1. No category

Anti-muscle atrophy peptides/proteins
The University of Tokushima Graduate School,
Institute of Health Biosciences,
Department of Physiological Nutrition
Takeshi Nikawa, MD and PhD
([email protected])
How do we inhibit ubiquitin ligase Cbl-b?
We focus on competitively inhibitory function of oligopeptides.
Ubiquitination
Ub Ub
Ub Ub
Ub
Ub
Ub
Ub
Ub
Ub
Ub Ub
Ubiqutin ligase (E3)
Ub
Ub
Ub
E2
IRS-1
E2
IRS-1
IRS-1
Degradation
by proteasome
Muscle atrophy
Insulin signaling
Inhibitory
oligopeptide
Increased protein synthesis
Hypertrophy
Cbl-b preferentially interacts with phospho-tyrosine of IRS-1
f
IRS-1
a
b
c
d
e
PI3K
VGYLR Peptide
‘f’
608
18
IRS-1：
PH
DGYMP Peptide
‘a’
EEYMN Peptide
‘c’
C-fyn
939
PTB
895
GEYVN Peptide
Grb-2
‘b’
1172
1222
LNYID Peptide ‘d’ STYAS Peptide ‘e’
SHP2
Inhibition of Cbl-b-mediated ubiquitination
Inhibitory effect of oligopeptides on IRS-1 ubiquitination
（Cell free ubiquitination system）
WB: IRS-1
Ubiquitinated
IRS-1
We named peptide ‘a’ Cblin as Cbl-b inhibitor.
IRS-1
Oligopeptides
ー
ー
E1
UbcH7
GST-Ub
Ubc-CHO
ALP inhibitor
Peptide
－
－
＋
＋
＋
－
＋
＋
＋
＋
＋
－
a
b
c
d
＋
＋
＋
＋
＋
＋
e
f
（Patent pending 2006-145944）
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
Day 0 Day 2 Day 4 Day 6 Day 8 Day 10
→
←1week→
breeding start
(normal diet)
administration of oligopeptides
by intramuscular injection
Dose of oligopeptides：6 mg/mouse
Day10
Administration of oｌigopeptides
after 6 hr
Isolation of gastrocnemius muscles
Denervation
= Cutting sciatic nerve
Inhibitory effect of Cblin on Cbl-b-mediated ubiquitination in vivo
Ubiquitinated
IRS-1
IP: IRS-1
IB: Ub
IRS-1
IB: IRS-1
IB: b-Actin
i. m. :
Cblin :
Randomized peptide :
DGFMP :
-
+
+
+
-
+
-
+
+
-
+
+
+
-
+
-
+
+
-
-
-
+
-
-
-
+
-
-
DGYMP : -
-
-
+
-
-
-
+
-
Sham-ope.
Denervation
Effects of Cblin on atrogene expression and muscle weight
= Atrogin-1
= MuRF-1
= Cbl-b
Wet muscle weight (%)
Expression
15
10
5
1
0
To investigate the synergystic effects of Cblin with a HSP inducer,
100
we collaborate with Professor Choi at Yonsei University.
50
0
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.
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
Glycinine (11S)
39～43％
36～40％
b-Conglycinine (7S)
18～19％
Amino acid sequences of soy glycinine protein
Cblin
(DGpYMP)
≒
DXYXP
Abe T. et al. Int J Endoclin, in press
Inhibitory effect of Cblin-like peptide in glycinine on
Cbl-b-mediated IRS-1 ubiquitination
IRS-1-V5 (Substrate):
+
+
+
+
+
+
+
+
synthetic Cblin-like peptide
Cbl-b-HA (E3):
-
+
+
+
+
+
+
+
DIpYNP
Ubiquitin-FLAG:
+
+
+
+
+
+
+
+
0 600 0
0
0
0
0
0
0
0
Control peptide (mM): 0
Cblin (mM):
0
0
0 120 0
Cblin-like synthetic peptide (mM):
0
0
0
Patent No. 2015679, 2012
Phosphorylation of glycinine
0 100 200 400 600
IB: P-Tyrosine
MMSTD
(kDa)
97
IP: V5
IB: FLAG
180
IP: V5
IB: HA
21
IP: V5
IP: V5
Abe T. et al. Int J Endoclin, in press
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
Day 0
Day ４
←7days→
→
Preliminary
feeding
Experimental Diet
Sampling
For
・Measurement of hindlimb
muscle wet weights
・Biochemical analysis
・Histochemical analysis
75
50
25
0
#
100
75
50
25
0
*
100
75
50
25
0
Sol
% of denervated muscle weight
100 *
EDL
Gast
% of denervated muscle weight
TA
% of denervated muscle weight
% of denervated muscle weight
Inhibitory effects of dietary soy glycinine on
denervation-mediated muscle wet weight loss
100
75
*
*
*
50
25
0
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
Inhibitory effect of dietary soy glycinine
on denervation-mediated muscle atrophy
20% SPI
20% Casein
10% Glycinin
20% Glycinin
Sham
Denervation
Bar = 100 mm
CSA (%)
#
100
*
50
0
Abe T. et al. Int J Endoclin, in press
Effects of dietary soy glycinine on muscle IGF-1 signaling
A
B
Denervation
0
1
3
Control
4 (day)
IB: IRS-1
IB: IRS-1
IB: Actin
IB: Actin
20% Glycinin
ShamDenervatioSham Denervation
n
D
C
Control
MMSTD
(kDa)
Control
20% Glycinin
Sham Denervation Sham
20% Glycinin
ShamDenervatioSham Denervation
n
Denervation
IB: P-Akt-1
IP: IRS-1
IB: Ubiquitin
P-Akt-1/Akt-1
IB: Akt-1
1
*, #
0.5
*
0
Control
180
Sham Denervation
20% Glycinin
Sham Denervation
Abe T. et al. Int J Endoclin, in press
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。
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
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
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。
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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
~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.
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
Acknowledgement
NCNP
Shin’ichi Takeda
Ikuya Nonaka
Tokushima Bunri Univ.
Kazumi Ishidoh
The Univ. of Tokushima
Dept. of Nutritional Physiology
Reiko Nakao, Katsuya Hirasaka,
Shohei Kohno, Tomoki Abe,
Akiko Harada, Shigetada Kondoh,
Ayako Maita, Yuushi Okumura
Dept. attention!!
of Orhopology
Thank
youInst.
for your
Tokyo Metropolitan
Keiji Tanaka
Natsuo Yasui
Jyuntendo Univ.
Eiki Kominami
Dept. of Food Science
Junji Terao
Univ. of California, Arvine
KM Baldwin
Dept of Molecular Nutrition
Kenichi Miyamoto
Univ. of Texas, Austin
Mills Edward
Dept. of Pharmacology
Hisao Nemoto
JAXA
Akira Higashibata
Team ‘Myo’
Otsuka Pharmaceutical Co.
Ajinomoto Co.
Fuji Oil Co.