BMP-1 site. Further deletion constructs from Pro11, including Pro12-14 also showed lower inhibitory effects on myostatin activity compared with Pro11. The inhibitory effect of Pro11 was consistent in A204 human rhabdomyosarcoma cells. We conclude that Pro11 consisting of 29 amino acid residues is the inhibitory core of the myostatin prodomain. 5 / 18 The Inhibitory Core of the Myostatin Prodomain Fig 2. The identified inhibitory core of the myostatin prodomain specifically suppresses myostatin and its analog, GDF11, and includes an AH that is evolutionarily conserved among several other TGF- family members. The full-length myostatin prodomain and its inhibitory core inhibited the transcriptional activities of myostatin and GDF11, but not of TGF-1 or activin A, in HEK293 cells. Sequence alignment of the prodomains of myostatin in nine species and nine TGF- family members. Red indicates the identified inhibitory core of the myostatin prodomain, consisting of 29 amino acids. The AH structure of the TGF-1 prodomain has been shown to bind to both its ligand and TSP-1. Crystallographic analyses of TGF-1 and its receptors have predicted that the random coiled structure and the AH are located closely to its type I receptor, whereas the latency lasso structure is located close to its type II receptor. doi:10.1371/journal.pone.0133713.g002 The inhibitory core of the myostatin prodomain preferentially inhibits the biological activity of myostatin and its analog, GDF11 A previous study revealed that the full-length myostatin prodomain specifically suppresses myostatin and its analog, GDF11, but not activin. We thus examined which TGF- family members can be suppressed by the identified inhibitory core of the myostatin prodomain in vitro. As shown in Fig 2A, the inhibitory core inhibited not only myostatin, but also GDF11 to the same extent. In contrast, the activities of activin and TGF-1 were not affected by the inhibitory core, suggesting certain specificity of the inhibitory core for suppression of TGF- family members. The inhibitory core of the prodomain suppresses myostatin activity by interacting with myostatin ligand and two distinct receptors By creating a multiple sequence alignment from a protein BLAST, we found that the 29-amino acid inhibitory core in the human myostatin prodomain was well conserved not only in other species, but 
also among other TGF- family members. Of note, a recent mutational STA 4783 web analysis demonstrated that the corresponding region in the TGF-1 prodomain includes PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19747578 an -helix that binds to and suppresses the TGF-1 ligand. As shown in Fig 2C, the AH almost corresponds to the TSP-1 site. Importantly, more recent three-dimensional crystallographic analyses predicted that the inhibitory core of TGF-1 was closely located to both type I and II receptors for TGF-1 and the TGF-1 ligand. 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 6 / 18 The Inhibitory Core of the Myostatin Prodomain Fig 3. Interaction of the inhibitory core of myostatin with its ligand and receptors. Co-localization and co-immunoprecipitation of the inhibitory core of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19748686 the myostatin prodomain and its ligand, its type I receptors, and its type II receptors in COS-7 embryonic kidney cells expressing FLAG-tagged IC and V5- or HA-tagged ligand