Department of Chemistry, Syracuse University, Syracuse, NY 13244, USA.
Anal Biochem. 2013 Jun 1;437(1):68-76. doi: 10.1016/j.ab.2013.02.013. Epub 2013 Feb 27.
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.
胃饥饿素是一种参与调节食欲、葡萄糖稳态和一系列其他生理过程的肽激素。胃饥饿素需要一种独特的翻译后修饰，即丝氨酸侧链的辛酰化，以与其同源受体结合并激活信号转导。催化这种修饰的酶，即胃饥饿素 O-酰基转移酶（GOAT），作为治疗肥胖症、糖尿病和其他被认为与胃饥饿素信号有关的疾病的潜在药物靶点，受到越来越多的关注。在这项研究中，我们报告了一种用于 GOAT 活性的新型荧光测定法的开发，并利用该测定法研究了 GOAT 抑制和人类 GOAT（hGOAT）底物选择性的基础相互作用。通过对我们的荧光肽底物进行一系列突变和化学修饰，我们已经确定了胃饥饿素前两个氨基酸上的特定基团，这些基团可能有助于 hGOAT 识别胃饥饿素。这些数据提供了关于胃饥饿素-hGOAT 复合物内相互作用的第一个分子水平信息。定义 hGOAT 结合和识别胃饥饿素所使用的相互作用将有助于了解 hGOAT 活性位点的结构，有助于设计和优化靶向 hGOAT 抑制剂，并有助于评估除胃饥饿素之外的新型 hGOAT 底物的可能性。
