Analysis of negative regulator of necroptosis

• Project/Area Number: 16K15423
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Gastroenterology
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: OSHIMA Shigeru 東京医科歯科大学, 医学部附属病院, 助教 (50376787)
• Project Period (FY): 2016-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000); Fiscal Year 2017: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2016: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
• Keywords: オートファジー / ユビキチン / ネクロプトーシス

Outline of Final Research Achievements

To identify novel GFP-LC3-interacting proteins in intestinal epithelial cells (IECs), we performed immunoprecipitation with a GFP antibody and then analyzed co- immunoprecipitates by mass spectrometry. HADHA was identified as an LC3-interacting protein. Given that HADHA catalyzes the last three steps of mitochondrial beta-oxidation of long-chain fatty acids, we investigated whether long-chain fatty acids induce autophagy. We found that palmitic acid- induced autophagy supports the survival of IECs.
Ubiquitination is frequently a prerequisite for substrate recognition and determines selectivity in autophagy. We developed a polyubiquitin-mediated fluorescence complementation (PolyUb-FC) assay. The PolyUb- FC assay has the advantage that chain-specific polyubiquitination can be directly visualized in living cells without using antibodies. We applied the PolyUb-FC assay to examine RIPK3 polyubiquitination. We demonstrated that RIPK3 colocalized with PolyUb-FC.

Research Products

• [Journal Article] Novel polyubiquitin imaging system, PolyUb-FC, reveals that K33-linked polyubiquitin is recruited by p62/SQSTM1. (2017)
  • Author(s): Yoichi Nibe, Shigeru Oshima, Masanori Kobayashi, Chiaki Maeyashiki, Yu Matsuzawa, Kana Otsubo, Hiroki Matsuda, Emi Aonuma, Yasuhiro Nemoto, Takashi Nagaishi, Ryuichi Okamoto, Kiichiro Tsuchiya, Tetsuya Nakamura, Shinichiro Nakada, Mamoru Watanabe
  • Journal Title: Autophagy. Volume: 22 Issue: 2 Pages: 1-43
  • DOI: 10.1080/15548627.2017.1407889
  • Peer Reviewed / Open Access
• [Journal Article] HADHA, the alpha subunit of the mitochondrial trifunctional protein,is involved in long-chain fatty acid-induced autophagy in intestinal epithelial cells. (2017)
  • Author(s): Maeyashiki C, Oshima S, Otsubo K, Kobayashi M, Nibe Y, Matsuzawa Y, Onizawa M, Nemoto Y, Nagaishi T, Okamoto R, Tsuchiya K, Nakamura T, Watanabe M
  • Journal Title: Biochem Biophys Res Commun. Volume: 484 Issue: 3 Pages: 636-641
  • DOI: 10.1016/j.bbrc.2017.01.159
  • Peer Reviewed
• [Presentation] Novel polyubiquitin imaging system, PolyUb-FC, visualized atypical polyubiquitin chain in living cells (2018)
  • Author(s): Nibe Y, Matsuda H, Aonuma E, Otsubo K, Maeyashiki C, Kobayashi M, Matsuzawa Y, Nakada S, Watanabe M, and Oshima S
  • Organizer: Keystone Symposia - Ubiquitin Signaling (A8)
  • Int'l Joint Research
• [Presentation] Necroptosis stimulation regulates SQSTM1/p62-LC3 complex formation and polyubiquitination. (2018)
  • Author(s): Aonuma E, Nibe Y, Matsuda H, Otsubo K, Maeyashiki C, Kobayashi M, Matsuzawa Y, Watanabe M, and Oshima S
  • Organizer: Keystone Symposia - Ubiquitin Signaling (A8)
  • Int'l Joint Research
• [Presentation] Novel polyubiquitin imaging reveals that atypical polyubiquitin contribute to autophagy (2017)
  • Author(s): Nibe Y, Oshima S, Aonuma E, Matsuda H, Otsubo K, Maeyashiki C, Kobayashia M, Matsuzawa Y , Nakada S, Watanabe M.
  • Organizer: GI research Academy
  • Int'l Joint Research
• [Presentation] HADHA, the alpha subunit of the mitochondrial trifunctional protein, is involved in long-chain fatty acid-induced autophagy in intestinal epithelial cells. (2017)
  • Author(s): Otsubo K, Oshima S, Maeyashiki C, Aonuma E, Matsuda H, Kobayashi M, Nibe Y, Matsuzawa Y, Watanabe M
  • Organizer: The 8th International Symposium on Autophagy
  • Int'l Joint Research
• [Presentation] Novel polyubiquitin imaging reveals that atypical polyubiquitin contribute to autophagy via p62 (2017)
  • Author(s): Nibe Y, Oshima S, Aonuma E, Matsuda H, Otsubo K, Maeyashiki C, Kobayashi M, Matsuzawa Y, Nakada S, Watanabe M.
  • Organizer: The 8th International Symposium on Autophagy
  • Int'l Joint Research
• [Presentation] Indentification of autophagic regulators as therapeutic targets in intestinal epithelial cells. (2016)
  • Author(s): Chiaki Maeyashiki, Shigeru Oshima, Yoishi Nibe, Masanori Kobayashi, Mamoru Watanabe.
  • Organizer: DDW
  • Place of Presentation: San Diego (USA)
  • Year and Date: 2016-05-23
  • Int'l Joint Research