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Notum deacylates octanoylated ghrelin

DOI: 10.1016/j.molmet.2021.101201 DOI Help

Authors: Yuguang Zhao (Wellcome Centre for Human Genetics, Oxford University) , Laura-Nadine Schuhmacher (The Francis Crick Institute) , Morgan Roberts (The Francis Crick Institute) , Satoshi Kakugawa (The Francis Crick Institute) , Ganka Bineva-Todd (The Francis Crick Institute) , Steve Howell (The Francis Crick Institute) , Nicola O'Reilly (The Francis Crick Institute) , Christine Perret (Institut Cochin) , Ambrosius P. Snijders (The Francis Crick Institute) , Jean-Paul Vincent (The Francis Crick Institute) , E. Yvonne Jones (Wellcome Centre for Human Genetics, Oxford University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Molecular Metabolism

State: Published (Approved)
Published: February 2021
Diamond Proposal Number(s): 14744

Open Access Open Access

Abstract: 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 shows the octanoyl lipid is accommodated in the hydrophobic pocket of Notum. The knock-in allele expressing HA-tagged Notum reveals that Notum is produced in the liver and present in the bloodstream, albeit at a low level. Liver-specific inactivation of Notum in animals fed with a high fat diet leads to a small but significant increase in acylated ghrelin in the circulation, while no such increase is seen in wildtype animals on the same diet. Conclusions: Overall our data demonstrate Notum can act as a ghrelin deacylase, and that this may be physiologically relevant under high fat diet conditions. Our work therefore adds Notum to the list of enzymes, including butylcholineasterase 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.

Journal Keywords: Notum; ghrelin; deacylation; crystal structure; metabolism

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)

Added On: 03/03/2021 09:16


Discipline Tags:

Biochemistry Chemistry Structural biology Organic Chemistry Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)