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(GDF8) Latency, Activation, and Antagonism

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(GDF8) Latency, Activation, and Antagonism Molecular mechanisms of growth differentiation factor 8 (GDF8) latency, activation, and antagonism A dissertation submitted to the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy In the Department of Molecular Genetics, Biochemistry, and Microbiology of the College of medicine by Jason C. McCoy B.S. Miami University August 2020 Committee Chair: Thomas Thompson, Ph.D. i Abstract Growth differentiation factor 8 (GDF8), a.k.a. myostatin, is a member of the activin subclass within the larger TGFβ 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. TGFβ 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 TGFβ 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 TGFβ1 procomplex. Using the TGFβ1 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 ii 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. iii iv Dedication This work was made possible due to many people: To my parents, John and Judy McCoy. They have always pushed and supported me in every aspect of my life. Without them behind me I would not be where I am today. I cannot thank them enough for their love and compassion in every aspect of my life. I owe them more than I could ever repay. To my sister Jennifer. Although at times we have not seen eye to eye we both know that no matter what we will be there for each other. I could not ask for a better role model and sister to look up to. To the many friends I have made along the way. Graduate school is both mentally and emotionally challenging. I have made too many friends to list them all here. They kept me sane and lifted me up when I was down. Thank you all. A special thanks to Erich Goebel and his wife Jessica Goebel. I was the best man at their wedding, and they will be the best man/maid of honor in mine. I have known them since day 1 of graduate school or longer and I could not ask for a better couple to spend these years and the future with. To my fiancée, Victoria Jensen. Anyone who knows me knows that I sometimes float a little too much, Victoria is my rock. She keeps me grounded, focused, and gives me the motivation to keep moving forward. Without her support this journey would have been infinitely harder. I cannot wait for our future together and to overcome every challenge in our way. v Acknowledgements First and foremost, I would like to thank my mentor, Tom Thompson, for giving me an amazing opportunity within his laboratory. Having known many of his previous and now current graduate students one thing remains consistent, Tom will push you. Not over an edge, but to greater heights. He motivates his students to not only be better scientist, but writers through involvement in grants, communicators through lab meetings and conferences, and overall, more well-rounded members of the scientific community. Without his guidance and constant support, I would not be writing this today. What is more, while pushing me to be a better scientist, he gave me room. Room to grow, make mistakes, and learn from those many, many mistakes. He provided the tools, the guidance, and the motivation to be better and for that I cannot thank him enough. I would also like to thank my committee members: Sean Davidson, Jeff Molkentin, and David Wieczorek for their valuable insight, feedback, constructive criticism, and overall support during my academic pursuits. Two other professors deserve recognition alone: Rhett Kovall and Bill Miller. While Tom was my primary mentor Rhett and Bill always supported me. Offering any expertise, reagents, or advice at any time. I cannot thank them enough for being another source of excellent mentorship during this journey. The Thompson Lab is full of great scientists, friends, and peers: Erich Goebel, Greg Gipson, Kaitlin Hart, Emily Kappes, Chandra Kattamuri, and Magda Czepnik. Every member of the current lab and two past members, Ryan Walker and Kristoff Nolan, have been integral to my success. Whether it is the exchange of ideas or the release of stress I owe them all a debt of gratitude. I would like to give a special thanks to Magda, her and I have worked very closely on many experiments and projects. Her dedication and work ethic are unparalleled. I cannot thank her enough for helping me finish a multitude of experiments and projects. vi Lastly, I would like to thank my soon-to-be wife Victoria. More than anyone else she has pushed me to where I am. We both went through graduate school together, supporting one another and succeeding together. Her strength is unrivaled, and I cannot wait for our future together. vii Table of Contents Abstract……………………………………………………………………………………………………………………………………………………ii Dedication…………………………………………………………………………………………………………………..………………………….iii Acknowledgements. …………………………………………………………………………………………………………………….………….v Table of Contents. ………………………………………………………………………………………………………………………………..viii List of Figures and Tables. ……………………………………………………………………………………………………………………...xi Chapter I: Introduction………………………………………………………………………………………………………………………..1 Introduction………………………………………………………………………………………………………………………………..2 Ligand domain architecture and signaling…………………………………………………………………………………...3 TGFβ superfamily ligand prodomains facilitate proper complex formation and regulation………….6 Structure of the TGFβ1 procomplex and mechanisms of activation……………………………………………..7 GDF8/11 procomplexes and mechanisms of activation……………………………………………………………...10 Extracellular antagonism of the TGFβ superfamily……………………………………………………………………..11 Aims of this dissertation..14 Chapter II: Molecular characterization of latent GDF8……………………………………………………………………… 16 Abstract…………………………………………………………………………………………………………………………………… 17 Significance……………………………………………………………………………………………………………………………… 17 Introduction…………………………………………………………………………………………………………………………….. 18 Results…………………………………………………………………………………………………………………………………….. 19 -Prodomain-GDF8 can exist in a latent and active complex…………………………………………. 19 -SAXS analysis reveals conformational differences between GDF8 and other TGFβ prodomain-ligand complexes…………………………………………………………………………………….… 22 -Specific mutation within the prodomain enhance GDF8 activity………………………………… 25 -GDF8 prodomain mutations exhibit reduced antagonism………………………………………….. 31 -Reformed complexes using the GDF8 prodomain mutants are more active and exhibit decreased thermal stability…………………………………………………………………………………………. 32 -GDF8 mutants enhance muscle atrophy compared with WT GDF8……………………………. 34 Discussion……………………………………………………………………………………………………………………………….. 36 Materials and methods…………………………………………………………………………………………………..………. 42 Acknowledgements………………………………………………………………………………………………………………… 49 viii Chapter III: Residues adjacent to the tolloid cut site of GDF8 and important for Tolloid mediated Activation………………………………………………………………………………………………………………………………………… 50 Abstract……………………………………………………………………………………………………………………………………
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