Date of Award

August 2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

James Hougland

Second Advisor

Jason Fridley

Keywords

Ghrelin, Ghrelin maturation, GOAT, GOAT inhibition, Protein acylation, Protein expression

Subject Categories

Physical Sciences and Mathematics

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.

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