Proc Natl Acad Sci U S A 115(5): E866-E875

PMC: PMC5798348

PMID: 29348202

Molecular characterization of latent GDF8 reveals mechanisms of activation

Richard Lee+3 authors

Supplementary Material

Supplementary File

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^{Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, OH, 45267;}

^{Harvard Stem Cell Institute, Harvard University, Cambridge, MA, 02138;}

^{Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, 02138;}

^{Biomedicine Discovery Institute, Department of Physiology, Monash University, Clayton, VIC 3800, Australia;}

^{Baker Heart and Diabetes Institute, Melbourne, VIC 3004, Australia;}

^{Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom;}

^{Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia;}

^{Department of Physiology, University of Melbourne, Parkville, VIC 3010, Australia;}

^{Department of Neurology, University of Washington School of Medicine, Seattle, WA, 98195;}

^{Hudson Institute of Medical Research, Clayton, VIC 3168, Australia}

^{To whom correspondence may be addressed. Email:}ude.hsanom@nosirrah.giarc or ude.cu@nospmoht.mot.

Edited by Se-Jin Lee, Johns Hopkins University, Baltimore, MD, and approved December 7, 2017 (received for review August 22, 2017)

Author contributions: R.G.W., C.A.H., and T.B.T. conceived the idea and designed the research; R.G.W., J.C.M., M.C., M.J.M., A.H., K.L.W., T.R.C., and M.H. performed research; R.G.W., J.C.M., K.L.W., T.R.C., M.H., R.T.L., P.G., C.A.H., and T.B.T. analyzed data; and R.G.W., J.C.M., C.A.H., and T.B.T. wrote the paper.

^{R.G.W. and J.C.M. contributed equally to this work.}

Edited by Se-Jin Lee, Johns Hopkins University, Baltimore, MD, and approved December 7, 2017 (received for review August 22, 2017)

Published under the PNAS license.

Significance

GDF8 is a signaling protein that inhibits muscle mass. Inhibitors of GDF8 are highly sought as therapeutics for the treatment of muscle-wasting diseases. During synthesis, GDF8 is made as a precursor where the signaling segment is cleaved from the N-terminal prodomain, which remains associated and inhibits signaling. Activation involves an additional cleavage of the prodomain. We demonstrate GDF8 signaling could be gained through a conformational change where the prodomain remains associated with the signaling segment. Alteration of the prodomain can weaken the interactions causing GDF8 to signal, thus alleviating inhibition by the prodomain. This study illuminates how GDF8 transitions from an inhibited state to an active state—information that will help to understand the mechanism of GDF8 signaling.

Keywords: GDF8, latent activation, GDF8 prodomain, myostatin activation, myostatin prodomain

Significance

Abstract

Growth/differentiation factor 8 (GDF8), or myostatin, negatively regulates muscle mass. GDF8 is held in a latent state through interactions with its N-terminal prodomain, much like TGF-β. Using a combination of small-angle X-ray scattering and mutagenesis, we characterized the interactions of GDF8 with its prodomain. Our results show that the prodomain:GDF8 complex can exist in a fully latent state and an activated or “triggered” state where the prodomain remains in complex with the mature domain. However, these states are not reversible, indicating the latent GDF8 is “spring-loaded.” Structural analysis shows that the prodomain:GDF8 complex adopts an “open” configuration, distinct from the latency state of TGF-β and more similar to the open state of Activin A and BMP9 (nonlatent complexes). We determined that GDF8 maintains similar features for latency, including the alpha-1 helix and fastener elements, and identified a series of mutations in the prodomain of GDF8 that alleviate latency, including I56E, which does not require activation by the protease Tolloid. In vivo, active GDF8 variants were potent negative regulators of muscle mass, compared with WT GDF8. Collectively, these results help characterize the latency and activation mechanisms of GDF8.

Abstract

One of the most thoroughly described negative regulators of skeletal muscle mass is the TGF-β superfamily ligand growth/differentiation factor 8 (GDF8), also known as myostatin (1, 2). Genetic disruption of Gdf8 results in substantial skeletal muscle growth (1, 2). Further, a significant increase in muscle fiber size is also observed when adult animals are treated with agents that bioneutralize GDF8 (reviewed in ref. 3). As such, targeted inhibition of GDF8 is currently being pursued for the treatment of skeletal muscle-related disorders and associated symptoms (4, 5).

