Date of Award

12-24-2025

Date Published

January 2026

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

James Hougland

Subject Categories

Chemistry | Physical Sciences and Mathematics

Abstract

Post-translational modifications (PTMs) are essential for the diversification of the human proteome. Prenylation is a PTM defined by the addition of a 15-carbon or 20 carbon isoprenoid group by FTase and GGTase-I, respectively, onto a cysteine near the C-terminus of proteins. This modification is the first step within the prenylation pathway where proteolysis and carboxymethylation occur afterwards, aiding in proteins localization to the cellular membrane. These prenyltransferases canonically recognize a “CaaX” box motif which has been determined through biochemical and structural studies. Recently, sequences following the C(x)3X and Cxx patterns have been shown to be prenylated as well as an alternate route for prenylated proteins known as the shunt pathway. These new developments indicate that there is more to learn about prenylation than previously thought. In this work, we investigate the reactivity of -CKQX peptides with mammalian FTase to broaden the scope of known prenylated sequences. Analysis and characterization of an array of prenyl donor analogues with mammalian prenyltransferases validates the incorporation of these analogues and therefore increases the techniques in which prenylation can be observed. Octanoylation is another PTM that is carried out by ghrelin O-acyltransferase (GOAT), member of the membrane bound O-acyltransferase (MBOAT) enzyme family. This modification adds an octanoyl moiety onto the third serine of the peptide hormone ghrelin, also known as the “hunger hormone”. This serine octanoylation is essential for ghrelin to be able to bind and activate the growth hormone secretagogue receptor (GHSR). GOAT presents an avenue to regulate ghrelin signaling as ghrelin is its only known substrate. Analysis of an integral membrane protein poses challenges due to the requirement of an added surfactant to maintain enzyme structure when out of the membrane. Considering this, we screened the solubilization of GOAT in various detergents and explored several purification techniques to obtain a pure form. Such work allowed for the accumulation of a database of knowledge on the activity of GOAT across different detergent groups. A combination of this analysis with computational studies has revealed dynamics within GOAT that are essential for enzyme catalytic function. This work lays a foundation for future structure determination and understanding of GOAT’s mechanism.

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