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The Hedgehog pathway is a key developmental signaling pathway but is also implicated in many types of cancer. The extracellular signaling protein Sonic hedgehog (Shh) requires dual lipidation for functional signaling, whereby N-terminal palmitoylation is performed by the enzyme Hedgehog acyltransferase (Hhat). Hhat is an attractive target for small-molecule inhibition to arrest Hedgehog signaling, and methods for assaying Hhat activity are central to understanding its function. However, all existing assays to quantify lipidation of peptides suffer limitations, such as safety hazards, high costs, extensive manual handling, restriction to stopped-assay measurements, or indirect assessment of lipidation. To address these limitations, we developed a microfluidic mobility shift assay (MSA) to analyze Shh palmitoylation. MSA allowed separation of fluorescently labeled Shh amine-substrate and palmitoylated Shh amide-product peptides based on differences in charge and hydrodynamic radius, coupled with online fluorescence intensity measurements for quantification. The MSA format was employed to study Hhat-catalyzed reactions, investigate Hhat kinetics, and determine small-molecule inhibitor IC50 values. Both real-time and stopped assays were performed, with the latter achieved via addition of excess unlabeled Shh peptide. The MSA format therefore allows direct and real-time fluorescence-based measurement of acylation and represents a powerful alternative technique in the study of N-lipidation.

Original publication

DOI

10.1177/2472555216689529

Type

Journal article

Journal

SLAS Discov

Publication Date

2017

Volume

22

Pages

418 - 424

Keywords

Acyltransferases/antagonists & inhibitors/genetics/*metabolism Amino Acid Sequence Electrophoretic Mobility Shift Assay/instrumentation/*methods Enzyme Assays Enzyme Inhibitors/pharmacology HEK293 Cells Hedgehog Proteins/genetics/*metabolism Humans Kinetics Lipoylation/drug effects Microfluidics/instrumentation/*methods *Protein Processing, Post-Translational Recombinant Proteins/genetics/metabolism *enzyme assays or enzyme kinetics *fluorescence methods *lipids or lipid metabolism *medicinal chemistry