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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.

Original publication

DOI

10.1016/j.ab.2015.08.025

Type

Journal article

Journal

Anal Biochem

Publication Date

01/12/2015

Volume

490

Pages

66 - 72

Keywords

Click chemistry, Hedgehog acyltransferase, MBOAT, Protein palmitoylation, Acyltransferases, Biotinylation, Click Chemistry, Detergents, Enzyme Inhibitors, Enzyme-Linked Immunosorbent Assay, Fatty Acids, Unsaturated, HEK293 Cells, Hedgehog Proteins, Humans, Immobilized Proteins, Lipoylation, Maltose, Oligopeptides, Palmitoyl Coenzyme A, Peptide Fragments, Protein Processing, Post-Translational, Recombinant Fusion Proteins, Solubility, Streptavidin, Substrate Specificity