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1 Anti-muscle atrophy peptides/proteins The University of Tokushima Graduate School, Institute of Health Biosciences, Department of Physiological Nutrition Takeshi Nikawa, MD and PhD
2 How do we inhibit ubiquitin ligase Cbl-b? We focus on competitively inhibitory function of oligopeptides. iqutin ligase (E3) E2 iquitination IRS-1 IRS-1 Degradation by proteasome E2 IRS-1 Insulin signaling Muscle atrophy Inhibitory oligopeptide Increased protein synthesis Hypertrophy
3 Cbl-b preferentially interacts with phospho-tyrosine of IRS-1 IRS-1 f a c b d e VGYLR Peptide f 18 PI3K DGYMP Peptide a EEYMN Peptide c C-fyn IRS-1: PH PTB Grb GEYVN Peptide b LNYID Peptide d STYAS Peptide e SHP2
4 Inhibitory effect of oligopeptides on IRS-1 ubiquitination WB: IRS-1 Inhibition of Cbl-b-mediated ubiquitination (Cell free ubiquitination system) We named peptide a Cblin as Cbl-b inhibitor. iquitinated IRS-1 IRS-1 Oligopeptides ーー E1 ch7 GST- c-cho ALP inhibitor Peptide a b c d e f (Patent pending )
5 Effect of Cblin on IRS-1 ubiquitination in denervated muscle Experimental design C57/BL6 mice (7 weeks old, male) Group Control [peptide administration (-)] Loading control [Peptide a or control peptide] Denervation [Peptide a or control peptide] Denervation = Cutting sciatic nerve 1week denervation Day 0 Day 2 Day 4 Day 6 Day 8 Day 10 breeding start (normal diet) Day10 administration of oligopeptides by intramuscular injection Dose of oligopeptides:6 mg/mouse Administration of oligopeptides after 6 hr Isolation of gastrocnemius muscles
6 Inhibitory effect of Cblin on Cbl-b-mediated ubiquitination in vivo IP: IRS-1 IB: iquitinated IRS-1 IRS-1 IB: IRS-1 IB: b-actin i. m. : Cblin : Randomized peptide : DGFMP : DGYMP : Sham-ope Denervation
7 Wet muscle weight (%) Expression Effects of Cblin on atrogene expression and muscle weight = Atrogin-1 = MuRF-1 = Cbl-b To investigate the synergystic effects of Cblin with a HSP inducer, 100 we collaborate with Professor Choi at Yonsei University i. m. : Cblin : Randomized peptide : DGFMP : DGYMP : Sham-ope Denervation *P <0.05, injected versus non-injected animals, #P <0.05, Peptide a versus Control peptide treatment in denervated mice.
8 Inhibitory effects of soy-derived peptides on muscle atrophy "Soy glycinin contains a functional inhibitory sequence against muscle atrophy-associated ubiquitin ligase Cbl-b," Tomoki Abe et al. International Journal of Endocrinology, in press. Soy Proteins Lipid proteins 36~40% Glycinine (11S) 39~43% b-conglycinine (7S) 18~19%
9 Amino acid sequences of soy glycinine protein Cblin (DGpYMP) DXYXP Abe T. et al. Int J Endoclin, in press
10 Inhibitory effect of Cblin-like peptide in glycinine on Cbl-b-mediated IRS-1 ubiquitination IRS-1-V5 (Substrate): Cbl-b-HA (E3): - iquitin-flag: Control peptide (mm): Cblin (mm): Cblin-like synthetic peptide (mm): IP: V5 IB: FLAG synthetic Cblin-like peptide DIpYNP Patent No , 2012 Phosphorylation of glycinine IB: P-Tyrosine MMSTD (kda) 97 IP: V5 IB: HA IP: V5 IP: V Abe T. et al. Int J Endoclin, in press
