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Inflammatory stimuli regulate caspase substrate profiles.

ABSTRACT: The inflammatory caspases, human caspases-1, -4, and -5, proteolytically modulate diverse physiological outcomes in response to proinflammatory signals. Surprisingly, only a few substrates are known for these enzymes, including other caspases and the interleukin-1 family of cytokines. To more comprehensively characterize inflammatory caspase substrates, we combined an enzymatic N-terminal enrichment method with mass spectrometry-based proteomics to identify newly cleaved proteins. Analysis of THP-1 monocytic cell lysates treated with recombinant purified caspases identified 82 putative caspase-1 substrates, three putative caspase-4 substrates, and no substrates for caspase-5. By contrast, inflammatory caspases activated in THP-1 cells by mimics of gout (monosodium urate), bacterial infection (lipopolysaccharide and ATP), or viral infection (poly(dA.dT)) were found to cleave only 27, 16, and 22 substrates, respectively. Quantitative stable isotope labeling with amino acids in cell culture (SILAC) comparison of these three inflammatory stimuli showed that they induced largely overlapping substrate profiles but different extents of proteolysis. Interestingly, only half of the cleavages found in response to proinflammatory stimuli were contained within our set of 82 in vitro cleavage sites. These data provide the most comprehensive set of caspase-1-cleaved products reported to date and indicate that caspases-4 and -5 have far fewer substrates. Comparisons between the in vitro and in vivo data highlight the importance of localization in regulating inflammatory caspase activity. Finally, our data suggest that inducers of inflammation may subtly alter caspase-1 substrate profiles.

SUBMITTER: Agard NJ

PROVIDER: S-EPMC2871421 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Inflammatory stimuli regulate caspase substrate profiles.

Agard Nicholas J NJ Maltby David D Wells James A JA

Molecular & cellular proteomics : MCP 20100220 5

The inflammatory caspases, human caspases-1, -4, and -5, proteolytically modulate diverse physiological outcomes in response to proinflammatory signals. Surprisingly, only a few substrates are known for these enzymes, including other caspases and the interleukin-1 family of cytokines. To more comprehensively characterize inflammatory caspase substrates, we combined an enzymatic N-terminal enrichment method with mass spectrometry-based proteomics to identify newly cleaved proteins. Analysis of TH ...[more]

PMID: 20173201

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Project description: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.

Project description:BackgroundAcute CNS damage is commonly studied using rat and mouse models, but increasingly, molecular analysis is finding species differences that might affect the ability to translate findings to humans. Microglia can undergo complex molecular and functional changes, often studied by in vitro responses to discrete activating stimuli. There is considerable evidence that pro-inflammatory (M1) activation can exacerbate tissue damage, while anti-inflammatory (M2) states help resolve inflammation and promote tissue repair. However, in assessing potential therapeutic targets for controlling inflammation, it is crucial to determine whether rat and mouse microglia respond the same.MethodsPrimary microglia from Sprague-Dawley rats and C57BL/6 mice were cultured, then stimulated with interferon-γ + tumor necrosis factor-α (I + T; M1 activation), interleukin (IL)-4 (M2a, alternative activation), or IL-10 (M2c, acquired deactivation). To profile their activation responses, NanoString was used to monitor messenger RNA (mRNA) expression of numerous pro- and anti-inflammatory mediators, microglial markers, immunomodulators, and other molecules. Western analysis was used to measure selected proteins. Two potential targets for controlling inflammation-inward- and outward-rectifier K+ channels (Kir2.1, Kv1.3)-were examined (mRNA, currents) and specific channel blockers were applied to determine their contributions to microglial migration in the different activation states.ResultsPro-inflammatory molecules increased after I + T treatment but there were several qualitative and quantitative differences between the species (e.g., iNOS and nitric oxide, COX-2). Several molecules commonly associated with an M2a state differed between species or they were induced in additional activation states (e.g., CD206, ARG1). Resting levels and/or responses of several microglial markers (Iba1, CD11b, CD68) differed with the activation state, species, or both. Transcripts for several Kir2 and Kv1 family members were detected in both species. However, the current amplitudes (mainly Kir2.1 and Kv1.3) depended on activation state and species. Treatment-induced changes in morphology and migratory capacity were similar between the species (migration reduced by I + T, increased by IL-4 or IL-10). In both species, Kir2.1 block reduced migration and Kv1.3 block increased it, regardless of activation state; thus, these channels might affect microglial migration to damage sites.ConclusionsCaution is recommended in generalizing molecular and functional responses of microglia to activating stimuli between species.

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Inflammatory stimuli regulate caspase substrate profiles.

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Inflammatory stimuli regulate caspase substrate profiles.

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