title: New chemical probes targeting cholesterylation of Sonic Hedgehog in human cells and zebrafish.
abstract: Sonic Hedgehog protein (Shh) is a morphogen molecule important in embryonic development and in the progression of many cancer types in which it is aberrantly overexpressed. Fully mature Shh requires attachment of cholesterol and palmitic acid to its C- and N-termini, respectively. The study of lipidated Shh has been challenging due to the limited array of tools available, and the roles of these posttranslational modifications are poorly understood. Herein, we describe the development and validation of optimised alkynyl sterol probes that efficiently tag Shh cholesterylation and enable its visualisation and analysis through bioorthogonal ligation to reporters. An optimised probe was shown to be an excellent cholesterol biomimetic in the context of Shh, enabling appropriate release of tagged Shh from signalling cells, formation of multimeric transport complexes and signalling. We have used this probe to determine the size of transport complexes of lipidated Shh in culture medium and expression levels of endogenous lipidated Shh in pancreatic ductal adenocarcinoma cell lines through quantitative chemical proteomics, as well as direct visualisation of the probe by fluorescence microscopy and detection of cholesterylated Hedgehog protein in developing zebrafish embryos. These sterol probes provide a set of novel and well-validated tools that can be used to investigate the role of lipidation on activity of Shh, and potentially other members of the Hedgehog protein family.
authors.affiliation: Department of Chemistry , Imperial College London , Exhibition Road , London SW7 2AZ , UK . Email: e.tate@imperial.ac.uk ; Institute of Chemical Biology , Imperial College London , Exhibition Road , London SW7 2AZ , UK . Email: t.magee@imperial.ac.uk.
authors.forename: Paulina
authors.initials: P
authors.lastname: Ciepla
authors.affiliation: National Heart and Lung Institute , Imperial College London , Exhibition Road , London SW7 2AZ , UK.
authors.forename: Antonios D
authors.initials: AD
authors.lastname: Konitsiotis
authors.affiliation: Department of Chemistry , Imperial College London , Exhibition Road , London SW7 2AZ , UK . Email: e.tate@imperial.ac.uk.
authors.forename: Remigiusz A
authors.initials: RA
authors.lastname: Serwa
authors.affiliation: Department of Chemistry , Imperial College London , Exhibition Road , London SW7 2AZ , UK . Email: e.tate@imperial.ac.uk ; Institute of Chemical Biology , Imperial College London , Exhibition Road , London SW7 2AZ , UK . Email: t.magee@imperial.ac.uk.
authors.forename: Naoko
authors.initials: N
authors.lastname: Masumoto
authors.affiliation: Department of Chemistry , Imperial College London , Exhibition Road , London SW7 2AZ , UK . Email: e.tate@imperial.ac.uk.
authors.forename: Wai P
authors.initials: WP
authors.lastname: Leong
authors.affiliation: Institute of Chemical Biology , Imperial College London , Exhibition Road , London SW7 2AZ , UK . Email: t.magee@imperial.ac.uk ; Department of Life Sciences , Imperial College London , Exhibition Road , London SW7 2AZ , UK.
authors.forename: Margaret J
authors.initials: MJ
authors.lastname: Dallman
authors.affiliation: Institute of Chemical Biology , Imperial College London , Exhibition Road , London SW7 2AZ , UK . Email: t.magee@imperial.ac.uk ; National Heart and Lung Institute , Imperial College London , Exhibition Road , London SW7 2AZ , UK.
authors.forename: Anthony I
authors.initials: AI
authors.lastname: Magee
authors.affiliation: Department of Chemistry , Imperial College London , Exhibition Road , London SW7 2AZ , UK . Email: e.tate@imperial.ac.uk ; Institute of Chemical Biology , Imperial College London , Exhibition Road , London SW7 2AZ , UK . Email: t.magee@imperial.ac.uk.
authors.forename: Edward W
authors.initials: EW
authors.lastname: Tate