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

• Verfasst von: Kupke, Thomas [VerfasserIn]
• Verfasst von: Götz, Rabea [VerfasserIn]
• Verfasst von: Richter, Florian M. [VerfasserIn]
• Verfasst von: Beck, Rainer [VerfasserIn]
• Verfasst von: Lolicato, Fabio [VerfasserIn]
• Verfasst von: Nickel, Walter [VerfasserIn]
• Verfasst von: Hopf, Carsten [VerfasserIn]
• Verfasst von: Brügger, Britta [VerfasserIn]
• Titel: In vivo characterization of the bacterial intramembrane-cleaving protease RseP using the heme binding tag-based assay iCliPSpy
• Verf.angabe: Thomas Kupke, Rabea M. Götz, Florian M. Richter, Rainer Beck, Fabio Lolicato, Walter Nickel, Carsten Hopf & Britta Brügger
• E-Jahr: 2023
• Jahr: 18 March 2023
• Umfang: 15 S.
• Fussnoten: Gesehen am 20.04.2023
• Titel Quelle: Enthalten in: Communications biology
• Ort Quelle: London : Springer Nature, 2018
• Jahr Quelle: 2023
• Band/Heft Quelle: 6(2023) vom: März, Artikel-ID 287, Seite 1-15
• ISSN Quelle: 2399-3642
• DOI: doi:10.1038/s42003-023-04654-z
• Datenträger: Online-Ressource
• Sprache: eng
• Sach-SW: Enzyme mechanisms
• Sach-SW: Molecular modelling
• Sach-SW: Proteases
• Sach-SW: Sensors and probes
• K10plus-PPN: 1843265893
• K10plus-PPN:
  Universitätsbibliothek
• Lokale URL UB: Zum Volltext

Abstract

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.