GDF8, like numerous TGF-β family members, is a disulfide-linked dimer that is synthesized as a precursor protein which requires cleavage by a furin-like protease to yield an N-terminal prodomain and a C-terminal mature, signaling domain (6). Interestingly, for a number of TGF-β ligands the role of the prodomain extends beyond ligand maturation and folding support (7, 8), remaining noncovalently associated with the mature ligand following secretion in either a low-affinity, noninhibitory or high-affinity, inhibitory fashion (reviewed in ref. 9). For example, the prodomains of TGF-β1, TGF-β2, TGF-β3, GDF11, and GDF8 hold the mature ligand in a latent or inactive state mediated by a noncovalent, yet high-affinity, ligand-specific interaction (6, 10 –13), whereas mature Activin A and BMP9 remain associated with, but are not inhibited by, their prodomain (14, 15). Activation of TGF-β1 and TGF-β3 requires covalent interactions with the extracellular matrix and cellular contractile forces to release the mature ligand (16 –18). In fact, resolution of the latent TGF-β1 crystal structure provided a molecular explanation for how latency is exerted by the prodomain via a coordinated interaction between the N-terminal alpha helix (alpha-1), latency lasso, and fastener of the prodomain with type I and type II receptor epitopes of the mature domain (18). However, GDF8 activation requires a second cleavage event within the prodomain via proteases from the BMP1/Tolloid (TLD) family of metalloproteases (13). However, the molecular and structural details of the GDF8 latent state have yet to be determined.

Based on sequence conservation and prior biochemical data describing the N-terminal portion of the GDF8 prodomain (10), it is plausible that the molecular interactions and overall structure of the GDF8 latent complex may be similar to that of TGF-β1. However, the prodomains of a number of TGF-β family members share similar sequence conservation, yet they do not regulate the mature ligand in the same fashion and also exhibit significant structural diversity (14, 15). Therefore, while one might expect that GDF8 and TGF-β1 would share certain elements for how the prodomain binds and confers latency, it is possible that significant structural and molecular differences in these interactions occur as they exhibit profoundly different mechanisms of activation. However, this comparison is hindered by a lack of understanding of the GDF8 latent complex at the molecular level.

In this study, we utilized small-angle X-ray scattering (SAXS) and mutagenesis to characterize the GDF8 latent complex. Interestingly, SAXS analysis reveals that the GDF8 latent complex adopts a more “open” conformation, similar to the overall structure of the BMP9 and Activin A prodomain complexes, which are not latent. The open conformation of the GDF8 latent complex is in stark contrast to the “closed” conformation adopted by the TGF-β1 latent complex. Furthermore, we identify key residues in the GDF8 prodomain that are responsible for promoting latency, indicating that GDF8 and TGF-β1 share similar features for latency including a latency lasso. We further show that certain mutations in the prodomain of GDF8 can reduce latency, producing a more active ligand both in vitro and in vivo. Overall, our data provide insight toward the molecular mechanisms of GDF8 latency and activation.

Supplementary File

Click here to view.^{(666K, pdf)}

Acknowledgments

We thank Monash Micro Imaging staff, Alfred Medical Research and Education Precinct (AMREP) campus, for technical guidance and AMREP Precinct Animal Centre staff for animal husbandry; Dr. Hongwei Qian (Baker Heart and Diabetes Institute) for assistance with production of recombinant AAV vectors; and Georgia Goodchild for help with the histological analysis. This work was supported, in part, by NIH; a National Health and Medical Research Council (NHMRC) grant; the Muscular Dystrophy Association; the University of Cincinnati Graduate Dean Fellowship; American Heart Association Grants R01AG047131, R01AG040019, R03AG049657 (to R.T.L.), and 1078907 (to C.A.H.); App 1117835 for Gregorevic Senior Research Fellowship from NHMRC; Graduate Dean Fellowship and Grant 12PRE11790027 (to R.G.W.); and Grant R01GM114640 and Muscular Dystrophy Association Grant 240087 (to T.B.T.). This work was also supported by the Integrated Diffraction Analysis Technologies Program of the Department of Energy Office of Basic Energy Sciences awarded to the Advanced Light Source at Lawrence Berkeley National Laboratory. The Baker Heart and Diabetes Institute is supported in part by the Operational Infrastructure Support Program of the Victorian Government.

Acknowledgments

Footnotes

Conflict of interest statement: T.B.T. is a consultant for Acceleron Pharma. The University of Cincinnati and Monash University have filed for intellectual property on GDF8 and GDF11 listing R.G.W., T.B.T., and C.A.H. as inventors. Harvard University and Brigham and Women’s Hospital have filed for intellectual property on GDF11 listing R.T.L. as an inventor.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1714622115/-/DCSupplemental.

