Evaluating Hedgehog Acyltransferase Activity and Inhibition Using the Acylation-coupled Lipophilic Induction of Polarization (Acyl-cLIP) Assay.
Evaluating Hedgehog Acyltransferase Activity and Inhibition Using the Acylation-coupled Lipophilic Induction of Polarization (Acyl-cLIP) Assay.
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DOI：
10.1007/978-1-0716-1701-4_2
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发表时间：
2022-01-01
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影响因子：
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通讯作者：
Lanyon-Hogg, Thomas
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作者：
Andrei, Sebastian A;Tate, Edward W;Lanyon-Hogg, Thomas
Hedgehog家族蛋白质的棕榈酰化是Hedgehog信号通路中的关键步骤，并由膜结合O-酰基转移酶Hedgehog酰基转移酶（HHAT）进行。HHAT活性的测量传统上依赖于放射性标记的脂肪酸底物，这对通量、成本和安全性施加了相当大的限制，从而阻碍了小分子HHAT抑制剂的有效鉴定和开发。我们实验室最近开发了酰化偶联亲脂性极化诱导（Acyl-cLIP）测定法，作为一种新型平台，用于真实的实时和高通量评价肽的脂化。在本章中，我们描述了从HEK 293 a细胞中分离活性HHAT和应用酰基-cLIP测定来表征HHAT抑制剂。我们的方法使用标准的化学生物学实验室设备，并从最小的样品体积产生高质量的动力学数据。该分析使用标准384孔板，并且很容易适应中等或高通量筛选格式。
Palmitoylation of the Hedgehog family of proteins is a critical step in the Hedgehog signaling pathway and is performed by the membrane-bound O-acyltransferaseenzyme 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.