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Molecular mechanisms of growth differentiation factor 8 (GDF8) latency, activation, and antagonism.

McCoy, Jason
http://orcid.org/0000-0003-4307-0466
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1601992984528774

Abstract Details

2020, PhD, University of Cincinnati, Medicine: Molecular Genetics, Biochemistry, and Microbiology.
Growth differentiation factor 8 (GDF8), a.k.a. myostatin, is a member of the activin subclass within the larger TGFß superfamily of signaling ligands that contains over 30 distinct members. Discovered in 1997, GDF8 quickly became characterized as a negative regulator of muscle mass because a highly active GDF8 caused muscle wasting or atrophy. In contrast, when GDF8 is rendered inactive through extracellular inhibitors or mutations, massive muscle gain was observed. This discovery generated massive interest within the pharmaceutical industry to manufacture inhibitors for GDF8 to combat a variety of muscle wasting diseases. A better understanding of how GDF8 is regulated in vivo is imperative to develop efficacious and novel inhibitors against GDF8. The activity of GDF8 is tightly regulated in vivo by several different processes. TGFß ligands are synthesized as large precursor proteins with a N-terminal signal sequence and prodomain followed by the mature signaling domain. The prodomain is cleaved from the mature domain, but unlike most other TGFß family members, GDF8 forms a high affinity interaction with its prodomain rendering it inactive or latent. Prior to our work, the molecular mechanisms dictating latency were not well understood but were hypothesized to be like the latent TGFß1 procomplex. Using the TGFß1 procomplex as a model, we identified residues critical for a stable latent GDF8 procomplex. Furthermore, the structure of the latent GDF8 procomplex was solved by another laboratory and our mutants could be further characterized to determine the molecular mechanisms of GDF8 latency. In order to signal, latent GDF8 needs to be activated by a member of the tolloid family of metalloproteases which proteolytically cleaves the prodomain. While the site of tolloid cleavage on the GDF8 prodomain has been identified, how tolloid recognized the prodomain as a substrate was unknown. Unlike other proteases, tolloid has no concrete consensus sequence required for cleavage. We sought to characterize what molecular features of the GDF8 cut site were required for tolloid processing. To this end, we identified several residues near the cut site that when mutated significantly reduced latent GDF8 activation by tolloid. Although GDF8 is tightly regulated through the formation of a latent procomplex, there are also extracellular antagonists that will bind to GDF8 and prevent it from signaling. Of the antagonists that target GDF8, the WFIKKN family is by far the most specific. However, the driver of this specificity was not well defined. Here, we characterized the follistatin domain (FSD) of WFIKKN2. Previously, the FSD had been identified as a primary driver of the high affinity interaction between GDF8 and WFIKKN. We solved the crystal structure of the WFIKKN2 FSD and identified key residues for antagonism and characterized how the FSD of WFIKKN contributes to antagonism. Together, our in vitro data provide valuable insight into the molecular mechanisms dictating GDF8 regulation in vivo and has the potential to be leveraged by the pharmaceutical industry to develop novel approaches for GDF8 inhibition to combat muscle wasting.
Thomas Thompson, Ph.D. (Committee Chair)
Sean Davidson, Ph.D. (Committee Member)
Rhett Kovall, Ph.D. (Committee Member)
Jeffery Molkentin, Ph.D. (Committee Member)
David Wieczorek, Ph.D. (Committee Member)
153 p.
Molecular Biology
GDF8; Prodomain; Latency; Activation; TGF-beta; Antagonism

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Citations

  • McCoy, J. (2020). Molecular mechanisms of growth differentiation factor 8 (GDF8) latency, activation, and antagonism. [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1601992984528774

    APA Style (7th edition)

  • McCoy, Jason. Molecular mechanisms of growth differentiation factor 8 (GDF8) latency, activation, and antagonism. 2020. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1601992984528774.

    MLA Style (8th edition)

  • McCoy, Jason. "Molecular mechanisms of growth differentiation factor 8 (GDF8) latency, activation, and antagonism." Doctoral dissertation, University of Cincinnati, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1601992984528774

    Chicago Manual of Style (17th edition)

ucin1601992984528774
299
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This open access ETD is published by University of Cincinnati and OhioLINK.