Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase

Analytical Biochemistry
Thomas Lanyon-HoggEdward W Tate

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 wher...Continue Reading

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Citations

Mar 16, 2017·SLAS Discovery·Thomas Lanyon-HoggEdward W Tate
May 22, 2018·Chemical Communications : Chem Comm·Amanda L Garner
Apr 7, 2021·Chemical Reviews·Kiall F SuazoMark D Distefano
Apr 16, 2019·Journal of Medicinal Chemistry·Ilya GalperinMatthias Lauth

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Datasets Mentioned

BETA
BC117130

Methods Mentioned

BETA
lipidation
enzyme-linked immunosorbent assay
transfection
electrophoresis
ELISAs
acylation
ELISA
enzyme-linked

Software Mentioned

ImageQuant

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