Ghrelin O-Acyltransferase Assays and Inhibition
ABSTRACT Ghrelin O-acyltransferase (GOAT) is responsible for catalyzing the attachment of the eight-carbon fatty acid octanoyl to the Ser3 side chain of the peptide ghrelin to generate the active form of this metabolic hormone. As such, GOAT is viewed as a potential therapeutic target for the treatment of obesity and diabetes mellitus. Here, we review recent progress in the development of cell and in vitro assays to measure GOAT action and the identification of several synthetic GOAT inhibitors. In particular, we discuss the design, synthesis, and characterization of the bisubstrate analog GO-CoA-Tat and its ability to modulate weight and blood glucose in mice. We also highlight current challenges and future research directions in our biomedical understanding of this fascinating ghrelin processing enzyme.
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ABSTRACT: Ghrelin O-AcylTransferase (GOAT) is a polytopic integral membrane protein required for activation of ghrelin, a secreted metabolism-regulating peptide hormone. Although GOAT is a potential therapeutic target for the treatment of obesity and diabetes and plays a key role in other physiologic processes, little is known about its structure or mechanism. GOAT is a member of the Membrane Bound O-AcylTransferase (MBOAT) family, a group of polytopic integral membrane proteins involved in lipid-biosynthetic and lipid-signaling reactions from prokaryotes to humans. Here, we use phylogeny and a variety of bioinformatic tools to predict the topology of GOAT. Using selective permeabilization indirect immunofluorescence microscopy in combination with glycosylation-shift immunoblotting, we demonstrate that GOAT contains 11 transmembrane helices and one reentrant loop. Development of the V5Glyc tag, a novel, small, and sensitive dual topology reporter, 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 for these enzymes.Journal of Biological Chemistry 09/2013; DOI:10.1074/jbc.M113.510313 · 4.60 Impact Factor
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ABSTRACT: Ghrelin, a gastric hormone, is a growth hormone-releasing peptide. Its serine-3 acylation with octanoic acid is essential for its orexigenic activity, and therefore, inhibition of the acylation of ghrelin may help in decreasing appetite and preventing obesity. This study aimed to establish a human gastric cell-based assay system to evaluate candidate inhibitors of octanoylated ghrelin production. In human gastric carcinoma AGS cells, obligatory factors for the posttranslational modification of ghrelin, such as certain prohormone convertases responsible for processing of proghrelin to the mature ghrelin and the enzyme-catalyzing acyl-modification of ghrelin, were well expressed, but ghrelin was expressed at low levels. Accordingly, we transfected a ghrelin-expressing vector into AGS cells and isolated a stable ghrelin-expressing cell line (AGS-GHRL8). AGS-GHRL8 cells secreted octanoylated ghrelin in accordance with the concentrations of octanoic acid in the culture medium. Given that ingested heptanoic acid is used for the acyl-modification of ghrelin, we evaluated whether heptanoic acid inhibits production of octanoylated ghrelin in AGS-GHRL8 cells. Butyric acid was used as a control. Indeed, heptanoic acid predictably decreased the secretion of octanoylated ghrelin, whereas butyric acid did not. The AGS-GHRL8 line established in this study will facilitate the screening of inhibitors of octanoylated ghrelin production.Journal of Biomolecular Screening 05/2013; 18(9). DOI:10.1177/1087057113489349 · 2.01 Impact Factor
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ABSTRACT: Ghrelin is a peptide hormone involved in regulation of appetite, glucose homeostasis, and a range of other physiological processes. Ghrelin requires a unique posttranslational modification, octanoylation of a serine side chain, to bind its cognate receptor to activate signaling. The enzyme that catalyzes this modification, ghrelin O-acyltransferase (GOAT), is receiving increased interest as a potential drug target for the treatment of obesity, diabetes, and other diseases proposed to be linked to ghrelin signaling. In this study, we report the development of a novel fluorescence-based assay for GOAT activity and the use of this assay to investigate GOAT inhibition and interactions underlying human GOAT (hGOAT) substrate selectivity. Using a series of mutations and chemical modifications of our fluorescent peptide substrate, we have identified specific groups on the first two amino acids of ghrelin that potentially contribute to ghrelin recognition by hGOAT. These data provide the first molecular-level information regarding interactions within the ghrelin-hGOAT complex. Defining the interactions used by hGOAT to bind and recognize ghrelin will provide insight into the structure of the hGOAT active site, aid in the design and optimization of targeted hGOAT inhibitors, and help to assess the possibility of novel hGOAT substrates beyond ghrelin.Analytical Biochemistry 02/2013; 437(1). DOI:10.1016/j.ab.2013.02.013 · 2.31 Impact Factor