| Autor: |
Taylor MS; From the Department of Pharmacology and Molecular Sciences,; the High Throughput Biology Center and Department of Molecular Biology and Genetics., Ruch TR; the Department of Cell Biology., Hsiao PY; From the Department of Pharmacology and Molecular Sciences., Hwang Y; From the Department of Pharmacology and Molecular Sciences., Zhang P; the Program in Gene Expression and Regulation, The Wistar Institute, Philadelphia, Pennsylvania 19104., Dai L; the High Throughput Biology Center and Department of Molecular Biology and Genetics., Huang CRL; the High Throughput Biology Center and Department of Molecular Biology and Genetics,; the McKusick-Nathans Institute of Genetic Medicine., Berndsen CE; the Howard Hughes Medical Institute and Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205., Kim MS; the McKusick-Nathans Institute of Genetic Medicine., Pandey A; the McKusick-Nathans Institute of Genetic Medicine., Wolberger C; the Howard Hughes Medical Institute and Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205., Marmorstein R; the Program in Gene Expression and Regulation, The Wistar Institute, Philadelphia, Pennsylvania 19104; the Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104., Machamer C; the Department of Cell Biology,. Electronic address: machamer@jhmi.edu., Boeke JD; the High Throughput Biology Center and Department of Molecular Biology and Genetics,. Electronic address: machamer@jhmi.edu., Cole PA; From the Department of Pharmacology and Molecular Sciences,. Electronic address: pcole@jhmi.edu. |
| Abstrakt: |
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 His-338 is in the ER lumen, consistent with other family members, but conserved Asn-307 is cytosolic, making it unlikely that both are involved in catalysis. Photocross-linking of synthetic ghrelin analogs and inhibitors demonstrates binding to the C-terminal region of GOAT, consistent with a role of His-338 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 advance the discovery of selective inhibitors for these enzymes. |
