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A Direct in vitro Fatty Acylation Assay for Hedgehog Acyltransferase
Adina R Schonbrun1,2, Marilyn D Resh1,2,3
1Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
Bio-Protocol
|January 9, 2023
Summary
Researchers developed a direct, fluorescent assay to measure Hedgehog acyltransferase (Hhat) activity. This safer and more efficient method uses a fluorescent substrate for easier detection of Hhat enzyme function in cellular pathways.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Hedgehog acyltransferase (Hhat) is crucial for the Hedgehog signaling pathway.
- Existing Hhat activity assays often use radioactive substrates, are multi-step, or require specialized equipment, limiting safety and efficiency.
Purpose of the Study:
- To develop a direct, fluorescent in vitro assay for monitoring Hhat catalytic activity.
- To provide a safer, more efficient, and convenient alternative to existing Hhat assays.
Main Methods:
- Utilized purified Hhat enzyme.
- Employed a fluorescently labeled fatty acyl-CoA donor substrate.
- Used a Sonic hedgehog (Shh) peptide as the recipient substrate for fatty acylation.
Main Results:
- Developed a straightforward protocol for direct Hhat activity monitoring.
- Achieved direct readout of fatty acylated Shh peptide via fluorescence detection.
- Validated the assay protocol in a relevant scientific publication.
Conclusions:
- The developed fluorescent assay offers a significant improvement in safety, efficiency, and convenience for measuring Hhat activity.
- This assay facilitates research into the Hedgehog signaling pathway by providing a robust tool for enzyme activity assessment.
