New chemical probes targeting cholesterylation of Sonic Hedgehog in human cells and zebrafish
File(s)
Published version
Author(s)
Type
Journal Article
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
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
Date Issued
2014-07-28
Date Acceptance
2014-07-25
Citation
Chemical Science, 2014, 5, pp.4249-4259
ISSN
2041-6520
Publisher
RSC
Start Page
4249
End Page
4259
Journal / Book Title
Chemical Science
Volume
5
Copyright Statement
This journal is © The Royal Society of Chemistry 2014. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence
License URL
Publication Status
Published