Caspase-4 and caspase-5, members of the ICE/CED-3 family of cysteine proteases, are CrmA-inhibitable proteases

Proteases of the caspase family are implicated in mammalian apoptosis and constitute a protease cascade. We characterized caspase-4 (TX/ICH-2/ICErelII) and caspase-5 (ICErelIII/TY), which are most closely related to caspase-1 (ICE) among the caspase family. Although overexpression of caspase-4 and caspase-5 induced apoptosis, confirming previous observations, this apoptosis was not inhibited by a caspase-1-specific tetrapeptide inhibitor (Ac-YVAD-CHO), suggesting that caspase-4 and caspase-5 have different substrate specificities from caspase-1 and also that caspase-4- and caspase-5-induced apoptosis is not mediated by caspase-1. CrmA, a cowpox virus-derived caspase-1 inhibitor that prevents apoptosis induced by various stimuli, was cleaved by caspase-4 and caspase-5, and inhibited their proteolytic activity as assessed by cleavage of pro-caspase-3 (pro-CPP32/Yama/apopain). Thus, caspase-4 and caspase-5 are CrmA-inhibitable proteases like caspase-1 and might be involved in apoptosis.     

Caspase-7 mediates caspase-1-induced apoptosis independently of Bid

Inflammasomes are innate immune mechanisms that activate caspase-1 in response to a variety of stimuli, including Salmonella infection. Active caspase-1 has a potential to induce two different types of cell death, depending on the expression of the pyroptosis mediator gasdermin D (GSDMD); following caspase-1 activation, GSDMD-sufficient and GSDMD-null/low cells undergo pyroptosis and apoptosis, respectively. Although Bid, a caspase-1 substrate, plays a critical role in caspase-1 induction of apoptosis in GSDMD-null/low cells, an additional mechanism that mediates this cell death independently of Bid has also been suggested. This study investigated the Bid-independent pathway of caspase-1-induced apoptosis. Caspase-1 has been reported to process caspase-6 and caspase-7. Silencing of caspase-7, but not caspase-6, significantly reduced the activation of caspase-3 induced by caspase-1, which was activated by chemical dimerization, in GSDMD/Bid-deficient cells. CRISPR/Cas9-mediated depletion of caspase-7 had the same effect on the caspase-3 activation. Moreover, in the absence of GSDMD and Bid, caspase-7 depletion reduced apoptosis induced by caspase-1 activation. Caspase-7 was activated following caspase-1 activation independently of caspase-3, suggesting that caspase-7 acts downstream of caspase-1 and upstream of caspase-3. Salmonella induced the activation of caspase-3 in GSDMD-deficient macrophages, which relied partly on Bid and largely on caspase-1. The caspase-3 activation and apoptotic morphological changes seen in Salmonella-infected GSDMD/Bid-deficient macrophages were attenuated by caspase-7 knockdown. These results suggest that in addition to Bid, caspase-7 can also mediate caspase-1-induced apoptosis and provide mechanistic insights into inflammasome-associated cell death that is one major effector mechanism of inflammasomes.     

Resurrection of an ancient inflammatory locus reveals switch to caspase-1 specificity on a caspase-4 scaffold

Pyroptosis is a mechanism of inflammatory cell death mediated by the activation of the prolytic protein gasdermin D by caspase-1, caspase-4, and caspase-5 in human, and caspase-1 and caspase-11 in mouse. In addition, caspase-1 amplifies inflammation by proteolytic activation of cytokine interleukin-1β (IL-1β). Modern mammals of the order Carnivora lack the caspase-1 catalytic domain but express an unusual version of caspase-4 that can activate both gasdermin D and IL-1β. Seeking to understand the evolutionary origin of this caspase, we utilized the large amount of data available in public databases to perform ancestral sequence reconstruction of an inflammatory caspase of a Carnivora ancestor. We expressed the catalytic domain of this putative ancestor in Escherichia coli, purified it, and compared its substrate specificity on synthetic and protein substrates to extant caspases. We demonstrated that it activates gasdermin D but has reduced ability to activate IL-1β. Our reconstruction suggests that caspase-1 was lost in a Carnivora ancestor, perhaps upon a selective pressure for which the generation of biologically active IL-1β by caspase-1 was detrimental. We speculate that later, a Carnivora encountered selective pressures that required the production of IL-1β, and caspase-4 subsequently gained this activity. This hypothesis would explain why extant Carnivora possess an inflammatory caspase with caspase-1 catalytic function placed on a caspase-4 scaffold.     

