Human Pro-Myostatin Precursor

Matthew Nguyen '26 and Julianna Granetzke '26

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I. Introduction

Myostatin, also known as Growth Differentiation Factor 8 (GDF8), is a paracrine signaling protein that serves as a negative regulator of skeletal muscle growth. It belongs to the Transforming Growth Factor-β superfamily of secreted growth and differentiation factors. The effects of myostatin are influenced by complex mechanisms such as transcriptional and epigenetic regulation and extracellular protein interactions.

Myostatin exists in two distinct forms: pro-myostatin and active myostatin. Pro-myostatin is a precursor protein or a larger, inactivate protein form that requires post-translational modification for activation. Whereas active myostatin has already undergone proteolytic cleavages and has the ability to activate its receptors or bind with its antagonists.

Follistatin is a natural, extracellular antagonist of myostatin that competitively binds to prevent receptor activation. An isoform of follistatin, follistatin 288 (Fst288), can bind and form a complex with active myostatin. In complex with Fst288, a unique electropositive cell surface interaction is created and myostatin degradation is increased, allowing for an increase in muscle development. 

II. General Structure

Pro-myostatin precursor or 5NTU is made up of 670 residues and has a total structural weight of 77.14 kDa. 5NTU is a disulfide linked homodimer made up of two chains each containing a N-terminal pro-domain and a C-terminal growth factor (GF) domain. The N-terminal pro-domains are made up of a helix-loop-helix “forearm” region with α1 and α2 helices and a latency lasso and a spherical “arm” region that sits above the GF domain with a short α-helix and two antiparallel β-sheets, similar to other pro-TGF-β superfamily polypeptides. Interactions between the N-terminal pro-domain and helices and the GF domain hold the terminals together.
The links the C-terminal GF domains together by a between identical Cys339 residues. Together, these interactions form a V-shaped structure.  

III. Pro-Myostatin Activation

Pro-myostatin is composed of an inhibitory N-terminal pro-domain, two protease cleavage sites, and C-terminal growth factor (GF) domain. Myostatin must dimerize at the C-terminal to become active. This C-terminal dimer remains non-covalently bound to the inhibitory N-terminal pro-domain, forming a latent or pro-myostatin complex. The pro-myostatin complex is inactive until cleavage initiation of the pro-domain from the complex by a furin protease. Next, a secondary cleavage takes place on the inhibitory N-terminal pro-domain by tolloid metalloproteases.
The mature myostatin growth factor dimer is released by these cleavages and is now available for binding with its receptors on the cell membranes of target skeletal muscle cells.

IV. Receptor Binding

Following the activation of pro-myostatin, a signaling cascade begins when the mature myostatin growth factor dimer binds with two activin type II receptors (ActIIRA or primarily ActIIRB), phosphorylating the receptors and inducing a conformational change. This allows for the subsequent recruitment and phosphorylation of two activin or TGF-β type I receptors (ALK4 or ALK5 respectively), forming a stable, heterotetrameric signaling complex. The SMAD downstream signaling pathway is activated through the phosphorylation of SMAD 2 and 3 by activated type I receptors. SMAD 2/3 forms a complex with SMAD 4 which is translocated from the extracellular matrix to the nucleus and activates the expression of target genes affecting muscle fiber size. Additionally, SMAD 7 functions as an inhibitory protein, when bound to activated type I and II receptors it will block the activation of SMAD 2/3. Screenshot

Figure 1: Myostatin signaling pathway (Adapted from Huang et al. 2011).

 

V. Antagonist Binding

The Myostatin:Follistatin 288 complex made up of two Fst288 molecules wrapped around an active myostatin dimer, blocking the four receptor-binding sites (FSD-1,2,3). FSD1 and FSD2 will contact one myostatin monomer and bury the type II receptor-binding site. Additionally, the N-terminal domain will contact both monomers and bind the type I receptor-binding site. At the type I interface, the fingertip loops of myostatin clamp on the N-terminal domain helix to form three new hydrogen bonds. The side chains of and rearrange to help stabilize the helical conformation causing and to be displayed outwards in different directions. This allows to insert between them which permits the N-terminal domain to interact more closely with myostatin.

VI. Biological Application

The complex regulatory role of myostatin in muscle growth has opened many doors towards medical innovation. Myostatin has been explored in regards to potential therapy for muscle disorders. A common muscle disorder, muscular dystrophy, has been readily studied and scientists have looked at gene therapy as a potential fix. Myostatin antagonist binding can lead to an increase in muscle mass. Thus scientists have used this knowledge to develop antagonist gene clones delivered through adeno-associated vectors or small, non-pathogenic viruses capable of transferring genetic material into target cells by intramuscular injection. This is believed to have a long-lasting therapeutic effect.


VII. References

Z. Huang, X. Chen, D. Chen, Myostatin: A novel insight into its role in metabolism, signal pathways, and expression regulation. Cellular Signalling. 23, 1441–1446 (2011).

Rodgers BD, Ward CW. Myostatin/Activin Receptor Ligands in Muscle and the Development Status of Attenuating Drugs. Endocr Rev. 2022 Mar 9;43(2):329-365. doi: 10.1210/endrev/bnab030. PMID: 34520530; PMCID: PMC8905337.

Lee SJ, McPherron AC. Regulation of myostatin activity and muscle growth. Proc Natl Acad Sci U S A. 2001 Jul 31;98(16):9306-11. doi: 10.1073/pnas.151270098. Epub 2001 Jul 17. PMID: 11459935; PMCID: PMC55416.

Cash JN, Rejon CA, McPherron AC, Bernard DJ, Thompson TB. The structure of myostatin:follistatin 288: insights into receptor utilization and heparin binding. EMBO J. 2009 Sep 2;28(17):2662-76. doi: 10.1038/emboj.2009.205. Epub 2009 Jul 30. PMID: 19644449; PMCID: PMC2738701.

Carnac G, Vernus B, Bonnieu A. Myostatin in the pathophysiology of skeletal muscle. Curr Genomics. 2007 Nov;8(7):415-22. doi: 10.2174/138920207783591672. PMID: 19412331; PMCID: PMC2647158.

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