Date of Award

6-27-2025

Date Published

August 2025

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

James Hougland

Keywords

ghrelin, ghrelin o-acyltransferase, ligand-conjugate, membrane bound o-acyltransferase, peptide mimetic inhibitor, post translational modification

Subject Categories

Biochemistry | Biochemistry, Biophysics, and Structural Biology | Life Sciences

Abstract

Ghrelin is a 28 amino acid peptide hormone responsible for a variety of physiological functions. Ghrelin signals these functions via its cognate receptor, GHSR, after an essential post translational modification which acylates its third serine. The acylation of ghrelin is facilitated by the enzyme ghrelin O-acyltransferase (GOAT) for which ghrelin is the only known substrate. Canonically, GOAT acylates ghrelin in the endoplasmic reticulum before it is further processed and secreted in circulation. Recent studies, however, have detected GOAT in the cytoplasm, plasma membrane, and in circulation in extracellular vesicles. Furthermore, GOAT has been identified as a potential prostate cancer (PCa) biomarker due to its overexpression in several aggressive PCa cell lines and detection in patient urine and plasma. These data combined suggest GOAT may be an attractive target for potential PCa detection and treatment. The work presented in this dissertation aimed to target GOAT and understand its function. Using antibody-drug conjugates as a model, we developed a customizable ligand-conjugate (LC) system with a nanomolar affinity for GOAT. After confirming these LCs inhibited GOAT, we gathered evidence capturing these LC-GOAT interactions by developing several binding assays including microplate reader assays and photo cross-linking. We also evaluated LC interactions with PCa cells overexpressing GOAT and showed evidence of GOAT-specific LC internalization tolerant of several fluorescent cargoes. Based on extracellular interactions with our LCs and evidence of cell surface GOAT expression, we began work to detect cell surface acylation of ghrelin by using new LCs capable of being acylated and acyl donor derivatives capable of click chemistry. These studies are vital in identifying biochemical and biological GOAT interactions with extracellular LCs, understanding the potentially expanded role of GOAT in local ghrelin re-acylation, and assessing the potential for GOAT to be a novel PCa biomarker.

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