Caspase-1 activates nuclear factor of the kappa-enhancer in B cells independently of its enzymatic activity

The proteolytic activity of caspases is involved in apoptosis and inflammation. In this regard, caspase-1 is required for pro-interleukin (IL)-1beta and pro-IL-18 maturation. We report here on a novel function of caspase-1 as an activator of nuclear factor of the kappa-enhancer in B-cells (NF-kappaB) and p38 mitogen-activated protein kinase (MAPK). This function is not shared by the murine caspase-1 homologues caspase-11 and -12. In contrast to pro-IL-1beta maturation, caspase-1-induced NF-kappaB activation is not inhibited by the virus-derived caspase-1 inhibitor cytokine response modifier A and is equally induced by the enzymatically inactive caspase-1 C285A mutant. Although the general NF-kappaB-inhibiting protein A20 inhibits caspase-1-derived activation of NF-kappaB, dominant-negative forms of TRAF2 and RIP1 have no effect. We demonstrate that caspase-1 interacts with RIP2 and that dominant-negative forms of RIP2 and IkappaB kinase complex-beta inhibit caspase-1-mediated NF-kappaB activation. Structure-function analysis shows that the caspase recruitment domain of caspase-1 mediates the activation of NF-kappaB and p38 MAPK. These data demonstrate that caspase-1 contributes to inflammation by two distinct pathways: proteolysis of pro-IL-1beta, and RIP2-dependent activation of NF-kappaB and p38 MAPK mediated by the caspase recruitment domain.     

Flightless-I regulates proinflammatory caspases by selectively modulating intracellular localization and caspase activity

Caspase-1 and caspase-11 are proinflammatory caspases that regulate cytokine production and leukocyte migration during pathogen infection. In an attempt to identify new intracellular regulators of caspase-11, we found that Flightless-I, a member of the gelsolin superfamily of actin-remodeling proteins, interacts and regulates both caspase-11 and caspase-1. Flightless-I targets caspase-11 to the Triton X-100-insoluble cytoskeleton fraction and the cell leading edge. In addition, Flightless-I inhibits caspase-1 activation and caspase-1-mediated interleukine-1beta (IL-1beta) maturation. The physiological relevance of these findings is supported by the opposing effects of Flightless-I overexpression and knockdown on caspase-1 activity and IL-1beta maturation. Our results suggest that Flightless-I may be a bona fide caspase-1 inhibitor that acts through a mechanism similar to that of cytokine response modifier A, a potent caspase-1 inhibitor from the cowpox virus. Our study provides a new mechanism controlling the localization and activation of proinflammatory caspases.     

Monocyte Caspase-1 Is Released in a Stable,Active High Molecular Weight Complex Distinct from the Unstable Cell Lysate-Activated Caspase-1

Mononuclear phagocytes utilize caspase-1 activation as a means to respond to danger signals. Although caspase-1 was discovered using highly concentrated cell extracts that spontaneously activate caspase-1, it is now clear that in live cell models caspase-1 activation occurs in the process of its cellular release and is not an intracellular event. Therefore, we compared the characteristics of caspase-1 activation in the cell lysate model to that of caspase-1 that is released in response to exogenous inflammasome activation. Whereas both models generated active caspase-1, the cell-lysate induced caspase-1 required highly concentrated cell lysates and had a short half-life (~15 min) whereas, the activation induced released caspase-1 required 2–3 log fold fewer cells and was stable for greater than 12 h. Both forms were able to cleave proIL-1beta but unexpectedly, the released activity was unable to be immunodepleted by caspase-1 antibodies. Size exclusion chromatography identified two antigenic forms of p20 caspase-1 in the activation induced released caspase-1: one at the predicted size of tetrameric, p20/p10 caspase-1 and the other at >200 kDa. However, only the high molecular weight form had stable functional activity. These results suggest that released caspase-1 exists in a unique complex that is functionally stable and protected from immunodepletion whereas cell-extract generated active caspase-1 is rapidly inhibited in the cytosolic milieu.     

