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The Inhibitory Core of the Myostatin Prodomain: Its Interaction with Both Type I and II Membrane Receptors, and Potential to Treat Muscle Atrophy.

Ohsawa Y, Takayama K, Nishimatsu S, Okada T, Fujino M, Fukai Y, Murakami T, Hagiwara H, Itoh F, Tsuchida K, Hayashi Y, Sunada Y - PLoS ONE (2015)

Bottom Line: We identified a 29-amino acid region that inhibited myostatin-induced transcriptional activity by 79% compared with the full-length prodomain.Moreover, intramuscular injection of p29 alleviated muscle atrophy and decreased the absolute force in caveolin 3-deficient limb-girdle muscular dystrophy 1C model mice.The injection suppressed activation of myostatin signaling and restored the decreased numbers of muscle precursor cells caused by caveolin 3 deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Kawasaki Medical School, Kurashiki, Okayama, 701-0192, Japan.

ABSTRACT
Myostatin, a muscle-specific transforming growth factor-β (TGF-β), negatively regulates skeletal muscle mass. The N-terminal prodomain of myostatin noncovalently binds to and suppresses the C-terminal mature domain (ligand) as an inactive circulating complex. However, which region of the myostatin prodomain is required to inhibit the biological activity of myostatin has remained unknown. We identified a 29-amino acid region that inhibited myostatin-induced transcriptional activity by 79% compared with the full-length prodomain. This inhibitory core resides near the N-terminus of the prodomain and includes an α-helix that is evolutionarily conserved among other TGF-β family members, but suppresses activation of myostatin and growth and differentiation factor 11 (GDF11) that share identical membrane receptors. Interestingly, the inhibitory core co-localized and co-immunoprecipitated with not only the ligand, but also its type I and type II membrane receptors. Deletion of the inhibitory core in the full-length prodomain removed all capacity for suppression of myostatin. A synthetic peptide corresponding to the inhibitory core (p29) ameliorates impaired myoblast differentiation induced by myostatin and GDF11, but not activin or TGF-β1. Moreover, intramuscular injection of p29 alleviated muscle atrophy and decreased the absolute force in caveolin 3-deficient limb-girdle muscular dystrophy 1C model mice. The injection suppressed activation of myostatin signaling and restored the decreased numbers of muscle precursor cells caused by caveolin 3 deficiency. Our findings indicate a novel concept for this newly identified inhibitory core of the prodomain of myostatin: that it not only suppresses the ligand, but also prevents two distinct membrane receptors from binding to the ligand. This study provides a strong rationale for the use of p29 in the amelioration of skeletal muscle atrophy in various clinical settings.

No MeSH data available.


Related in: MedlinePlus

Interaction of the inhibitory core of myostatin with its ligand and receptors.Co-localization (Upper) and co-immunoprecipitation (Lower) of the inhibitory core (IC) of the myostatin prodomain and its ligand (A), its type I receptors (ALK4 and ALK5, B), and its type II receptors (ActRIIA and ActRIIB, C) in COS-7 embryonic kidney cells expressing FLAG-tagged IC and V5- or HA-tagged ligand or receptors. Scale bar, 20 μm. Whole cell extracts (WCE) were immunoprecipitated with anti-FLAG, anti-V5, or anti-HA agarose and then immunoblotted using anti-FLAG, anti-V5, or anti-HA antibodies, respectively.
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pone.0133713.g003: Interaction of the inhibitory core of myostatin with its ligand and receptors.Co-localization (Upper) and co-immunoprecipitation (Lower) of the inhibitory core (IC) of the myostatin prodomain and its ligand (A), its type I receptors (ALK4 and ALK5, B), and its type II receptors (ActRIIA and ActRIIB, C) in COS-7 embryonic kidney cells expressing FLAG-tagged IC and V5- or HA-tagged ligand or receptors. Scale bar, 20 μm. Whole cell extracts (WCE) were immunoprecipitated with anti-FLAG, anti-V5, or anti-HA agarose and then immunoblotted using anti-FLAG, anti-V5, or anti-HA antibodies, respectively.

Mentions: To explore the precise molecular mechanism by which the inhibitory core of the myostatin prodomain suppresses the biological activity of myostatin, we first examined whether the inhibitory core of the prodomain can interact with the type I and II receptors, and the myostatin ligand. COS-7 monkey kidney cells were co-transfected with constructs harboring the C-terminal FLAG-tagged inhibitory core and V5-tagged full-myostatin (prodomain + ligand) or C-terminal mature myostatin (ligand). As expected, individually tagged proteins co-localized and co-immunoprecipitated with each other (Fig 3A). Similar results were observed for the FLAG-tagged inhibitory core and HA-tagged full-GDF11 (prodomain + ligand) or C-terminal mature GDF11 (ligand) (S3 Fig). To further explore the intriguing interactions of the inhibitory core with receptors, we co-transfected COS-7 cells with constructs harboring a FLAG-tagged inhibitory core and either HA-tagged type I (ALK5, ALK4) or type II (ActRIIB, ActRIIA) receptors [21]. Fig 3 shows co-localization and co-immunoprecipitation of the inhibitory core and each receptor (Fig 3B and 3C). These results suggest that the identified inhibitory core can interact not only with its ligand, but also the two receptors as previously predicted by TGF-β1 crystal structure analyses [19,20].


