title: Measuring IL-1β Processing by Bioluminescence Sensors II: The iGLuc System.
abstract: Inflammasomes are multimeric protein complexes that proteolytically activate caspase-1, which subsequently matures cytokines of the IL-1 family and initiates the induction of pyroptotic cell death. Although this process is central both to pathogen defense and sterile inflammatory processes, there is currently no standard readout available for inflammasome activation which would be suitable for high-throughput applications. We have recently developed a new method for measuring inflammasome activation via the use of a novel proteolytic reporter iGLuc, an IL-1β Gaussia luciferase (iGLuc) fusion protein. Here, we provide detailed protocols for the use of iGLuc in transiently transfected or stably transduced cell lines. Using these protocols, IL-1β maturation as the result of inflammasome activation or other processes can be indirectly measured via the gain of Gaussia luciferase activity of cleaved iGLuc, allowing for rapid inflammasome reconstitution assays and high-throughput screening of inflammasome activity.
authors.affiliation: Institute of Molecular Medicine, University Hospital, University of Bonn, Sigmund-Freud-Straße 25, Bonn, Germany.|Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Sigmund-Freud-Straße 25, Bonn, Germany.
authors.forename: Eva
authors.initials: E
authors.lastname: Bartok
authors.affiliation: Institute of Molecular Medicine, University Hospital, University of Bonn, Sigmund-Freud-Straße 25, Bonn, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Kampes
authors.affiliation: Institute of Molecular Medicine, University Hospital, University of Bonn, Sigmund-Freud-Straße 25, Bonn, Germany. veit.hornung@uni-bonn.de.|Gene Center and Department of Biochemistry, Ludwig-Maximilians-University Munich, Feodor-Lynen-Str. 25, Munich, Germany. veit.hornung@uni-bonn.de.
authors.forename: Veit
authors.initials: V
authors.lastname: Hornung
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D058847:Inflammasomes; D007249:Inflammation / Q000378:metabolism*; D053583:Interleukin-1beta / Q000235:genetics / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D000069292:Pyroptosis; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D058847:Inflammasomes; D053583:Interleukin-1beta; D011993:Recombinant Fusion Proteins; D008156:Luciferases
keywords: Gaussia luciferase; High-throughput screening; IL-1β; Inflammasome