5JI1 : Myostatin (GDF8)

Myostatin was discovered in 1997 by geneticists Se-Jin Lee and Alexandra McPherron ^[1] who demonstrated that a phenotype of exaggerated muscle hypertrophy correlated with mutations in the myostatin gene. It was at first associated with the role it played in the regulation of muscular mass of mice. This new growth factor since then been completely sequenced, and the primary sequences obtained in different animals have been compared. The results showed that there were an important correlation between the sequences, whatever their origin. ^[2]

Classification

This protein was firstly named Growth/Differentiation Factor 8 (GDF8) because it belongs to the group of growth factors. Growth factors constitute a group of proteins that regulate the number of cells, increasing or decreasing their multiplication according to the needs. Then the nomenclature changed and, nowadays, we refer to myostatine as MSTN. Progressively, the myostatin has been affiliated to the TGF-beta family (transforming growth factor beta) ^[2].

Function ^[3]

Myostatin is a strong endogenous, negative regulator of muscle growth determining both muscle fiber number and size. The number of fibers is defined during the development of the animal while their size changes while they live, depending on activity, nutrition and aging. Myostatin acts on this by providing regulation on the growth of muscles. It has been found first in mice which, having their gene encoding for myostatin knocked-out, developed overgrowth of muscles, due to hyperplasia and hypertrophy, which effects are persistent throughout the life of animals. Therefore, myostatin appears to act at the level of fiber number during embryogenesis and its growth in adult life.

Myostatin processing and signal transduction ^[3]

The mechanism of myostatin action is similar to those of the members of TGF-beta family. The mature peptide binds to one of the two activin type II receptors which recruits, phosphorylates and activates the activin type I receptor, propagating signals along the Smad pathway. (Smad are receptor-associated proteins) Phosphorylated Smad2 and 3 form heterodimeric complex with Smad4(common mediator) and they activate the functions of the smad as mediators of signalling for myostatin : translocating into the nucleus and activating the transcription of the target genes (through interaction with DNA and other nuclear factors).

Inhibition of myostatin’s function^[3]

The mechanism of activation of myostatin remains to be fully determined but the function of myostatin appears to be dependent of a network of protein interactions. Indeed, several proteins have been identified as inhibitory binding proteins of myostatin : follistatin, hSGT, Titin cap, decorin were defined in muscles and FLRG and GASP (myostatin propeptide, follistatin related proteins) have been found to create a complex with myostatin in serum. All of these proteins negatively regulate myostatin activity (inhibiting its activation, secretion, or reception binding). Some of them (propeptide, follistatin and FLRG) are able to increase muscle mass when expressed as a transgene in skeletal muscle of wild-type mice. The increase in muscle mass is greater in follistatin transgenics than in the myostatin null mice and when both are combined, the increase of muscle mass is quadrupled. Therefore, it has been deduced that other ligands cooperate with myostatin to control muscle growth.

Myostatin and satellite cells ^[3]

Satellite cells’ main role concerns the repair of skeletal muscles. They are activated and proliferate then to respond to tissue damage. However a small part doesn’t differentiate and return to quiescence to maintain the pool of satellite cells. This is where myostatin steps in : it represents a key molecule signalling the quiescence of satellite cells. In myostatin mutant mouse the number of satellite cells increases a lot compared to non mutated mice. Basically, myostatin maintains satellite cells in a quiescent state during regeneration or muscle growth. And so, it negatively regulates muscle regeneration after injury.

Structure and synthesis ^[2]

Primary and secondary structures

Myostatin is a 42,7 kDa protein composed of only 108 residues in its mature form. It contains 7 residues in its C-terminal domain, all of which are involved in disulfide bridges. The secondary structure of myostatin is composed of two strands, both made of short . The structure is also made of 3  :

- Helix α-1 : containing between 4 and 7 residues (non-visible on the structure)

- Helix α-2 : containing between 24 and 28 residues

- Helix α-3 : containing between 58 and 68 residues

The folding of these structures gives myostatin a slightly bent, hand-like shape, with 2 fingers formed by the strands described above. The palm of the hand is formed by the helix alpha-3. The N- and C-terminal ends are situated very close to the palm and the last 10 residues on the N-terminal side form the thumb of the hand.

