Methods for Studying Wnt Protein Modifications/Inactivations by Extracellular Enzymes, Tiki and Notum

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Wnt proteins are crucial signaling molecules involved in development and disease.
• Extracellular antagonists Tiki and Notum regulate Wnt protein activity.
• Tiki functions as a metalloprotease, while Notum acts as a deacylase, both inactivating Wnt proteins.

Purpose of the Study:

• To provide detailed protocols for preparing enzymatically active Tiki and Notum proteins.
• To outline in vitro enzymatic reaction conditions for Tiki and Notum.
• To describe a method for detecting Wnt protein acylation.

Main Methods:

• Purification of recombinant Tiki and Notum proteins.
• Establishment of in vitro enzymatic assays for Wnt inactivation.
• Metabolic labeling of Wnt proteins with clickable alkyne-containing lipids.
• Detection of acylated Wnt proteins using click chemistry and Western blotting.

Main Results:

• Successful preparation of catalytically active Tiki and Notum proteins.
• Optimization of in vitro conditions for Wnt cleavage by Tiki and deacylation by Notum.
• Demonstration of a sensitive method for detecting Wnt protein acylation.

Conclusions:

• The provided protocols enable robust biochemical studies of Wnt antagonists.
• The described metabolic labeling and click chemistry approach facilitates Wnt acylation analysis.
• These methods are valuable tools for investigating Wnt signaling regulation.