Syracuse University SURFACE Dissertations - ALL SURFACE August 2018 Ghrelin Processing and Maturation: Developing a Molecular-Level Framework for Hormone Activation and Biological Function Elizabeth Rose Cleverdon Syracuse University Follow this and additional works at: https://surface.syr.edu/etd Part of the Physical Sciences and Mathematics Commons Recommended Citation Cleverdon, Elizabeth Rose, "Ghrelin Processing and Maturation: Developing a Molecular-Level Framework for Hormone Activation and Biological Function" (2018). Dissertations - ALL. 913. https://surface.syr.edu/etd/913 This Dissertation is brought to you for free and open access by the SURFACE at SURFACE. It has been accepted for inclusion in Dissertations - ALL by an authorized administrator of SURFACE. For more information, please contact [email protected]. Abstract Ghrelin is a 28 amino acid hormone involved in appetite stimulation, maintenance of energy balance, and a range of other neuroendocrine functions. Over the course of its expression and maturation, proghrelin (the prohormone of ghrelin) undergoes a unique posttranslational modification whereby a serine side chain is esterified with octanoic acid. Proghrelin then undergoes subsequent proteolysis to yield ghrelin. This octanoylation modification has been demonstrated to be required for ghrelin to activate its cognate receptor. Since acylated ghrelin has been linked with a variety of disease states, ghrelin signaling is a prime target for inhibition and inhibitor development. Biochemical and structural studies of the enzyme responsible for ghrelin octanoylation, ghrelin O-acyltransferase (GOAT), have identified features required for recognition of ghrelin by GOAT. A majority of these studies have utilized peptide mimetics of the N-terminal sequence of ghrelin. However, the impact of downstream elements in ghrelin and its 94 amino acid precursor proghrelin remains to be fully defined. To investigate this, we have developed bacterial expression systems to explore the role of both ghrelin and C-terminal ghrelin in proghrelin’s biological activity and maturation. The work presented in this dissertation is the first instance of expression and structural characterization of human proghrelin and C-ghrelin, as well as an unidentified self-cleavage behavior which has implications in hormone maturation. In complementary studies to characterize ghrelin binding to GOAT, ghrelin peptide mimetics incorporating an amine-substituted Dap residue at the site of acylation provided a superior system for exploring the molecular requirements for ghrelin recognition by GOAT. These studies have identified previously unidentified binding contacts and provides a comprehensive model of peptide binding in the hGOAT active site. The work utilizing Dap-substituted peptides provides a comprehensive peptide scaffold for future inhibitor design for targeting ghrelin signaling. Ghrelin Processing and Maturation: Developing a Molecular-Level Framework for Hormone Activation and Biological Function Elizabeth R. Cleverdon B.S., University of Wyoming, 2013 M.Phil., Syracuse University, 2015 Dissertation Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry Syracuse University August 2018 Copyright © Elizabeth R. Cleverdon 2018 All Rights Reserved Acknowledgements Firstly, I would like to thank my advisor Dr. James L. Hougland. I have valued his mentorship and scientific acumen throughout my time in his laboratory. His thirst for knowledge and inquisitive mind encouraged the same in me as well as his other students. He was always available for advice or to answer any questions that I had, which were many. He helped me to develop into the scientist that I am today, to be more skeptical and thorough when planning and evaluating experimental data. I thank him for always being very willing for discussion of theories, data, and of course the question of biological relevance. I would also like to thank my committee; Dr. Jason Fridley, Dr. Michael Cosgrove, Dr. Carlos Castañeda, Dr. Mathew Maye and Dr. Robert Doyle for their time and assistance for serving on my defense committee. I want to express my appreciation to Dr. Maye and Dr. Doyle as well as Dr. Dabrowiak for their assistance for serving on my graduate committee throughout my time at Syracuse University. Their help and input into my project was invaluable. I want to thank the Hougland lab, my family for the past five years. They were a pleasure to work with and I continue to appreciate their mentorship, fellowship and friendship. I want to thank Dr. Susan Flynn, Dr. Joe Darling, Dr. Soumya Gangopadhyay, Dr. Kayleigh McGovern- Gooch and Melanie Blanden for welcoming into the laboratory and for their support throughout my PhD. I also want to thank Maria Campana, Tasha Davis, Sudhat Ashok, Jacob Moose and Mariah Pierce for their support and friendship. Michelle Sieberg, our lab technician and lab mom, has been there throughout my PhD and has always been supportive, both emotionally and scientifically, as well as, a great teacher and resource. My undergraduates, Naomi Rivera-Robles and Casey Cabrinha gave me the opportunity to teach, to test my scientific knowledge and have taught me patience. I am very grateful to have been a member of this laboratory; everyone was iv always willing to teach, mentor and support each other. The environment was always friendly and encouraging; you guys were there on both good and bad days, when there was good data and when experiments weren’t going so well, and I am very thankful to everyone. Finally, I would like to thank my family. My parents have always fostered my inquisitive nature. They were always there to answer any questions I had, and if they couldn’t answer them they would help me to figure it out. They were always supportive and I know that it would have been much harder to achieve my goals had they not been there. I want to than my brother for his love and support throughout my studies; as well as, my grandparents for their love and encouragement not only during my training but throughout my life. As a first generation PhD there were many unknowns and a number of struggles were very present but they seemed much less apparent with my family’s love and support. Even though they had no idea what I was studying or researching throughout my education and training, it did not matter, they still championed me. v Table of Contents List of figures………………………………………………………………………………..….x List of tables…………………………………………………………………….……….……..xiii List of abbreviations…………………………………………………………………...………xiv Chapter 1: Introduction 1.1 Post-translational modifications................................................................................................ 2 1.2 Ghrelin ...................................................................................................................................... 5 1.2.1 Discovery ........................................................................................................................ 5 1.2.2 Tissue distribution ........................................................................................................... 6 1.2.3 Ghrelin processing .......................................................................................................... 6 1.2.4 Binding partners for ghrelin within the body .................................................................. 9 1.2.5 Physiological effects ..................................................................................................... 15 1.3 GOAT ..................................................................................................................................... 16 1.3.1 Discovery ...................................................................................................................... 16 1.3.2 GOAT conservation across species .............................................................................. 17 1.4 GOAT inhibitors ..................................................................................................................... 20 1.4.1 Ghrelin product mimetics as GOAT inhibitors ............................................................. 20 1.4.2 Small molecule GOAT inhibitors ................................................................................. 24 1.5 GOAT recognition of ghrelin- the role of the N-terminal “GSSF” sequence and beyond ..... 28 1.6 Obestatin ................................................................................................................................. 29 1.7 Aims of this work.................................................................................................................... 31 1.8 References ............................................................................................................................... 33 vi Chapter 2: Proghrelin and C-ghrelin: Expression, purification and structural analysis 2.1 Introduction ............................................................................................................................. 59 2.2 Results ..................................................................................................................................... 64 2.2.1 Human proghrelin expression system ........................................................................... 64 2.2.2 Induction method optimization for proghrelin-His6 ..................................................... 65 2.2.3 C-ghrelin insolubility utilizing autoinduction ..............................................................