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Chemical genetics reveals a kinase-independent role for protein kinase R in pyroptosis

Nature Chemical Biology volume 9, pages 398–405 (2013)Cite this article

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An Erratum to this article was published on 17 June 2013

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Abstract

Formation of the inflammasome, a scaffolding complex that activates caspase-1, is important in numerous diseases. Pyroptotic cell death induced by anthrax lethal toxin (LT) is a model for inflammasome-mediated caspase-1 activation. We discovered 7-desacetoxy-6,7-dehydrogedunin (7DG) in a phenotypic screen as a small molecule that protects macrophages from LT-induced death. Using chemical proteomics, we identified protein kinase R (PKR) as the target of 7DG and show that RNAi knockdown of PKR phenocopies treatment with 7DG. Further, we show that PKR's role in ASC assembly and caspase-1 activation induced by several different inflammasome stimuli is independent of PKR's kinase activity, demonstrating that PKR has a previously uncharacterized role in caspase-1 activation and pyroptosis that is distinct from its reported kinase-dependent roles in apoptosis and inflammasome formation in lipopolysaccharide-primed cells. Remarkably, PKR has different roles in two distinct cell death pathways and has a broad role in inflammasome function relevant in other diseases.

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Figure 1: Chemical HTS identifies 7DG as an inhibitor of LT-induced cell death.
Figure 2: 7DG does not directly inhibit proteasome or caspase-1 activity, but does prevent the activation of caspase-1 by LT.
Figure 3: 7DG pulldown identifies PKR as an interacting protein important for LT-induced cell death.
Figure 4: PKR knockdown phenocopies treatment with 7DG.
Figure 5: 7DG inhibits a kinase-independent function of PKR.
Figure 6: Pyroptosis versus apoptosis models.

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  • 21 May 2013

    In the print version of this article, two labels were omitted from the graphical abstract on the Table of Contents page. The error has been corrected in the HTML and PDF versions of the Table of Contents.

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Acknowledgements

We would like to thank J. Collier, S. Chiang, B. Kraybill, L. Whitesell, S. Lindquist, S. Santagata, S. Patel-Hett, R. King, N. Dimova, D. Patel and R. Avraham-Atzil for helpful discussions; V. Gelev (Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School) for PKR constructs and for a valiant attempt to solve the structure of PKR with 7DG; A. Clatworthy for experimental help and discussions; A. Barker for help with production and purification of protective antigen and lethal factor; Y. Wang for help with chemistry; J. Lee for technical assistance; H. Aktas for PKR constructs (Laboratory for Translational Research, Harvard Medical School); A. Barczak for assistance with C57Bl/6 macrophages and discussions; and the Broad Institute chemical screening platform for technical help. We thank E. Fava, P. Denner and A. Kitanovic from the laboratory automation laboratory at the German Center for Neurodegenerative Diseases for the analysis of the ASC speck assays. E.C.H. was funded by an US National Institutes of Health (NIH) National Research Service Award fellowship F32AI084323. This work was supported in part by NIH U54 AI057159 to the New England Center of Excellence–Biodefense and Emerging Infectious Diseases (to D.T.H.).

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  1. Erik C Hett

    Present address: Present address: Chemical Biology Group, Worldwide Medicinal Chemistry, Pfizer, Cambridge, Massachusetts, USA.,

Authors and Affiliations

  1. Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA

    Erik C Hett, Louise H Slater, Kevin G Mark, Tomohiko Kawate & Deborah T Hung

  2. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Erik C Hett, Louise H Slater, Kevin G Mark, Tomohiko Kawate & Deborah T Hung

  3. Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA

    Erik C Hett, Louise H Slater, Kevin G Mark, Tomohiko Kawate & Deborah T Hung

  4. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA

    Erik C Hett, Louise H Slater, Kevin G Mark, Tomohiko Kawate & Deborah T Hung

  5. Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Brian G Monks & Eicke Latz

  6. Institute of Innate Immunity, University of Bonn, Bonn, Germany

    Brian G Monks, Andrea Stutz & Eicke Latz

  7. German Center of Neurodegenerative Diseases (DZNE), Bonn, Germany

    Eicke Latz

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Contributions

E.C.H. prepared the manuscript, designed and conducted experiments; L.H.S., K.G.M., B.G.M., A.S. and E.L. designed and conducted experiments; T.K. designed and synthesized compounds; and D.T.H. prepared the manuscript and designed experiments.

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Correspondence to Erik C Hett or Deborah T Hung.

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E.C.H. is currently employed by Pfizer Inc., which develops therapies for immunological diseases.

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Hett, E., Slater, L., Mark, K. et al. Chemical genetics reveals a kinase-independent role for protein kinase R in pyroptosis. Nat Chem Biol 9, 398–405 (2013). https://doi.org/10.1038/nchembio.1236

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