TY - JOUR
T1 - A fluorescent peptide substrate facilitates investigation of ghrelin recognition and acylation by ghrelin O-acyltransferase
AU - Darling, Joseph E.
AU - Prybolsky, Edward P.
AU - Sieburg, Michelle
AU - Hougland, James L.
N1 - Funding Information:
This work was funded by Syracuse University and a National Science Foundation (NSF) predoctoral fellowship (DGE-1247399) to J.E.D. We thank Robert Doyle, Kevin Sweder, and members of the Hougland laboratory for helpful comments and discussion. We also thank the Korendovych laboratory (Syracuse University) for assistance with peptide synthesis.
PY - 2013/6/1
Y1 - 2013/6/1
N2 - 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.
AB - 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.
KW - Fluorescent assay
KW - Ghrelin
KW - Ghrelin O-acyltransferase
KW - Posttranslational modification
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U2 - 10.1016/j.ab.2013.02.013
DO - 10.1016/j.ab.2013.02.013
M3 - Article
C2 - 23453974
AN - SCOPUS:84875962170
SN - 0003-2697
VL - 437
SP - 68
EP - 76
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -