Notum deacylates octanoylated ghrelin
Objectives: The only proteins known to be modified by O-linked lipidation are Wnts and ghrelin, and enzymatic removal of this post-translational modification inhibits ligand activity. Indeed, the Wnt-deacylase activity of Notum is the basis of its ability to act as a feedback inhibitor of Wnt signal...
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doaj-c594c909a4894de098721bcad79aabdb2021-07-01T04:33:24ZengElsevierMolecular Metabolism2212-87782021-07-0149101201Notum deacylates octanoylated ghrelinYuguang Zhao0Laura-Nadine Schuhmacher1Morgan Roberts2Satoshi Kakugawa3Ganka Bineva-Todd4Steve Howell5Nicola O'Reilly6Christine Perret7Ambrosius P. Snijders8Jean-Paul Vincent9E. Yvonne Jones10Division of Structural Biology, Wellcome Centre for Human Genetics, Oxford University, Oxford, OX3 7BN, UKFrancis Crick Institute, London, NW1 1AT, UKFrancis Crick Institute, London, NW1 1AT, UKFrancis Crick Institute, London, NW1 1AT, UKFrancis Crick Institute, London, NW1 1AT, UKFrancis Crick Institute, London, NW1 1AT, UKFrancis Crick Institute, London, NW1 1AT, UKUniversité de Paris, Institut Cochin, INSERM, CNRS, F75014 Paris, FranceFrancis Crick Institute, London, NW1 1AT, UKFrancis Crick Institute, London, NW1 1AT, UK; Corresponding author. Francis Crick Institute, London, NW1 1AT, UK.Division of Structural Biology, Wellcome Centre for Human Genetics, Oxford University, Oxford, OX3 7BN, UK; Corresponding author. Division of Structural Biology, Wellcome Centre for Human Genetics, Oxford University, Oxford, OX3 7BN, UK.Objectives: The only proteins known to be modified by O-linked lipidation are Wnts and ghrelin, and enzymatic removal of this post-translational modification inhibits ligand activity. Indeed, the Wnt-deacylase activity of Notum is the basis of its ability to act as a feedback inhibitor of Wnt signalling. Whether Notum also deacylates ghrelin has not been determined. Methods: We used mass spectrometry to assay ghrelin deacylation by Notum and co-crystallisation to reveal enzyme–substrate interactions at the atomic level. CRISPR/Cas technology was used to tag endogenous Notum and assess its localisation in mice while liver-specific Notum knock-out mice allowed us to investigate the physiological role of Notum in modulating the level of ghrelin deacylation. Results: Mass spectrometry detected the removal of octanoyl from ghrelin by purified active Notum but not by an inactive mutant. The 2.2 Å resolution crystal structure of the Notum-ghrelin complex showed that the octanoyl lipid was accommodated in the hydrophobic pocket of the Notum. The knock-in allele expressing HA-tagged Notum revealed that Notum was produced in the liver and present in the bloodstream, albeit at a low level. Liver-specific inactivation of Notum in animals fed a high-fat diet led to a small but significant increase in acylated ghrelin in the circulation, while no such increase was seen in wild-type animals on the same diet. Conclusions: Overall, our data demonstrate that Notum can act as a ghrelin deacylase, and that this may be physiologically relevant under high-fat diet conditions. Our study therefore adds Notum to the list of enzymes, including butyrylcholinesterase and other carboxylesterases, that modulate the acylation state of ghrelin. The contribution of multiple enzymes could help tune the activity of this important hormone to a wide range of physiological conditions.http://www.sciencedirect.com/science/article/pii/S2212877821000417NotumGhrelinDeacylationCrystal structureMetabolism |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yuguang Zhao Laura-Nadine Schuhmacher Morgan Roberts Satoshi Kakugawa Ganka Bineva-Todd Steve Howell Nicola O'Reilly Christine Perret Ambrosius P. Snijders Jean-Paul Vincent E. Yvonne Jones |
spellingShingle |
Yuguang Zhao Laura-Nadine Schuhmacher Morgan Roberts Satoshi Kakugawa Ganka Bineva-Todd Steve Howell Nicola O'Reilly Christine Perret Ambrosius P. Snijders Jean-Paul Vincent E. Yvonne Jones Notum deacylates octanoylated ghrelin Molecular Metabolism Notum Ghrelin Deacylation Crystal structure Metabolism |
author_facet |
Yuguang Zhao Laura-Nadine Schuhmacher Morgan Roberts Satoshi Kakugawa Ganka Bineva-Todd Steve Howell Nicola O'Reilly Christine Perret Ambrosius P. Snijders Jean-Paul Vincent E. Yvonne Jones |
author_sort |
Yuguang Zhao |
title |
Notum deacylates octanoylated ghrelin |
title_short |
Notum deacylates octanoylated ghrelin |
title_full |
Notum deacylates octanoylated ghrelin |
title_fullStr |
Notum deacylates octanoylated ghrelin |
title_full_unstemmed |
Notum deacylates octanoylated ghrelin |
title_sort |
notum deacylates octanoylated ghrelin |
publisher |
Elsevier |
series |
Molecular Metabolism |
issn |
2212-8778 |
publishDate |
2021-07-01 |
description |
Objectives: The only proteins known to be modified by O-linked lipidation are Wnts and ghrelin, and enzymatic removal of this post-translational modification inhibits ligand activity. Indeed, the Wnt-deacylase activity of Notum is the basis of its ability to act as a feedback inhibitor of Wnt signalling. Whether Notum also deacylates ghrelin has not been determined. Methods: We used mass spectrometry to assay ghrelin deacylation by Notum and co-crystallisation to reveal enzyme–substrate interactions at the atomic level. CRISPR/Cas technology was used to tag endogenous Notum and assess its localisation in mice while liver-specific Notum knock-out mice allowed us to investigate the physiological role of Notum in modulating the level of ghrelin deacylation. Results: Mass spectrometry detected the removal of octanoyl from ghrelin by purified active Notum but not by an inactive mutant. The 2.2 Å resolution crystal structure of the Notum-ghrelin complex showed that the octanoyl lipid was accommodated in the hydrophobic pocket of the Notum. The knock-in allele expressing HA-tagged Notum revealed that Notum was produced in the liver and present in the bloodstream, albeit at a low level. Liver-specific inactivation of Notum in animals fed a high-fat diet led to a small but significant increase in acylated ghrelin in the circulation, while no such increase was seen in wild-type animals on the same diet. Conclusions: Overall, our data demonstrate that Notum can act as a ghrelin deacylase, and that this may be physiologically relevant under high-fat diet conditions. Our study therefore adds Notum to the list of enzymes, including butyrylcholinesterase and other carboxylesterases, that modulate the acylation state of ghrelin. The contribution of multiple enzymes could help tune the activity of this important hormone to a wide range of physiological conditions. |
topic |
Notum Ghrelin Deacylation Crystal structure Metabolism |
url |
http://www.sciencedirect.com/science/article/pii/S2212877821000417 |
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