24580152
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Applied biochemistry
• Research Institution: Tokyo Metropolitan Institute of Medical Science
• Principal Investigator: YOSHIDA Yukiko 公益財団法人東京都医学総合研究所, 生体分子先端研究分野, 主任研究員 (90271543)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000); Fiscal Year 2014: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2013: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2012: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: ユビキチン / ユビキチンリガーゼ / F-boxタンパク質 / プロテオミクス / FBXO21 / タンパク質分解

Outline of Final Research Achievements

The Skp1-Cul1-F-box protein (SCF) complex, one of the best-characterized ubiquitin ligases, is composed of three invariable components and a variable component F-ox protein that serves as the substrate recognition subunit. Among the over 70 F-box proteins found in humans, less than half have been characterized. To identify substrates for uncharacterized F-box proteins, I developed a comprehensive method for identification of ubiquitinated substrates using the trypsin-resistant tandem ubiquitin-binding entity (TR-TUBE). The coexpression of TR-TUBE with a F-box protein stabilizes the ubiquitinated substrates by masking the ubiquitin chains. Using a combination of two strategies for enriching ubiquitinated substrates, TR-TUBE and ubiquitin Lys-e-Gly-Gly (diGly) remnant antibody, I successfully identified specific ubiquitin ligase-substrate pairs. Using this method, I identified target substrates of FBXO21, an uncharacterized and ubiquitiously expressed F-box protein.

Research Products

• [Journal Article] A comprehensive method for detecting ubiquitinated substrates using TR-TUBE. (2015)
  • Author(s): Yoshida, Y., Saeki, Y., Murakami, A., Kawawaki, J., Tsuchiya, H., Yoshihara, H., Shindo, M, Tanaka, K.
  • Journal Title: Proc Natl Acad Sci U S A. Volume: 112 Pages: 4630-4635
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] ユビキチン結合プローブUb-catcherを用いたユビキチン化基質の検出と同定 (2014)
  • Author(s): 吉田雪子、佐伯泰、村上有沙、川脇純子、土屋光、吉原英人、進藤真由美、田中啓二
  • Organizer: 第37回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（神奈川・横浜）
  • Year and Date: 2014-11-26
• [Presentation] FBXO17 is involved in formation of stress granule. (2013)
  • Author(s): Yoshida, Y., Murakami, A., Shindo, M., and Tanaka, K.
  • Organizer: 第86回日本生化学会大会
  • Place of Presentation: 横浜
• [Presentation] ポリユビキチン結合タンパク質を利用したポリユビキチン化基質の効率的同定法の確立． (2013)
  • Author(s): 吉田雪子、佐伯泰、村上有沙、土屋光、田中啓二．
  • Organizer: 第36回日本分子生物学会年会
  • Place of Presentation: 神戸
• [Presentation] Skp1変異体を用いたF-box蛋白質の基質の同定． (2012)
  • Author(s): 吉田雪子、村上有沙、田中啓二．
  • Organizer: 第８５回日本生化学会大会
  • Place of Presentation: 福岡
• [Remarks] http://www.igakuken.or.jp/protein/
• [Remarks] 東京都医学総合研究所・蛋白質代謝研究室・吉田チーム
  • URL: http://www.igakuken.or.jp/pro-meta/studies/yoshida.html
• [Patent(Industrial Property Rights)] ポリユビキチン化基質の同定方法 (2014)
  • Inventor(s): 吉田雪子、佐伯泰、土屋光、村上有沙、田中啓二
  • Industrial Property Rights Holder: 吉田雪子、佐伯泰、土屋光、村上有沙、田中啓二
  • Industrial Property Rights Type: 特許
  • Filing Date: 2014-11-13
  • Overseas
• [Patent(Industrial Property Rights)] ポリユビキチン化基質の同定方法 (2013)
  • Inventor(s): 吉田雪子、佐伯泰、土屋光、村上有沙、田中啓二．
  • Industrial Property Rights Holder: 吉田雪子、佐伯泰、土屋光、村上有沙、田中啓二．
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2013-237362
  • Filing Date: 2013-11-15