Targeted peptidecentric proteomics reveals caspase-7 as a substrate of the caspase-1 inflammasomes

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.     

Caspase-1 promiscuity is counterbalanced by rapid inactivation of processed enzyme

Members of the caspase family of cysteine proteases coordinate the highly disparate processes of apoptosis and inflammation. However, although hundreds of substrates for the apoptosis effector caspases (caspase-3 and caspase-7) have been identified, only two confirmed substrates for the key inflammatory protease (caspase-1) are known. Whether this reflects intrinsic differences in the substrate specificity of inflammatory versus apoptotic caspases or their relative abundance in vivo is unknown. To address this issue, we have compared the specificity of caspases-1, -3, and -7 toward peptide and protein substrates. Contrary to expectation, caspase-1 displayed concentration-dependent promiscuity toward a variety of substrates, suggesting that caspase-1 specificity is maintained by restricting its abundance. Although endogenous concentrations of caspase-1 were found to be similar to caspase-3, processed caspase-1 was found to be much more labile, with a half-life of ~9 min. This contrasted sharply with the active forms of caspase-3 and caspase-7, which exhibited half-lives of 8 and 11 h, respectively. We propose that the high degree of substrate specificity displayed by caspase-1 is maintained through rapid spontaneous inactivation of this protease.     

TNF-alpha induces two distinct caspase-8 activation pathways

The inflammatory response of mammalian cells to TNF-alpha can be switched to apoptosis either by cotreatment with a protein synthesis inhibitor, cycloheximide, or Smac mimetic, a small molecule mimic of Smac/Diablo protein. Cycloheximide promotes caspase-8 activation by eliminating endogenous caspase-8 inhibitor, c-FLIP, while Smac mimetic does so by triggering autodegradation of cIAP1 and cIAP2 (cIAP1/2), leading to the release of receptor interacting protein kinase (RIPK1) from the activated TNF receptor complex to form a caspase-8-activating complex consisting of RIPK1, FADD, and caspase-8. This process also requires the action of CYLD, a RIPK1 K63 deubiquitinating enzyme. RIPK1 is critical for caspase-8 activation-induced by Smac mimetic but dispensable for that triggered by cycloheximide. Moreover, Smac mimetic-induced caspase-8 activation is not blocked by endogenous c-FLIP. These findings revealed that TNF-alpha is able to induce apoptosis via two distinct caspase-8 activation pathways that are differentially regulated by cIAP1/2 and c-FLIP.     

Substrate and Inhibitor-induced Dimerization and Cooperativity in Caspase-1 but Not Caspase-3

Caspases are intracellular cysteine-class proteases with aspartate specificity that is critical for driving processes as diverse as the innate immune response and apoptosis, exemplified by caspase-1 and caspase-3, respectively. Interestingly, caspase-1 cleaves far fewer cellular substrates than caspase-3 and also shows strong positive cooperativity between the two active sites of the homodimer, unlike caspase-3. Biophysical and kinetic studies here present a molecular basis for this difference. Analytical ultracentrifugation experiments show that mature caspase-1 exists predominantly as a monomer under physiological concentrations that undergoes dimerization in the presence of substrate; specifically, substrate binding shifts the K_D for dimerization by 20-fold. We have created a hemi-active site-labeled dimer of caspase-1, where one site is blocked with the covalent active site inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. This hemi-labeled enzyme is about 9-fold more active than the apo-dimer of caspase-1. These studies suggest that substrate not only drives dimerization but also, once bound to one site in the dimer, promotes an active conformation in the other monomer. Steady-state kinetic analysis and modeling independently support this model, where binding of one substrate molecule not only increases substrate binding in preformed dimers but also drives the formation of heterodimers. Thus, the cooperativity in caspase-1 is driven both by substrate-induced dimerization as well as substrate-induced activation. Substrate-induced dimerization and activation seen in caspase-1 and not in caspase-3 may reflect their biological roles. Whereas caspase-1 cleaves a dramatically smaller number of cellular substrates that need to be concentrated near inflammasomes, caspase-3 is a constitutively active dimer that cleaves many more substrates located diffusely throughout the cell.     

