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.
Highlights
Sonic Hedgehog protein (Shh) is a vertebrate morphogen, mitogen and differentiation factor belonging to the Hedgehog protein (Hh) family
We previously reported the rst azide-tagged cholesterol probe (AzChol)[22] that can be used to label ShhN in cells; AzChol has several signi cant limitations as a tool: it is incorporated at
Our original design for AzChol bene tted from straightforward synthetic access to this class of probe molecules; we and others have observed that azide-tagged probes can o en result in ampli ed background labelling in cells relative to comparable alkyne-tagged counterparts.[31,32]
Summary
Sonic Hedgehog protein (Shh) is a vertebrate morphogen, mitogen and differentiation factor belonging to the Hedgehog protein (Hh) family. 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.
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