Footnotes

References

1. McPherron AC, Lawler AM, Lee SJRegulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature. 1997;387:83–90.[PubMed][Google Scholar]
2. McPherron AC, Lee S-JDouble muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci USA. 1997;94:12457–12461.[Google Scholar]
3. Lee S-JExtracellular regulation of myostatin: A molecular rheostat for muscle mass. Immunol Endocr Metab Agents Med Chem. 2010;10:183–194.[Google Scholar]
4. Suragani RNVS, et al Transforming growth factor-β superfamily ligand trap ACE-536 corrects anemia by promoting late-stage erythropoiesis. Nat Med. 2014;20:408–414.[PubMed][Google Scholar]
5. Lach-Trifilieff E, et al An antibody blocking activin type II receptors induces strong skeletal muscle hypertrophy and protects from atrophy. Mol Cell Biol. 2014;34:606–618.[Google Scholar]
6. Lee S-J, McPherron ACRegulation of myostatin activity and muscle growth. Proc Natl Acad Sci USA. 2001;98:9306–9311.[Google Scholar]
7. Gray AM, Mason AJRequirement for activin A and transforming growth factor–beta 1 pro-regions in homodimer assembly. Science. 1990;247:1328–1330.[PubMed][Google Scholar]
8. Harrison CA, Al-Musawi SL, Walton KLProdomains regulate the synthesis, extracellular localisation and activity of TGF-β superfamily ligands. Growth Factors. 2011;29:174–186.[PubMed][Google Scholar]
9. Hinck AP, Mueller TD, Springer TAStructural biology and evolution of the TGF-β family. Cold Spring Harb Perspect Biol. 2016;8:a022103.[Google Scholar]
10. Thies RS, et al GDF-8 propeptide binds to GDF-8 and antagonizes biological activity by inhibiting GDF-8 receptor binding. Growth Factors. 2001;18:251–259.[PubMed][Google Scholar]
11. Zimmers TA, et al Induction of cachexia in mice by systemically administered myostatin. Science. 2002;296:1486–1488.[PubMed][Google Scholar]
12. Hill JJ, et al The myostatin propeptide and the follistatin-related gene are inhibitory binding proteins of myostatin in normal serum. J Biol Chem. 2002;277:40735–40741.[PubMed][Google Scholar]
13. Wolfman NM, et al Activation of latent myostatin by the BMP-1/tolloid family of metalloproteinases. Proc Natl Acad Sci USA. 2003;100:15842–15846.[Google Scholar]
14. Mi L-Z, et al Structure of bone morphogenetic protein 9 procomplex. Proc Natl Acad Sci USA. 2015;112:3710–3715.[Google Scholar]
15. Wang X, Fischer G, Hyvönen MStructure and activation of pro-activin A. Nat Commun. 2016;7:12052.[Google Scholar]
16. Annes JP, Chen Y, Munger JS, Rifkin DBIntegrin alphaVbeta6-mediated activation of latent TGF-beta requires the latent TGF-beta binding protein-1. J Cell Biol. 2004;165:723–734.[Google Scholar]
17. Wipff P-J, Hinz BIntegrins and the activation of latent transforming growth factor β1–An intimate relationship. Eur J Cell Biol. 2008;87:601–615.[PubMed][Google Scholar]
18. Shi M, et al Latent TGF-β structure and activation. Nature. 2011;474:343–349.[Google Scholar]
19. Lawrence DA, Pircher R, Jullien PConversion of a high molecular weight latent beta-TGF from chicken embryo fibroblasts into a low molecular weight active beta-TGF under acidic conditions. Biochem Biophys Res Commun. 1985;133:1026–1034.[PubMed][Google Scholar]
20. Schultz-Cherry S, Murphy-Ullrich JEThrombospondin causes activation of latent transforming growth factor-beta secreted by endothelial cells by a novel mechanism. J Cell Biol. 1993;122:923–932.[Google Scholar]
21. Cash JN, et al Structure of myostatin·follistatin-like 3: N-terminal domains of follistatin-type molecules exhibit alternate modes of binding. J Biol Chem. 2012;287:1043–1053.[Google Scholar]
22. Cash JN, Angerman EB, Keutmann HT, Thompson TBCharacterization of follistatin-type domains and their contribution to myostatin and activin A antagonism. Mol Endocrinol. 2012;26:1167–1178.[Google Scholar]
23. Cash JN, et al Development of a small-molecule screening method for inhibitors of cellular response to myostatin and activin A. J Biomol Screen. 2013;18:837–844.[PubMed][Google Scholar]
