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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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References

  1. Martinon, F., Mayor, A. & Tschopp, J. The inflammasomes: guardians of the body. Annu. Rev. Immunol. 27, 229–265 (2009).

    Article  CAS  Google Scholar 

  2. Bauernfeind, F. et al. Inflammasomes: current understanding and open questions. Cell. Mol. Life Sci. 68, 765–783 (2011).

    Article  CAS  Google Scholar 

  3. Dinarello, C.A. Immunological and inflammatory functions of the interleukin-1 family. Annu. Rev. Immunol. 27, 519–550 (2009).

    Article  CAS  Google Scholar 

  4. Hoffman, H.M., Mueller, J.L., Broide, D.H., Wanderer, A.A. & Kolodner, R.D. Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle-Wells syndrome. Nat. Genet. 29, 301–305 (2001).

    Article  CAS  Google Scholar 

  5. Masters, S.L., Simon, A., Aksentijevich, I. & Kastner, D.L. Horror autoinflammaticus: the molecular pathophysiology of autoinflammatory disease. Annu. Rev. Immunol. 27, 621–668 (2009).

    Article  CAS  Google Scholar 

  6. Martinon, F., Petrilli, V., Mayor, A., Tardivel, A. & Tschopp, J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 440, 237–241 (2006).

    Article  CAS  Google Scholar 

  7. Duewell, P. et al. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 464, 1357–1361 (2010).

    Article  CAS  Google Scholar 

  8. Masters, S.L. et al. Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1β in type 2 diabetes. Nat. Immunol. 11, 897–904 (2010).

    Article  CAS  Google Scholar 

  9. Halle, A. et al. The NALP3 inflammasome is involved in the innate immune response to amyloid-β. Nat. Immunol. 9, 857–865 (2008).

    Article  CAS  Google Scholar 

  10. Bauernfeind, F. et al. Cutting edge: reactive oxygen species inhibitors block priming, but not activation, of the NLRP3 inflammasome. J. Immunol. 187, 613–617 (2011).

    Article  CAS  Google Scholar 

  11. Bauernfeind, F.G. et al. Cutting edge: NF-κB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J. Immunol. 183, 787–791 (2009).

    Article  CAS  Google Scholar 

  12. Franchi, L., Eigenbrod, T. & Nunez, G. Cutting edge: TNF-α mediates sensitization to ATP and silica via the NLRP3 inflammasome in the absence of microbial stimulation. J. Immunol. 183, 792–796 (2009).

    Article  CAS  Google Scholar 

  13. Dodé, C. et al. New mutations of CIAS1 that are responsible for Muckle-Wells syndrome and familial cold urticaria: a novel mutation underlies both syndromes. Am. J. Hum. Genet. 70, 1498–1506 (2002).

    Article  Google Scholar 

  14. Kim, S. et al. Listeria monocytogenes is sensed by the NLRP3 and AIM2 inflammasome. Eur. J. Immunol. 40, 1545–1551 (2010).

    Article  CAS  Google Scholar 

  15. Lazebnik, Y.A., Kaufmann, S.H., Desnoyers, S., Poirier, G.G. & Earnshaw, W.C. Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature 371, 346–347 (1994).

    Article  CAS  Google Scholar 

  16. Garcia-Calvo, M. et al. Purification and catalytic properties of human caspase family members. Cell Death Differ. 6, 362–369 (1999).

    Article  CAS  Google Scholar 

  17. Takenaka, Y. et al. Two forms of secreted and thermostable luciferases from the marine copepod crustacean, Metridia pacifica. Gene 425, 28–35 (2008).

    Article  CAS  Google Scholar 

  18. Hornung, V. et al. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 458, 514–518 (2009).

    Article  CAS  Google Scholar 

  19. Mahajan, N.P., Harrison-Shostak, D.C., Michaux, J. & Herman, B. Novel mutant green fluorescent protein protease substrates reveal the activation of specific caspases during apoptosis. Chem. Biol. 6, 401–409 (1999).

    Article  CAS  Google Scholar 

  20. Fan, F. et al. Novel genetically encoded biosensors using firefly luciferase. ACS Chem. Biol. 3, 346–351 (2008).

    Article  CAS  Google Scholar 

  21. Demon, D. et al. Caspase substrates: easily caught in deep waters? Trends Biotechnol. 27, 680–688 (2009).

    Article  CAS  Google Scholar 

  22. Timmer, J.C. & Salvesen, G.S. Caspase substrates. Cell Death Differ. 14, 66–72 (2007).

    Article  CAS  Google Scholar 

  23. Kutner, R.H., Zhang, X.Y. & Reiser, J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat. Protoc. 4, 495–505 (2009).

    Article  CAS  Google Scholar 

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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

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  1. Eva Bartok
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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.

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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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