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Palmitoylation of the Hedgehog family of proteins is a critical step in the Hedgehog signaling pathway and is performed by the membrane-bound O-acyltransferase enzyme Hedgehog acyltransferase (HHAT). Measurement of HHAT activity has traditionally relied on radiolabeled fatty acid substrates, which imposes considerable constraints on throughput, cost, and safety, consequently hindering the efficient identification and development of small-molecule HHAT inhibitors. The Acylation-coupled Lipophilic Induction of Polarisation (Acyl-cLIP) assay was recently developed in our lab as a novel platform to evaluate lipidation of peptides in real time and high throughput. In this chapter, we describe the isolation of active HHAT from HEK293a cells and application of the Acyl-cLIP assay to characterize HHAT inhibitors. Our methodology uses standard chemical biology lab equipment and yields high-quality kinetic data from minimal sample volumes. The assay uses standard 384-well plates and is easily adapted to medium- or high-throughput screening formats.

Original publication

DOI

10.1007/978-1-0716-1701-4_2

Type

Chapter

Publication Date

2022

Volume

2374

Pages

13 - 26

Keywords

Biochemical assay, Drug discovery, Hedgehog acyltransferase, Palmitoylation, Acylation, Acyltransferases, Hedgehog Proteins, Lipoylation, Surgical Instruments