Date of Award
Doctor of Philosophy (PhD)
James L. Hougland
Physical Sciences and Mathematics
Ghrelin is a peptide hormone primarily secreted by the stomach that acts as the endogenous ligand for the GHSR1a G-protein-coupled receptor. This 28 amino acid peptide stimulates appetite and has been associated with the cardiovascular, gastrointestinal, neuroendocrine, and immune systems. Ghrelin requires a unique posttranslational modification to be biological active. Ghrelin O-acyltransferase (GOAT) is responsible for catalyzing octanoylation of the 3rd serine side chain of ghrelin to generate the active form of this hormone. Thus, GOAT is considered as a potential therapeutic target for treatment of obesity and other disorders linked to the peptide. Yet, the active site architecture and mechanism for GOAT catalyzed ghrelin acylation are presently undetermined. Purification of an active form of GOAT has been challenging, impeding structural studies of this integral membrane protein. To improve on these challenges, we have synergistically combined computational modeling and biochemical validation to construct the first structural model of the eukaryotic membrane-bound human GOAT protein. Our structure revealed an unforeseen strategy for transmembrane protein acylation with catalysis occurring in an internal channel connecting the lumen and cytoplasm. Structure-guided studies used in this work, have revealed essential amino acid responsible for important substrate-enzyme interactions. Moreover, by using a tight-binding fluorescent ghrelin derived peptide, we demonstrate GOAT’s interaction with extracellular ghrelin and facilitation of ligand cell internalization. Our work provides a new understanding of GOAT’s catalytic mechanism, establishes key substrate-enzyme interactions, and advances structure-guided inhibitor design to target therapeutically important but experimentally intractable membrane proteins.
Campana, Maria, "Structural and Functional Insights into Ghrelin Acylation by Ghrelin O-Acyltransferase" (2020). Dissertations - ALL. 1197.