title: Targeted peptidecentric proteomics reveals caspase-7 as a substrate of the caspase-1 inflammasomes.
abstract: 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.
authors.affiliation: Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Lamkanfi
authors.forename: Thirumala-Devi
authors.initials: TD
authors.lastname: Kanneganti
authors.forename: Petra
authors.initials: P
authors.lastname: Van Damme
authors.forename: Tom
authors.initials: T
authors.lastname: Vanden Berghe
authors.forename: Isabel
authors.initials: I
authors.lastname: Vanoverberghe
authors.forename: Joël
authors.initials: J
authors.lastname: Vandekerckhove
authors.forename: Peter
authors.initials: P
authors.lastname: Vandenabeele
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
authors.forename: Gabriel
authors.initials: G
authors.lastname: Núñez
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D020170:Caspase 1 / Q000737:chemistry / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000172:deficiency / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D016207:Cytokines / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D007249:Inflammation / Q000201:enzymology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000382:microbiology; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009550:Nigericin / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D020543:Proteome / Q000378:metabolism; D040901:Proteomics; D012475:Salmonella / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D016207:Cytokines; D008070:Lipopolysaccharides; D010455:Peptides; D020543:Proteome; D001224:Aspartic Acid; D000255:Adenosine Triphosphate; D053148:Caspase 3; D053179:Caspase 7; D020170:Caspase 1; D009550:Nigericin