Ghrelin is a 28-amino acid hormone secreted primarily by the stomach and gastrointestinal tract. The only known blood-circulating hormone to stimulate appetite, ghrelin is post-translationally modified with an 8-carbon acyl group by the enzyme ghrelin O-acyltransferase (GOAT). Octanoylation of ghrelin’s 3rd serine residue by GOAT is required for physiological activity, and this 8-carbon modification is unique within the human proteome. Approaches to control ghrelin signaling via inhibiting GOAT have been proposed as novel treatments targeting type II diabetes, obesity, and other diseases impacted by ghrelin. GOAT is a membrane-bound enzyme, whose purification and structural determination have not been possible, limiting our understanding of how GOAT binds and modifies ghrelin. Developing such an understanding of GOAT’s structure, active site, and catalytic mechanism is critical towards the future goal of creating GOAT inhibitors as potential therapeutics targeting ghrelin-related diseases. Based on a recent finding in our laboratory that at least one sulfur-containing cysteine residue is necessary for enzyme function in human GOAT (hGOAT), this research focuses on locating functionally essential cysteine(s) within hGOAT. Toward this goal, a block cysteine mutagenesis was used to identify domains within hGOAT which contain cysteine(s) essential for ghrelin acylation. To pinpoint the specific cysteine(s) involved in enzyme function, site-directed mutagenesis was performed on a total of eleven cysteine residues within hGOAT. Five of these hGOAT cysteine mutants exhibited severely compromised acylation activity with a ghrelin-mimetic peptide substrate. Finally, studies using cysteine-modifying agents, hGOAT point-mutants, and fluorescently-labeled substrate mimics were developed to locate catalytically essential cysteines in hGOAT. Identifying and locating catalytic residues will provide an essential contribution towards understanding the structure and function of ghrelin O-acyltransferase.

Investigation of functionally essential cysteine residues within human ghrelin O-acyltransferase

256th National Meeting (2018)

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