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Molecular & Cellular Proteomics 7:2350-2363, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Targeted Peptidecentric Proteomics Reveals Caspase-7 as a Substrate of the Caspase-1 Inflammasomes ^*,S

Mohamed Lamkanfi^,,¶, Thirumala-Devi Kanneganti^,,||, Petra Van Damme^**,, Tom Vanden Berghe^,¶¶, Isabel Vanoverberghe^,¶¶, Joël Vandekerckhove^**,, Peter Vandenabeele^,¶¶, Kris Gevaert^**, and Gabriel Núñez^,||||

From the Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan 48109, ^** Department of Medical Protein Research, Flanders Institute for Biotechnology, B-9000 Ghent, Belgium, Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium, Molecular Signalling and Cell Death Unit, Department for Molecular Biomedical Research, Flanders Institute for Biotechnology, B-9052 Zwijnaarde, Belgium, and ^¶¶ Molecular Signalling and Cell Death Unit, Department of Molecular Biology, Ghent University, B-9052 Zwijnaarde, Belgium

The aspartate-specific cysteine protease caspase-1 is activated by the inflammasomes and is responsible for the proteolytic maturation of the cytokines IL-1β and IL-18 during infection and inflammation. To discover new caspase-1 substrates, we made use of a proteome-wide gel-free differential peptide sorting methodology that allows unambiguous localization of the processing site in addition to identification of the substrate. Of the 1022 proteins that were identified, 20 were found to be specifically cleaved after Asp in the setup incubated with recombinant caspase-1. Interestingly, caspase-7 emerged as one of the identified caspase-1 substrates. Moreover half of the other identified cleavage events occurred at sites closely resembling the consensus caspase-7 recognition sequence DEVD, suggesting caspase-1-mediated activation of endogenous caspase-7 in this setup. Consistently recombinant caspase-1 cleaved caspase-7 at the canonical activation sites Asp²³ and Asp¹⁹⁸, and recombinant caspase-7 processed a subset of the identified substrates. In vivo, caspase-7 activation was observed in conditions known to induce activation of caspase-1, including Salmonella infection and microbial stimuli combined with ATP. Interestingly Salmonella- and lipopolysaccharide + ATP-induced activation of caspase-7 was abolished in macrophages deficient in caspase-1, the pattern recognition receptors Ipaf and Cryopyrin, and the inflammasome adaptor ASC, demonstrating an upstream role for the caspase-1 inflammasomes in caspase-7 activation in vivo. In contrast, caspase-1 and the inflammasomes were not required for caspase-3 activation. In conclusion, we identified 20 new substrates activated downstream of caspase-1 and validated caspase-1-mediated caspase-7 activation in vitro and in knock-out macrophages. These results demonstrate for the first time the existence of a nucleotide binding and oligomerization domain-like receptor/caspase-1/caspase-7 cascade and the existence of distinct activation mechanisms for caspase-3 and -7 in response to microbial stimuli and bacterial infection.

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