24. Walker RG, et al Alternative binding modes identified for growth and differentiation factor-associated serum protein (GASP) family antagonism of myostatin. J Biol Chem. 2015;290:7506–7516.[Google Scholar]
25. Walker RG, et al Structural basis for potency differences between GDF8 and GDF11. BMC Biol. 2017;15:19.[Google Scholar]
26. Schneidman-Duhovny D, Hammel M, Sali AFoXS: A web server for rapid computation and fitting of SAXS profiles. Nucleic Acids Res. 2010;38:W540–W544.[Google Scholar]
27. Walton KL, et al A common biosynthetic pathway governs the dimerization and secretion of inhibin and related transforming growth factor beta (TGFbeta) ligands. J Biol Chem. 2009;284:9311–9320.[Google Scholar]
28. Lee S-JGenetic analysis of the role of proteolysis in the activation of latent myostatin. PLoS One. 2008;3:e1628.[Google Scholar]
29. Lee S, Solow-Cordero DE, Kessler E, Takahara K, Greenspan DSTransforming growth factor-beta regulation of bone morphogenetic protein-1/procollagen C-proteinase and related proteins in fibrogenic cells and keratinocytes. J Biol Chem. 1997;272:19059–19066.[PubMed][Google Scholar]
30. Leighton M, Kadler KEPaired basic/furin-like proprotein convertase cleavage of Pro-BMP-1 in the trans-Golgi network. J Biol Chem. 2003;278:18478–18484.[PubMed][Google Scholar]
31. Jasuja R, et al Bone morphogenetic protein 1 prodomain specifically binds and regulates signaling by bone morphogenetic proteins 2 and 4. J Biol Chem. 2007;282:9053–9062.[PubMed][Google Scholar]
32. Szláma G, Trexler M, Buday L, Patthy LK153R polymorphism in myostatin gene increases the rate of promyostatin activation by furin. FEBS Lett. 2014;589:295–301.[PubMed][Google Scholar]
33. Ge G, Hopkins DR, Ho W-B, Greenspan DSGDF11 forms a bone morphogenetic protein 1-activated latent complex that can modulate nerve growth factor-induced differentiation of PC12 cells. Mol Cell Biol. 2005;25:5846–5858.[Google Scholar]
34. Chen JL, et al Elevated expression of activins promotes muscle wasting and cachexia. FASEB J. 2014;28:1711–1723.[PubMed][Google Scholar]
35. Szláma G, Trexler M, Patthy LLatent myostatin has significant activity and this activity is controlled more efficiently by WFIKKN1 than by WFIKKN2. FEBS J. 2013;280:3822–3839.[Google Scholar]
36. Young GD, Murphy-Ullrich JEMolecular interactions that confer latency to transforming growth factor-beta. J Biol Chem. 2004;279:38032–38039.[PubMed][Google Scholar]
37. Cotton TR, et al. 2017. Structure of the human pro-myostatin precursor and determinants of growth factor latency. bioRxiv:153403.
38. Jiang M-S, et al Characterization and identification of the inhibitory domain of GDF-8 propeptide. Biochem Biophys Res Commun. 2004;315:525–531.[PubMed][Google Scholar]
39. Ohsawa Y, et al The inhibitory core of the myostatin prodomain: Its interaction with both type I and II membrane receptors, and potential to treat muscle atrophy. PLoS One. 2015;10:e0133713.[Google Scholar]
40. Takayama K, et al Identification of the minimum peptide from mouse myostatin prodomain for human myostatin inhibition. J Med Chem. 2015;58:1544–1549.[PubMed][Google Scholar]
41. Le VQ, et al. 2017. Tolloid cleavage activates latent GDF8 by priming the pro-complex for dissociation. bioRxiv:154823.
42. Jones DC, et al Identification of a κ-opioid agonist as a potent and selective lead for drug development against human African trypanosomiasis. Biochem Pharmacol. 2010;80:1478–1486.[Google Scholar]
43. Cash JN, Rejon CA, McPherron AC, Bernard DJ, Thompson TBThe structure of myostatin:follistatin 288: Insights into receptor utilization and heparin binding. EMBO J. 2009;28:2662–2676.[Google Scholar]
44. Lee Y-S, Lee S-JRegulation of GDF-11 and myostatin activity by GASP-1 and GASP-2. Proc Natl Acad Sci USA. 2013;110:E3713–E3722.[Google Scholar]