A Direct in vitro Fatty Acylation Assay for Hedgehog Acyltransferase.

Several assays have been developed to monitor the in vitro catalytic activity of Hedgehog acyltransferase (Hhat), an enzyme critical to the Hedgehog signaling pathway in cells. However, the majority of these previously reported assays involve radioactive fatty acyl donor substrates, multiple steps to achieve product readout, or specialized equipment. To increase safety, efficiency, and convenience, we developed a direct, fluorescent in vitro assay to monitor Hhat activity.

Hedgehog acyltransferase catalyzes a random sequential reaction and utilizes multiple fatty acyl-CoA substrates.

Sonic hedgehog (Shh) signaling is a key component of embryonic development and is a driving force in several cancers. Hedgehog acyltransferase (Hhat), a member of the membrane-bound O-acyltransferase family of enzymes, catalyzes the attachment of palmitate to the N-terminal cysteine of Shh, a posttranslation modification critical for Shh signaling. The activity of Hhat has been assayed in cells and in vitro, and cryo-EM structures of Hhat have been reported, yet several unanswered questions remain regarding the enzyme's reaction mechanism, substrate specificity, and the impact of the latter on Shh signaling.

Open Biology: overview for special issue on dynamics of protein fatty acylation.

Fatty acylation is a widespread form of protein modification that occurs on specific intracellular and secreted proteins. Beyond increasing hydrophobicity and the affinity of the modified protein for lipid bilayers, covalent attachment of a fatty acid exerts effects on protein localization, inter- and intramolecular interactions and signal transduction. As such, research into protein fatty acylation has been embraced by an extensive community of biologists.

Palmitoylation of Hedgehog proteins by Hedgehog acyltransferase: roles in signalling and disease.

Hedgehog acyltransferase (Hhat), a member of the membrane-bound -acyltransferase (MBOAT) family, catalyses the covalent attachment of palmitate to the N-terminus of Hedgehog proteins. Palmitoylation is a post-translational modification essential for Hedgehog signalling. This review explores the mechanisms involved in Hhat acyltransferase enzymatic activity, similarities and differences between Hhat and other MBOAT enzymes, and the role of palmitoylation in Hedgehog signalling.

Hedgehog Acyltransferase Promotes Uptake of Palmitoyl-CoA across the Endoplasmic Reticulum Membrane.

Attachment of palmitate to the N terminus of Sonic hedgehog (Shh) is essential for Shh signaling. Shh palmitoylation is catalyzed on the luminal side of the endoplasmic reticulum (ER) by Hedgehog acyltransferase (Hhat), an ER-resident enzyme. Palmitoyl-coenzyme A (CoA), the palmitate donor, is produced in the cytosol and is not permeable across membrane bilayers.