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In vivo characterization of the bacterial intramembrane-cleaving protease RseP using the heme binding tag-based assay iCliPSpy

Regulated intramembrane proteolysis (RIP) describes the protease-dependent cleavage of transmembrane proteins within the hydrophobic core of cellular membranes. Intramembrane-cleaving proteases (I-CliPs) that catalyze these reactions are found in all kingdoms of life and are involved in a wide range...

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Main Authors: Kupke, Thomas (Author) , Götz, Rabea (Author) , Richter, Florian M. (Author) , Beck, Rainer (Author) , Lolicato, Fabio (Author) , Nickel, Walter (Author) , Hopf, Carsten (Author) , Brügger, Britta (Author)
Format: Article (Journal)
Language:English
Published: 18 March 2023
In: Communications biology
Year: 2023, Volume: 6, Pages: 1-15
ISSN:2399-3642
DOI:10.1038/s42003-023-04654-z
Online Access:Verlag, kostenfrei, Volltext: https://doi.org/10.1038/s42003-023-04654-z
Verlag, kostenfrei, Volltext: https://www.nature.com/articles/s42003-023-04654-z
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Author Notes:Thomas Kupke, Rabea M. Götz, Florian M. Richter, Rainer Beck, Fabio Lolicato, Walter Nickel, Carsten Hopf & Britta Brügger
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Summary:Regulated intramembrane proteolysis (RIP) describes the protease-dependent cleavage of transmembrane proteins within the hydrophobic core of cellular membranes. Intramembrane-cleaving proteases (I-CliPs) that catalyze these reactions are found in all kingdoms of life and are involved in a wide range of cellular processes, including signaling and protein homeostasis. I-CLiPs are multispanning membrane proteins and represent challenging targets in structural and enzyme biology. Here we introduce iCLiPSpy, a simple assay to study I-CLiPs in vivo. To allow easy detection of enzyme activity, we developed a heme-binding reporter based on TNFα that changes color after I-CLiP-mediated proteolysis. Co-expression of the protease and reporter in Escherichia coli (E. coli) results in white or green colonies, depending on the activity of the protease. As a proof of concept, we use this assay to study the bacterial intramembrane-cleaving zinc metalloprotease RseP in vivo. iCLiPSpy expands the methodological repertoire for identifying residues important for substrate binding or activity of I-CLiPs and can in principle be adapted to a screening assay for the identification of inhibitors or activators of I-CLiPs, which is of great interest for proteases being explored as biomedical targets.
Item Description:Gesehen am 20.04.2023
Physical Description:Online Resource
ISSN:2399-3642
DOI:10.1038/s42003-023-04654-z

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