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Structural and biochemical studies on ligands and antagonists within the transforming growth factor ß family

Walker, Ryan G
http://orcid.org/0000-0002-2155-389X
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1468337225

Abstract Details

2016, PhD, University of Cincinnati, Medicine: Molecular Genetics, Biochemistry, and Microbiology.
The transforming growth factor-ß (TGFß) superfamily of signaling molecules includes over 30 ligands that are essential both during development and in the adult. Here, they are known to intimately maintain tissue growth and homeostasis, regulate the reproductive axis, and aid in wound healing. As such, cellular mechanisms exist to fine-tune and regulate signaling. In this study, we have structurally and biochemically characterized the regulation of two particular TGFß ligands, growth/differentiation factor 8 (also known as myostatin) and GDF11, and their interactions with known extracellular antagonists, such as growth and differentiation factor associated serum factor (GASP) and follistatin (FS). GDF8 and GDF11 are the subject of intense interest within the field of skeletal muscle biology. While GDF8 has been recognized as a negative regulator of muscle growth and differentiation, GDF11 was recently shown to improve muscle regeneration and reverse age-related dysfunction. We identified that GDF11 is a much more potent signaling ligand than GDF8, despite sharing 90% sequence identity. Together with previous structural data and our newly resolved apo-GDF8 and GDF11:FS crystal structures, we identified unique structural features between the two ligands that directly impact ligand activity. Similar to other TGF-ß family ligands, GDF8 is neutralized by binding one of a number of structurally diverse antagonists. Included are the antagonists GASP-1 and GASP-2, which are unique in that they specifically antagonize GDF8 and GDF11. However, little is known from a structural standpoint describing the interactions of GASP antagonists with GDF8. Using small angle x-ray scattering, we determined the first low-resolution solution structures of the GDF8:GASP1 and GDF8:GASP2 complexes. Together with supporting biochemical studies, we determined that GASP-1 forms a unique asymmetric 1:1 complex with GDF8 whereas GASP-2 forms a 2:1 symmetrical complex. Further, we determined that the C-terminal domains of GASP-1 are the primary driving force behind the observed stoichiometric differences. Finally, we studied the interactions between follistatin 288 (FS288), an isoform of follistatin, and the polysaccharides, heparin and heparan sulfate. Follistatin neutralizes the ligands GDF8 (and GDF11) and activin A by forming a nearly irreversible non-signaling complex. FS288 is predominantly localized to the cell surface mediated through heparin/HS binding and the binding of ligands to FS288 can enhance heparin/HS interactions and facilitate ligand degradation. However, limited information is available for how heparin affects the binding of FS288 to ligands. To our surprise, we determined that pre-incubation of FS288 with heparin/HS significantly decreased the association of FS288 with GDF8 and activin A. Again, using small angle x-ray scattering, we determined the low resolution solution structures of FS288 and the FS288:heparin complex. Coupled with additional biophysical studies, we determined that heparin mediates dimerization of FS288, which likely explains why FS288 binding to GDF8 and activin A is attenuated in the presence of heparin. Overall, the results of these studies extend our knowledge within the field of basic TGFß biology. Specifically, these results provide new structural and biochemical insight that may aid in the development of TGFß therapeutics for the treatment of muscle related diseases such as muscular dystrophy.
Thomas Thompson, Ph.D. (Committee Chair)
Kenneth Greis, Ph.D. (Committee Member)
Andrew Herr, Ph.D. (Committee Member)
Rhett Kovall, Ph.D. (Committee Member)
Anil Menon, Ph.D. (Committee Member)
225 p.
Biochemistry
Structure; Myostatin; GDF11; Transforming growth factor; ligand; crystallography

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Citations

  • Walker, R. G. (2016). Structural and biochemical studies on ligands and antagonists within the transforming growth factor ß family [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1468337225

    APA Style (7th edition)

  • Walker, Ryan. Structural and biochemical studies on ligands and antagonists within the transforming growth factor ß family. 2016. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1468337225.

    MLA Style (8th edition)

  • Walker, Ryan. "Structural and biochemical studies on ligands and antagonists within the transforming growth factor ß family." Doctoral dissertation, University of Cincinnati, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1468337225

    Chicago Manual of Style (17th edition)

ucin1468337225
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© 2016, all rights reserved.
This open access ETD is published by University of Cincinnati and OhioLINK.