Alpha 1-Antitrypsin Does Not Inhibit Human Monocyte Caspase-1

BackgroundAlpha 1-antitrypsin (A1AT) is a 52 kDa serine protease inhibitor produced largely by hepatocytes but also by mononuclear phagocytes. A1AT chiefly inhibits neutrophil elastase and proteinase-3 but has also been reported to have immune modulatory functions including the ability to inhibit caspases. Its clinical availability for infusion suggests that A1AT therapy might modulate caspase related inflammation. Here we tested the ability of A1AT to modulate caspase-1 function in human mononuclear phagocytes.MethodsPurified plasma derived A1AT was added to active caspase-1 in a cell-free system (THP-1 lysates) as well as added exogenously to cell-culture models and human whole blood models of caspase-1 activation. Functional caspase-1 activity was quantified by the cleavage of the caspase-1 specific fluorogenic tetrapeptide substrate (WEHD-afc) and the release of processed IL-18 and IL-1β.ResultsTHP-1 cell lysates generated spontaneous activation of caspase-1 both by WEHD-afc cleavage and the generation of p20 caspase-1. A1AT added to this cell free system was unable to inhibit caspase-1 activity. Release of processed IL-18 by THP-1 cells was also unaffected by the addition of exogenous A1AT prior to stimulation with LPS/ATP, a standard caspase-1 activating signal. Importantly, the A1AT exhibited potent neutrophil elastase inhibitory capacity. Furthermore, A1AT complexed to NE (and hence conformationally modified) also did not affect THP-1 cell caspase-1 activation. Finally, exogenous A1AT did not inhibit the ability of human whole blood samples to process and release IL-1β.ConclusionsA1AT does not inhibit human monocyte caspase-1.     

Execution of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis by the mitochondria-dependent caspase activation

ASK1 activates JNK and p38 mitogen-activated protein kinases and constitutes a pivotal signaling pathway in cytokine- and stress-induced apoptosis. However, little is known about the mechanism of how ASK1 executes apoptosis. Here we investigated the roles of caspases and mitochondria in ASK1-induced apoptosis. We found that benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), a broad-spectrum caspase inhibitor, mostly inhibited ASK1-induced cell death, suggesting that caspases are required for ASK1-induced apoptosis. Overexpression of ASK1DeltaN, a constitutively active mutant of ASK1, induced cytochrome c release from mitochondria and activation of caspase-9 and caspase-3 but not of caspase-8-like proteases. Consistently, caspase-8-deficient (Casp8 (-/-)) cells were sensitive to ASK1-induced caspase-3 activation and apoptosis, suggesting that caspase-8 is dispensable for ASK1-induced apoptosis, whereas ASK1 failed to activate caspase-3 in caspase-9-dificient (Casp9 (-/-)) cells. Moreover, mitochondrial cytochrome c release, which was not inhibited by zVAD-fmk, preceded the onset of caspase-3 activation and cell death induced by ASK1. ASK1 thus appears to execute apoptosis mainly by the mitochondria-dependent caspase activation.     

Caspase-4 is required for activation of inflammasomes

IL-1β and IL-18 are crucial regulators of inflammation and immunity. Both cytokines are initially expressed as inactive precursors, which require processing by the protease caspase-1 for biological activity. Caspase-1 itself is activated in different innate immune complexes called inflammasomes. In addition, caspase-1 activity regulates unconventional protein secretion of many other proteins involved in inflammation and repair. Human caspase-4 is a poorly characterized member of the caspase family, which is supposed to be involved in endoplasmic reticulum stress-induced apoptosis. However, its gene is located on the same locus as the caspase-1 gene, which raises the possibility that caspase-4 plays a role in inflammation. In this study, we show that caspase-4 expression is required for UVB-induced activation of proIL-1β and for unconventional protein secretion by skin-derived keratinocytes. These processes require expression of the nucleotide-binding domain leucine-rich repeat containing, Pyrin domain containing-3 inflammasome, and caspase-4 physically interacts with its central molecule caspase-1. As the active site of caspase-4 is required for activation of caspase-1, the latter most likely represents a substrate of caspase-4. Caspase-4 expression is also essential for efficient nucleotide-binding domain leucine-rich repeat containing, Pyrin domain containing-3 and for absent in melanoma 2 inflammasome-dependent proIL-1β activation in macrophages. These results demonstrate an important role of caspase-4 in inflammation and innate immunity through activation of caspase-1. Therefore, caspase-4 represents a novel target for the treatment of (auto)inflammatory diseases.     

