The
aspartate-specific
cysteine protease caspase-1 is activated by the
inflammasomes and is responsible for the proteolytic maturation of the
cytokines IL-1 beta 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 Asp(23) and Asp(198), 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.