The structural analysis of myostatin^[4] was achieved thanks to the proton nuclear magnetic resonance method^[5] .

Main partners related to the structure

When in its homodimeric mature form, myostatin is able to link with several other partners. First of all, myostatin partners with two types of receptors : ALK4 and ALK5 ^[6](Activin receptor-Like Kinase). Moreover, the active form of the protein is able to link with the ActRIIB ^[7]protein (Activin type II Receptor). The action of myostatin actually requires the binding of it to these two types of membrane receptors.

Myostatin can also bind to diverse partners, ensuing different results :

- The Myostatin-associated protein hSGT ^[8](human Small Glutamine-rich Tetratricopeptide repeat-containing protein) binds to myostatin on its N-terminal end. Recent studies suggest that hSGT is involved in the regulation of the secretion and activation of myostatin.

- The association of myostatin to the Titin-Cap protein enables to regulate the secretion of pre-myostatin in pre-myogenic cells.

- Follistatin ^[9] is able to for complexes with myostatin, enabling the inhibition of the myostatin’s action on muscular development.

- Decorin binds to myostatin in the muscles and is responsible for the modulation of the activity of myostatin in myogenic cells.

- The binding of myostatin with proteins WFIKKN1 and WFIKKN2^[10](large extracellular multidomain proteins) is responsible for the inhibition of myostatin.

Synthesis and assembly

In mammals, mature myostatin consists in a small homodimer. The immature form, called pre-Myostatin, is made of a complex of two non-covalently bound N-terminal propeptides along with C-terminal ends linked by a disulfide bridge. Both C-terminal ends of the pro-peptides are similar regarding their composition in amino-acids, allowing the formation of a stabilized dimer thanks to a specific inter-chain disulfide bridge. Following its maturation, myostatin is eventually produced in a shortened form compared to its initial synthesized sequence.

Maturation process

The pre-Myostatin dimer is first cleaved by a protease from the Furin family, to the 266 and 267 amino-acids level, namely right before the beginning of the similar C-terminal end of each pro-peptide. This leads to the formation of a latent pre-myostatin complex, made of both disulfide-linked C-terminal ends with both N-terminal propeptide next to it. Then comes a protease specific of growth factors, which will provoke the degradation of the N-terminal ends, resulting in the formation of the mature myostatin homodimer.

Disease/Research

Myostatin ^[2] is a protein that has a part in muscle development : it is a negative regulator of the skeletal muscles. It has a very important role during the development of the organism, but also during its whole life. It is a very important protein with a highly conserved sequence from zebrafish to humans ^[3] and thus it has to be very well regulated. Indeed, there are many ways of regulation of the action of this protein and at many levels.

Myostatin is a growth factor^[3] implicated into muscle development in mammals. It is involved in the transmission of messages to the nucleus which will promote the expression of a gene, leading to the production of ubiquitin. Ubiquitin is a signal of degradation, meaning that the muscle cells will be destroyed. Indeed, it reduces the muscular mass as well as the quantity of Myosin ^[11] which is very important for the cohesion of the muscles and for their movement. Myosin actually forms filament, and when these filaments associate with Actin and consume ATP it results in muscle movement.

Related diseases

If the quantity of myosin is not well regulated in the human body, it can trigger many muscle related illnesses^[12] - especially when there is too much myostatin - such as heart or liver diseases for instance. We will take the example of COPD (Chronic Obstructive Pulmonary Disease) which is a lung disease. People suffering from this condition have difficulties to breathe due to the obstruction of the airflow ^[12] . Their muscles are not strong enough to enable them to breathe properly, such condition is called pulmonary cachexia. This disease is also characterized by many muscular complications into the whole body, including a global reduction of muscular mass. It has been proved that a high concentration of myostatin in the human body can promote this disease. Myostatin is also involved in several metabolic pathways, like in the blood glucose one for instance.^[12]Indeed, the higher the myostatin concentration, the more the organism is resistant to insulin. This is related to Type 2 diabetes and obesity because it induces the PID1 (Phosphotyrosine Interaction Domain containing 1) protein [9] in human muscle cells, which is known to be involved in the development of insulin resistance.