The Inhibitory Core of the Myostatin Prodomain: Its Interaction with Both Type I and II Membrane Receptors, and Potential to Treat Muscle Atrophy.

Ohsawa Y, Takayama K, Nishimatsu S, Okada T, Fujino M, Fukai Y, Murakami T, Hagiwara H, Itoh F, Tsuchida K, Hayashi Y, Sunada Y - PLoS ONE (2015)

Interaction of the inhibitory core of myostatin with its ligand and receptors.Co-localization (Upper) and co-immunoprecipitation (Lower) of the inhibitory core (IC) of the myostatin prodomain and its ligand (A), its type I receptors (ALK4 and ALK5, B), and its type II receptors (ActRIIA and ActRIIB, C) in COS-7 embryonic kidney cells expressing FLAG-tagged IC and V5- or HA-tagged ligand or receptors. Scale bar, 20 μm. Whole cell extracts (WCE) were immunoprecipitated with anti-FLAG, anti-V5, or anti-HA agarose and then immunoblotted using anti-FLAG, anti-V5, or anti-HA antibodies, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520684&req=5

pone.0133713.g003: Interaction of the inhibitory core of myostatin with its ligand and receptors.Co-localization (Upper) and co-immunoprecipitation (Lower) of the inhibitory core (IC) of the myostatin prodomain and its ligand (A), its type I receptors (ALK4 and ALK5, B), and its type II receptors (ActRIIA and ActRIIB, C) in COS-7 embryonic kidney cells expressing FLAG-tagged IC and V5- or HA-tagged ligand or receptors. Scale bar, 20 μm. Whole cell extracts (WCE) were immunoprecipitated with anti-FLAG, anti-V5, or anti-HA agarose and then immunoblotted using anti-FLAG, anti-V5, or anti-HA antibodies, respectively.
Mentions: To explore the precise molecular mechanism by which the inhibitory core of the myostatin prodomain suppresses the biological activity of myostatin, we first examined whether the inhibitory core of the prodomain can interact with the type I and II receptors, and the myostatin ligand. COS-7 monkey kidney cells were co-transfected with constructs harboring the C-terminal FLAG-tagged inhibitory core and V5-tagged full-myostatin (prodomain + ligand) or C-terminal mature myostatin (ligand). As expected, individually tagged proteins co-localized and co-immunoprecipitated with each other (Fig 3A). Similar results were observed for the FLAG-tagged inhibitory core and HA-tagged full-GDF11 (prodomain + ligand) or C-terminal mature GDF11 (ligand) (S3 Fig). To further explore the intriguing interactions of the inhibitory core with receptors, we co-transfected COS-7 cells with constructs harboring a FLAG-tagged inhibitory core and either HA-tagged type I (ALK5, ALK4) or type II (ActRIIB, ActRIIA) receptors [21]. Fig 3 shows co-localization and co-immunoprecipitation of the inhibitory core and each receptor (Fig 3B and 3C). These results suggest that the identified inhibitory core can interact not only with its ligand, but also the two receptors as previously predicted by TGF-β1 crystal structure analyses [19,20].

Bottom Line: We identified a 29-amino acid region that inhibited myostatin-induced transcriptional activity by 79% compared with the full-length prodomain.Moreover, intramuscular injection of p29 alleviated muscle atrophy and decreased the absolute force in caveolin 3-deficient limb-girdle muscular dystrophy 1C model mice.The injection suppressed activation of myostatin signaling and restored the decreased numbers of muscle precursor cells caused by caveolin 3 deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Kawasaki Medical School, Kurashiki, Okayama, 701-0192, Japan.

ABSTRACT
Myostatin, a muscle-specific transforming growth factor-β (TGF-β), negatively regulates skeletal muscle mass. The N-terminal prodomain of myostatin noncovalently binds to and suppresses the C-terminal mature domain (ligand) as an inactive circulating complex. However, which region of the myostatin prodomain is required to inhibit the biological activity of myostatin has remained unknown. We identified a 29-amino acid region that inhibited myostatin-induced transcriptional activity by 79% compared with the full-length prodomain. This inhibitory core resides near the N-terminus of the prodomain and includes an α-helix that is evolutionarily conserved among other TGF-β family members, but suppresses activation of myostatin and growth and differentiation factor 11 (GDF11) that share identical membrane receptors. Interestingly, the inhibitory core co-localized and co-immunoprecipitated with not only the ligand, but also its type I and type II membrane receptors. Deletion of the inhibitory core in the full-length prodomain removed all capacity for suppression of myostatin. A synthetic peptide corresponding to the inhibitory core (p29) ameliorates impaired myoblast differentiation induced by myostatin and GDF11, but not activin or TGF-β1. Moreover, intramuscular injection of p29 alleviated muscle atrophy and decreased the absolute force in caveolin 3-deficient limb-girdle muscular dystrophy 1C model mice. The injection suppressed activation of myostatin signaling and restored the decreased numbers of muscle precursor cells caused by caveolin 3 deficiency. Our findings indicate a novel concept for this newly identified inhibitory core of the prodomain of myostatin: that it not only suppresses the ligand, but also prevents two distinct membrane receptors from binding to the ligand. This study provides a strong rationale for the use of p29 in the amelioration of skeletal muscle atrophy in various clinical settings.

No MeSH data available.


Related in: MedlinePlus