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iGLuc: a luciferase-based inflammasome and protease activity reporter

Nature Methods volume 10, pages 147–154 (2013)Cite this article

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Abstract

Measurement of protease activity in living cells or organisms remains a challenging task. We here present a transgene-encoded biosensor that reports the proteolytic activity of caspase-1 in the course of inflammasome activation and that of other proteases in a highly sensitive and specific manner. This protease reporter is based on the biological activity of a pro–interleukin (IL)-1β–Gaussia luciferase (iGLuc) fusion construct, in which pro–IL-1β–dependent formation of protein aggregates renders GLuc enzyme inactive. Cleavage leads to monomerization of this biosensor protein, resulting in a strong gain in luciferase activity. Exchange of the canonical caspase-1 cleavage site in this reporter construct allows the generation of protease biosensors with additional specificities. The high sensitivity, signal-to-background ratio and specificity of the iGLuc system renders it a useful tool to study proteolytic events in mouse and human cells at high throughput and to monitor protease activity in mice in vivo.

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Figure 1: iGLuc is active upon caspase-1–mediated cleavage.
Figure 2: iGLuc reports inflammasome activation.
Figure 3: Building of other protease biosensors based on iGLuc.
Figure 4: Functionality of purified iGLuc.
Figure 5: iGLuc is inactive in the context of protein aggregates.
Figure 6: iGLuc as a tool to study in vivo inflammasome activation.

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Acknowledgements

We thank M. Rossner (Max Planck Institute for Experimental Medicine) for providing us with pCMV-TEV, B. Kümmerer (University Hospital, University of Bonn) for the pMetLuc control, W. Barchet (University Hospital, University of Bonn) for GM-CSF supernatant and Millennium Pharmaceuticals for NLRP3- and NLRC4-deficient mice. V.H. is member of the excellence cluster ImmunoSensation and is supported by grants from the German Research Foundation (SFB704) and the European Research Council (ERC-2009-StG 243046).

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Author notes

  1. Eva Bartok and Franz Bauernfeind: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Clinical Chemistry and Clinical Pharmacology, Unit for Clinical Biochemistry, University Hospital, University of Bonn, Bonn, Germany

    Eva Bartok, Franz Bauernfeind, Maria G Khaminets, Christopher Jakobs & Veit Hornung

  2. Department of Internal Medicine III, University Hospital, University of Bonn, Bonn, Germany

    Franz Bauernfeind

  3. Institute of Innate Immunity, University Hospital, University of Bonn, Bonn, Germany

    Brian Monks & Eicke Latz

  4. Division of Infectious Diseases & Immunology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Brian Monks, Katherine A Fitzgerald & Eicke Latz

Authors
  1. Eva Bartok
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  2. Franz Bauernfeind
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  3. Maria G Khaminets
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Contributions

E.B., F.B. and V.H. designed experiments, analyzed the data and wrote the manuscript. E.B., F.B., M.G.K., C.J., B.M., E.L. and V.H. performed experiments. K.A.F. and E.L. assisted with data collection and interpretation. V.H. conceived and supervised the project.

Corresponding author

Correspondence to Veit Hornung.

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The authors declare no competing financial interests.

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Supplementary Figures 1–18, Supplementary Tables 1 and 2 and Supplementary Notes 1–3 (PDF 37143 kb)

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Bartok, E., Bauernfeind, F., Khaminets, M. et al. iGLuc: a luciferase-based inflammasome and protease activity reporter. Nat Methods 10, 147–154 (2013). https://doi.org/10.1038/nmeth.2327

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