Date of Award

8-23-2024

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

James Hougland

Keywords

cellular trafficking;COPI;ghrelin;ghrelin O-acyltransferase;post-translational modifications

Subject Categories

Chemistry | Physical Sciences and Mathematics

Abstract

Ghrelin O-acyltransferase (GOAT) catalyzes the acylation and activation of ghrelin, a peptide hormone involved in a range of physiological pathways and therapeutic target for metabolism-linked diseases such as obesity and diabetes. GOAT was determined to be localized in the membrane of the endoplasmic reticulum (ER) in early cell-based studies, but recent patient studies detected this integral membrane enzyme in blood plasma and urine. These findings challenge the current understanding of GOAT’s cellular localization, and it may provide a potential mechanism for ghrelin re-acylation by plasma membrane-resident GOAT. The work described in this dissertation aimed to define the intracellular trafficking mechanism and subcellular localization of GOAT and provide insight on the biological function of the enzyme. We identified a conserved C-terminal dilysine (KxKxx) sequence in GOAT and hypothesized a potential trafficking mechanism mediated by coat protein complex I (COPI), which binds this dilysine motif and traffics target cargo proteins between the Golgi and endoplasmic reticulum (ER). We engineered novel GOAT fluorescent fusion proteins and generated a cell line stably expressing a B’-COPI fusion protein to co-express and visualize proteins in cells, we investigated the role of the dilysine motif in a cellular context. Co-localization analysis support a binding interaction between GOAT and B’-COPI. To profile subcellular distribution, we investigated a panel of cancer cell lines expected to overexpress GOAT and performed pair-wise co-immunostaining studies to assign GOAT distribution to organelles through the secretory pathway. These studies are key to advancing our understanding of GOAT, its cellular trafficking, and the impact of cellular localization on ghrelin signaling in different cellular and organismal contexts, such as that of a therapeutic target for cancer cells that express the enzyme.

Access

Open Access

Download

Included in

Chemistry Commons

Share

COinS