Inhibition, Structure, and Function of Ghrelin-O-Acyltransferase
Taylor, Martin Samuel
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Ghrelin-O-Acyltransferase (GOAT) is an 11-transmembrane integral membrane protein that octanoylates Ser3 of the metabolism-regulating gastric peptide hormone ghrelin, producing the active form of this hormone. Protein octanoylation is unique to ghrelin in humans, and GOAT may represent an attractive target for the treatment of type II diabetes and the metabolic syndrome. Ghrelin physiology is complex; it has often been called a “hunger hormone,” but a growing body of literature suggests that this is incorrect. Rather, ghrelin seems to have evolved to help vertebrates survive extreme starvation by controlling metabolism to maintain blood sugar, minimize energy expenditure, and maximize energy storage. Ghrelin is potently pro-kinetic in the enteric nervous system, and this combined with pro-storage metabolic effects has led to a number of promising agonists in clinical trials for cachexia. Additionally, ghrelin has been implicated in a variety of other physiologic processes, including cardiovascular function, learning, memory, reward behavior, and addiction. This work outlines the development of the first in vivo inhibitors of ghrelin octanoylation and their effects in mice, the determination of the topology of GOAT, and studies on the structure, mechanism, and enzymology of GOAT. Chapter 1 provides an introduction, reviewing the history of the ghrelin field, recent progress in the development of cell and in vitro assays to measure GOAT action, and the identification of several synthetic GOAT inhibitors. In Chapter 2, we describe the design, synthesis, and characterization of GO-CoA-Tat, a peptide-based bisubstrate analog that antagonizes GOAT. GO-CoA-Tat potently inhibits GOAT in vitro, in cultured cells, and in mice. Intraperitoneal administration of GO-CoA-Tat improves glucose tolerance and reduces weight gain in wild-type mice but not in ghrelin-deficient mice, supporting the concept that its beneficial metabolic effects are due specifically to GOAT inhibition. In addition to serving as a research tool for mapping ghrelin actions, GO-CoA-Tat may help pave the way for clinical targeting of GOAT in metabolic diseases. In Chapter 3, we use phylogeny and a variety of bioinformatic tools to predict the topology of GOAT, which was previously unknown. Using selective permeabilization indirect immunofluorescence microscopy in combination with glycosylation-shift immunoblotting, we demonstrate that GOAT contains 11 transmembrane helices and one reentrant loop. We report the development of the V5Glyc tag, a novel, small, and sensitive dual topology reporter, which facilitated these experiments. The MBOAT family invariant residue His338 is in the ER lumen, consistent with other family members, but conserved Asn307 is cytosolic, making it unlikely that both are involved in catalysis. Photocrosslinking of synthetic ghrelin analogs and inhibitors demonstrates binding to the C-terminal region of GOAT, consistent with a role of His338 in the active site. This knowledge of GOAT architecture is important for a deeper understanding of the mechanism of GOAT and other MBOATs and could ultimately enhance the discovery of selective inhibitors of these enzymes. In Chapter 4, we develop explore an in vitro microsomal ghrelin octanoylation assay to analyze its enzymologic features. Measurement of Km for 10-mer, 27-mer, and synthetic Tat-peptide-containing ghrelin substrates provide evidence for a role of charge interactions in substrate binding. Ghrelin substrates with amino-alanine in place of Ser3 demonstrate that GOAT can catalyze the formation of an octanoyl-amide bond at a similar rate compared with the natural reaction. A pH-rate comparison of these substrates reveals minimal differences in acyltransferase activity across pH 6.0-9.0. These data support a GOAT catalytic mechanism which is insensitive to substrate nucleophilicity. Limitations of the microsomal assay then led us to attempt to find conditions where solubilized GOAT retained activity; we report failure in this effort with 35 detergents and no activity of GOAT successfully packaged into phospholipid bilayer membrane scaffolds (nanodiscs).