Developing sensor-proteins to detect CAPN3 protease activity

• Project/Area Number: 23500477
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Neurophysiology and muscle physiology
• Research Institution: National Agriculture and Food Research Organization
• Principal Investigator: OJIMA Koichi 独立行政法人農業・食品産業技術総合研究機構, 畜産草地研究所 畜産物研究領域, 主任研究員 (60415544)
• Co-Investigator(Renkei-kenkyūsha): HATA Shoji (財)東京都医学研究機構, 東京都臨床医学総合研究所, 主席研究員 (10392375) ; ONO Yasuko (財)東京都医学研究機構, 東京都臨床医学総合研究所, 主席研究員 (20392376)
• Project Period (FY): 2011-04-28 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000); Fiscal Year 2013: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2012: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2011: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: 骨格筋 / カルパイン / プロテアーゼ

Outline of Final Research Achievements

Calpain-3 (CAPN3) is predominantly expressed in skeletal muscle and is Ca2+-requiring intracellular cysteine protease. In this study, we made sensor-proteins to detect CAPN3 protease activity using a fluorescence resonance energy transfer technique and examined whether CAPN3 protease activity was controlled by phosphorylation. Unfortunately, it was very tough to detect protease activity of CAPN3 in living muscle cells although sensor-proteins were expressed well and were cleaved by CAPN3 in vitro experiments. On the other hand, we showed that rapid and exhaustive autolysis of CAPN3 was slightly attenuated by the defect of phosphorylation of Ser residue at 629 of CAPN3.

Research Products

• [Journal Article] Muscle-specific calpain-3 is phosphorylated in its unique insertion region for enrichment in a myofibril fraction (2014)
  • Author(s): Ojima, K. Ono, Y. Hata, S. Noguchi, S. Nishino, I. Sorimachi, H.
  • Journal Title: Genes to Cells Volume: 19 Issue: 11 Pages: 830-841
  • DOI: 10.1111/gtc.12181
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] カルパイン3-タンパク質分解酵素の活性不全による骨格筋変性- (2014)
  • Author(s): 尾嶋孝一
  • Organizer: 第157回日本獣医学会学術集会 【日本獣医解剖学会シンポジウム】「細胞形態ダイナミクスをリードする分子群を求めて」
  • Place of Presentation: 札幌市
  • Year and Date: 2014-09-10
  • Invited
• [Presentation] Effects of phosphorylation of muscle-specific calpain-3 on its proteolytic activity. (2013)
  • Author(s): Koichi Ojima, Yasuko Ono, Shoji Hata, Oe Mika, Ikuyo Nakajima, Susumu Muroya, Koichi Chikuni, Hiroyuki Sorimachi
  • Organizer: 2013 Annual Meeting of the American Society for Cell Biology
  • Place of Presentation: NewOrleans, LA, USA
• [Presentation] カルパイン3のリン酸化が酵素活性に与える影響 (2013)
  • Author(s): 尾嶋孝一, 小野弥子, 秦勝志, 大江美香, 中島郁世, 千国幸一, 室谷進, 反町洋之
  • Organizer: 日本畜産学会第117回大会
  • Place of Presentation: 新潟市
• [Presentation] Identification of phosphorylation sites of muscle-specific calpain-3 (2013)
  • Author(s): Koichi Ojima, Yasuko Ono, Shoji Hata, Oe Mika, Ikuyo Nakajima, Susumu Muroya, Koichi Chikuni, Hiroyuki Sorimachi
  • Organizer: FASEB Science Research Conferences, The Biology of Calpains in Health and Disease
  • Place of Presentation: Saxton River, VT, USA
• [Presentation] 骨格筋特異的に発現するカルパイン3はNa+により活性化する (2012)
  • Author(s): 尾嶋孝一, 小野弥子, 中島郁世, 大江美香, 室谷進 , 反町洋之
  • Organizer: 日本畜産学会第115回大会
  • Place of Presentation: 名古屋大学（愛知県）
• [Presentation] Structural functions of skeletal muscle-specific calpain in Ca2+ efflux from the sarcoplasmic reticulum (2011)
  • Author(s): Koichi Ojima, Yasuko Ono, Coen Ottenheijm, Shoji Hata, Hidenori Suzuki, Mika Oe, Ikuyo Nakajima, Susumu Muroya, Henk Granzier, and Hiroyuki Sorimachi
  • Organizer: 2011 Annual Meeting of the American Society for Cell Biology.
  • Place of Presentation: Denver, CO. (USA)