Abstract
Ghrelin O-acyltransferase (GOAT) is an enzyme responsible for octanoylating and activating ghrelin, a peptide hormone that plays a key role in energy regulation and hunger signaling. Due to its nature as an integral membrane protein, GOAT has yet to be purified in active form which has complicated biochemical and structural studies of GOAT-catalyzed ghrelin acylation. In this chapter, we describe protocols for efficient expression and enrichment of GOAT in insect cell-derived microsomal fraction, HPLC-based assays for GOAT acylation activity employing fluorescently labeled peptides, and assessment of inhibitor potency against GOAT.
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References
Müller TD, Nogueiras R, Andermann ML et al (2015) Ghrelin. Mol Metab 4:437–460
Kojima M, Hosoda H, Date Y et al (1999) Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402:656–660
Hofmann K (2000) A superfamily of membrane-bound O-acyltransferases with implications for wnt signaling. Trends Biochem Sci 25:111–112
Gutierrez JA, Solenberg PJ, Perkins DR et al (2008) Ghrelin octanoylation mediated by an orphan lipid transferase. Proc Natl Acad Sci U S A 105:6320–6325
Yang J, Brown MS, Liang G et al (2008) Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell 132:387–396
Darling JE, Zhao F, Loftus RJ et al (2015) Structure-activity analysis of human ghrelin O-acyltransferase reveals chemical determinants of ghrelin selectivity and acyl group recognition. Biochemistry 54:1100–1110
Murtuza MI, Isokawa M (2018) Endogenous ghrelin-O-acyltransferase (GOAT) acylates local ghrelin in the hippocampus. J Neurochem 144:58–67
Hopkins AL, Nelson TAS, Guschina IA et al (2017) Unacylated ghrelin promotes adipogenesis in rodent bone marrow via ghrelin O-acyl transferase and GHS-R1a activity: evidence for target cell-induced acylation. Sci Rep. https://doi.org/10.1038/srep45541
Taylor MS, Ruch TR, Hsiao PY et al (2013) Architectural organization of the metabolic regulatory enzyme ghrelin O-acyltransferase. J Biol Chem 288:32211–32228
Taylor MS, Dempsey DR, Hwang Y et al (2015) Mechanistic analysis of ghrelin-O-acyltransferase using substrate analogs. Bioorg Chem 62:64–73
Yang J, Zhao TJ, Goldstein JL et al (2008) Inhibition of ghrelin O-acyltransferase (GOAT) by octanoylated pentapeptides. Proc Natl Acad Sci U S A 105:10750–10755
Ohgusu H, Shirouzu K, Nakamura Y et al (2009) Ghrelin O-acyltransferase (GOAT) has a preference for n-hexanoyl-CoA over n-octanoyl-CoA as an acyl donor. Biochem Biophys Res Commun 386:153–158
Darling JE, Prybolsky EP, Sieburg M et al (2013) A fluorescent peptide substrate facilitates investigation of ghrelin recognition and acylation by ghrelin O-acyltransferase. Anal Biochem 437:68–76
Barnett BP, Hwang Y, Taylor MS et al (2010) Glucose and weight control in mice with a designed ghrelin O-acyltransferase inhibitor. Science 330:1689–1692
McGovern-Gooch KR, Rodrigues T, Darling JE et al (2016) Ghrelin octanoylation is completely stabilized in biological samples by alkyl fluorophosphonates. Endocrinology 157:4330–4338
Riddles PW, Blakeley RL, Zerner B (1979) Ellman's reagent: 5,5′-dithiobis(2-nitrobenzoic acid)--a reexamination. Anal Biochem 94:75–81
Jordan KR, Crawford F, Kappler JW et al (2009) Vaccination of mice with baculovirus-infected insect cells expressing antigenic proteins. Curr Protoc Immunol 85:2.15.1–2.15.23
Acknowledgments
This work was supported by grants from the Foundation for Prader-Willi Research (FPWR) and the American Diabetes Association (1-16-JDF-042).
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Sieburg, M.A., Cleverdon, E.R., Hougland, J.L. (2019). Biochemical Assays for Ghrelin Acylation and Inhibition of Ghrelin O-Acyltransferase. In: Linder, M. (eds) Protein Lipidation. Methods in Molecular Biology, vol 2009. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9532-5_18
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DOI: https://doi.org/10.1007/978-1-4939-9532-5_18
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