A way to cure

Myostatin comes of use in the curation of some diseases : Research has shown that if the myostatin's action is inhibited, the muscular mass increases ^[2][3].Myostatin and mostly its inhibition could thus be a solution to cure muscle atrophy diseases. For example OPMD ^[13] (Oculo-Pharyngeal Muscular Dystrophy) is a disease in which the muscles affected show increased fibrosis and atrophy. It is a late-onset disease, affecting 1 over 80 000 people. It is characterized by dysphagia and ptosis, but also limb weakness when the disease has reached a very advanced stage. Researchers have noticed that the inhibition of myostatin increases the muscular mass, thus helping to reduce the symptoms of OPMD. During the research trials, a monoclonal antibiotic was injected to mice during 10 weeks and the results showed that the muscle strength and the muscle fiber diameter increased. Moreover, the expression of the markers of muscle fibrosis reduced. However, myostatin does not cure the disease because no change was noticed the in intra-nuclear inclusion density, which is a characteristic of OPMD spread. It is for now only a solution to treat the symptom. In other cases, it is also possible to introduce follistatin ^[14] to block myostatin because they will form a complex which will stop the myostatin' action.

References

1. ↑ Wikipedia. Myostatin. [1]
2. ↑ ^{2.0 2.1 2.2 2.3 2.4} Université de Montpellier. Physiologie expérimentale du coeur et des muscles : la myostatine/partenaires de la myostatine. [2]
3. ↑ ^{3.0 3.1 3.2 3.3 3.4 3.5 3.6} 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 doi:http://dx.doi.org/10.2174/138920207783591672
4. ↑ S. Daopin et al. Crystal structure of transforming growth-factor beta 2 : an unusual fold for the superfamily. Nature. 1992 Jul 17;257(5068):369-73 [3]
5. ↑ Wikipedia. Nuclear Magnetic Resonance.[4]
6. ↑ Rebbapragada A et al. Myostatin Signals through a Transforming Growth Factor β-Like Signaling Pathway To Block Adipogenesis. Mol Cell Biol. 2003;23(20):7230–7242. doi:10.1128/mcb.23.20.7230-7242.2003 [5]
7. ↑ SJ Lee, AC McPherron. Regulation of myostatin activity and muscle growth. Proc Natl Acad Sci U S A. 2001 Jul 31;98(16):9306-11. Epub 2001 Jul 17. [6]
8. ↑ H Wang, Q Zhang, D Zhu. hSGT interacts with the N-terminal region of myostatin. Biochem Biophys Res Commun. 2003 Nov 28;311(4):877-83.[7]
9. ↑ 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. Epub 2009 Jul 30. PMID:19644449 doi:10.1038/emboj.2009.205
10. ↑ K Kondas et al. Both WFIKKN1 and WFIKKN2 have high affinity for growth and differentiation factors 8 and 11. J Biol Chem. 2008 Aug 29;283(35):23677-84. doi: 10.1074/jbc.M803025200. Epub 2008 Jul 1. [8]
11. ↑ Jeffrey L. Corden,David Tollervey. Cell Biology, Chapter 36 Motor Proteins.2017 DOI:10.1016/B978-0-323-34126-4.00036-0
12. ↑ ^{12.0 12.1 12.2} Sharma, M., McFarlane, C., Kambadur, R., Kukreti, H., Bonala, S. and Srinivasan, S. (2015), Myostatin: Expanding horizons. IUBMB Life, 67: 589-600. [ https://doi.org/10.1002/iub.1392 DOI:10.1002/iub.1392]
13. ↑ Harish P, Malerba A, Lu-Nguyen N, Forrest L, Cappellari O, Roth F, Trollet C, Popplewell L, Dickson G. Inhibition of myostatin improves muscle atrophy in oculopharyngeal muscular dystrophy (OPMD). J Cachexia Sarcopenia Muscle. 2019 Oct;10(5):1016-1026. doi: 10.1002/jcsm.12438. , Epub 2019 May 7. PMID:31066242 doi:http://dx.doi.org/10.1002/jcsm.12438
14. ↑ 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. Epub 2009 Jul 30. PMID:19644449 doi:10.1038/emboj.2009.205