Abstract
Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click–ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl β-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click–ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click–ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation.
Highlights
Hedgehog signaling is critical for correct embryogenesis and tissue development
We have developed a click chemistry-based ELISA format for measuring hedgehog acyltransferase (Hhat)-catalyzed acylation of sonic hedgehog (Shh) using an alkynetagged palmitoyl-coenzyme A (CoA) substrate
Alkynylated product peptides were functionalized via click chemistry with azido-FLAG and probed with a-FLAG-horseradish peroxidase (HRP) to allow colorimetric readout of
Summary
The resulting alkynylated peptide is subjected to labeling via click chemistry with azido-HRP (horseradish peroxidase) to facilitate product detection through fluorogenic deacetylation of Amplex Red in the presence of hydrogen peroxide catalyzed by HRP [7] This methodology allowed the assessment of GOAT activity and measurement of kinetic parameters along with screening to identify small molecule inhibitors [8]. Many studies of Hhat activity have employed radiolabeled palmitate [14,16,20e23], which may impede both the analysis and development of existing inhibitors and the identification of new alternative series with improved properties To this end, we employed a click chemistry armed enzyme-linked immunosorbent assay (clickeELISA) format to study Hhat activity, kinetic parameters, and assessment of Hhat inhibitors
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