11 Experimental protocol for testing inhibitory effect of glycinine Animals: C57/BL6 mice (6 weeks old, male) Atrophy Model: Denervation Diet Groups Control (20% Casein) 20% SPI (Soy protein isolate) 10% Glycinine (10% Casein &10% Glycinine) 20% Glycinine Denervation Day -7 Preliminary feeding Day 0 Day 4 7days Experimental Diet Sampling For Measurement of hindlimb muscle wet weights Biochemical analysis Histochemical analysis
12 % of denervated muscle weight % of denervated muscle weight % of denervated muscle weight % of denervated muscle weight Inhibitory effects of dietary soy glycinine on denervation-mediated muscle wet weight loss TA Gast EDL Sol * # * * * * Mean ± SE (n=4-5). Significant differences evaluated by the shirley williams test (P<0.05). Abe T. et al. Int J Endoclin, in press
13 CSA (%) Inhibitory effect of dietary soy glycinine on denervation-mediated muscle atrophy 20% Casein 20% SPI 10% Glycinin 20% Glycinin Sham Denervation Bar = 100 mm 100 * # 50 0 Abe T. et al. Int J Endoclin, in press
14 P-Akt-1/Akt-1 Effects of dietary soy glycinine on muscle IGF-1 signaling A Denervation Control 20% Glycinin (day) Sham DenervatioSham Denervation n IB: IRS-1 IB: IRS-1 B IB: Actin IB: Actin C Control 20% Glycinin D Control 20% Glycinin MMSTD (kda) Sham Denervation Sham Denervation IB: P-Akt-1 Sham DenervatioSham Denervation n IB: Akt-1 IP: IRS-1 IB: iquitin * *, # Control 20% Glycinin Sham Denervation Sham Denervation Abe T. et al. Int J Endoclin, in press
15 独立行政法人農業 食品産業技術総合研究機構生物系特定産業技術研究支援センターの支援による 食品の臨床研究にご協力頂ける方を募集しています この研究の成果は 寝たきりや宇宙フライトによる筋萎縮の予防に役立ちます 本研究は稲次整形外科病院と徳島大学病院にて共同実施しています 大豆ペプチドの期待される作用 筋肉を分解する酵素の働きを防ぐ ケルセチン ( タマネギの成分 ) の期待される作用 酸化ストレスを抑えて筋肉の分解を防ぐ 研究の内容と参加期間 * 目的 : 車イスを使用されていたり ベッド上で過ごす時間が多い患者さんを対象に 大豆蛋白質食材 またはタマネギ加工品などを食べていただき 筋肉の衰えを防ぐ効果を調べます * 内容 :1 日 1 回 30 日間試験食を摂取していただき 血液 尿 MRI 検査 筋力測定で効果と安全性を確認します ただし 対照食 ( 有効性を評価する成分を含まないもの ) が当たる可能性があります 研究参加の基準があり ご参加頂けない場合があります ご了承下さい あなたのプライバシーは厳守いたします ご連絡 ご参加頂いた方の情報は外部に漏れることはありませんので 安心してお問い合わせ下さい ご興味のある方は院内スタッフにお声かけ下さい 稲次整形外科病院徳島大学病院整形外科徳島大学大学院ヘルスバイオサイエンス研究部生体栄養学分野徳島大学大学院ヘルスバイオサイエンス研究部食品機能学分野
16 ~The goal of our space experiments~ Development of countermeasures for muscle atrophy caused by bed-rest as well as spaceflight Change! Yes, we can!! By functional foods and drugs.
17 Space biological experiments are beneficial for health of astronauts as well as people on ground. Exploration for Moon, Mars and so on Prevention of agingassociated diseases Improvement for QOL of elderly people Long and safe living in space Space Biological Experiments New drugs Novel functional foods
18 NCNP Shin ichi Takeda Ikuya Nonaka Tokushima Bunri Univ. Kazumi Ishidoh Tokyo Metropolitan Inst. Keiji Tanaka Acknowledgement The Univ. of Tokushima Dept. of Nutritional Physiology Reiko Nakao, Katsuya Hirasaka, Shohei Kohno, Tomoki Abe, Akiko Harada, Shigetada Kondoh, Ayako Maita, Yuushi Okumura Thank you for your attention!! Dept. of Orhopology Natsuo Yasui Jyuntendo Univ. Eiki Kominami Univ. of California, Arvine KM Baldwin Univ. of Texas, Austin Mills Edward JAXA Akira Higashibata Team Myo Dept. of Food Science Junji Terao Dept of Molecular Nutrition Kenichi Miyamoto Dept. of Pharmacology Hisao Nemoto Otsuka Pharmaceutical Co. Ajinomoto Co. Fuji Oil Co.
本文/YA6152C
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