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Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo
Published on: November 7, 2013
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In vivo characterization of the bacterial intramembrane-cleaving protease RseP using the heme binding tag-based assay
Thomas Kupke1, Rabea M Götz2, Florian M Richter2
1Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, 69120, Heidelberg, Germany. thomas.kupke@bzh.uni-heidelberg.de.
Communications Biology
|March 19, 2023
Summary
Researchers developed iCLiPSpy, a novel assay to study intramembrane-cleaving proteases (I-CliPs) in vivo. This tool uses a color-changing reporter to easily detect I-CliP activity, aiding in understanding these vital enzymes.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzyme Kinetics
Background:
- Regulated intramembrane proteolysis (RIP) is crucial for cellular processes, involving proteases that cleave transmembrane proteins within membranes.
- Intramembrane-cleaving proteases (I-CliPs) are essential across life but are challenging to study due to their complex structure.
- Understanding I-CliP function is vital for cell signaling and protein homeostasis.
Purpose of the Study:
- To introduce iCLiPSpy, a new in vivo assay for studying intramembrane-cleaving proteases (I-CliPs).
- To enable straightforward detection of I-CliP activity using a colorimetric reporter system.
- To demonstrate the assay's utility by studying the bacterial metalloprotease RseP.
Main Methods:
- Development of a heme-binding reporter fused to TNFα, designed to change color upon I-CliP-mediated cleavage.
- Co-expression of I-CliPs and the reporter system in Escherichia coli (E. coli).
- Observation of colony color changes (white or green) indicative of protease activity.
Main Results:
- The iCLiPSpy assay successfully detected I-CliP activity in vivo.
- Colony color variation directly correlated with the activity levels of the studied I-CliPs.
- Proof-of-concept demonstrated with the bacterial intramembrane-cleaving zinc metalloprotease RseP.
Conclusions:
- iCLiPSpy provides a simple and effective method for studying I-CliPs in vivo.
- The assay facilitates the identification of key residues for substrate binding and enzyme activity.
- iCLiPSpy can be adapted for high-throughput screening of I-CliP inhibitors and activators, relevant for biomedical applications.