Distinct downstream pathways of caspase-11 in regulating apoptosis and cytokine maturation during septic shock response

Caspase-11 is an essential mediator of septic shock response and caspase-11-deficient mice are resistant to LPS-induced shock. Here we report that LPS-induced caspase-11 regulates lymphocyte apoptosis by activating both caspase-3 and caspase-7. The activation of caspase-11 preceded that of caspase-1 and caspases-3/-7, and in the absence of caspase-11, the activation of caspases-3/-7 was significantly reduced. The early activation of caspases-3/-7 by caspase-11 was not affected by blocking of caspase-1 activity and IL-1beta release, implying that caspase-11 activates caspases-3/-7 independently of caspase-1 activation. Furthermore, we show that caspase-11-mediated apoptosis under septic condition is Bid-independent. Our work suggests that the human homologue of caspase-11 may be an effective therapeutic target for treatment of septic shock.     

Caspase-1 Is an Apical Caspase Leading to Caspase-3 Cleavage in the AIM2 Inflammasome Response,Independent of Caspase-8

Canonical inflammasomes are multiprotein complexes that can activate both caspase-1 and caspase-8. Caspase-1 drives rapid lysis of cells by pyroptosis and maturation of interleukin (IL)-1β and IL-18. In caspase-1-deficient cells, inflammasome formation still leads to caspase-3 activation and slower apoptotic death, dependent on caspase-8 as an apical caspase. A role for caspase-8 directly upstream of caspase-1 has also been suggested, but here we show that caspase-8-deficient macrophages have no defect in AIM2 inflammasome-mediated caspase-1 activation, pyroptosis, and IL-1β cleavage. In investigating the inflammasome-induced apoptotic pathway, we previously demonstrated that activated caspase-8 is essential for caspase-3 cleavage and apoptosis in caspase-1-deficient cells. However, here we found that AIM2 inflammasome-initiated caspase-3 cleavage was maintained in Ripk3^?/? Casp8^?/? macrophages. Gene knockdown showed that caspase-1 was required for the caspase-3 cleavage. Thus inflammasomes activate a network of caspases that can promote both pyroptotic and apoptotic cell death. In cells where rapid pyroptosis is blocked, delayed inflammasome-dependent cell death could still occur due to both caspase-1- and caspase-8-dependent apoptosis. Initiation of redundant cell death pathways is likely to be a strategy for coping with pathogen interference in death processes.     

AIM2/ASC triggers caspase-8-dependent apoptosis in Francisella-infected caspase-1-deficient macrophages

The inflammasome is a signalling platform leading to caspase-1 activation. Caspase-1 causes pyroptosis, a necrotic-like cell death. AIM2 is an inflammasome sensor for cytosolic DNA. The adaptor molecule ASC mediates AIM2-dependent caspase-1 activation. To date, no function besides caspase-1 activation has been ascribed to the AIM2/ASC complex. Here, by comparing the effect of gene inactivation at different levels of the inflammasome pathway, we uncovered a novel cell death pathway activated in an AIM2/ASC-dependent manner. Francisella tularensis, the agent of tularaemia, triggers AIM2/ASC-dependent caspase-3-mediated apoptosis in caspase-1-deficient macrophages. We further show that AIM2 engagement leads to ASC-dependent, caspase-1-independent activation of caspase-8 and caspase-9 and that caspase-1-independent death is reverted upon caspase-8 inhibition. Caspase-8 interacts with ASC and active caspase-8 specifically colocalizes with the AIM2/ASC speck thus identifying the AIM2/ASC complex as a novel caspase-8 activation platform. Furthermore, we demonstrate that caspase-1-independent apoptosis requires the activation of caspase-9 and of the intrinsic pathway in a typical type II cell manner. Finally, we identify the AIM2/ASC-dependent caspase-1-independent pathway as an innate immune mechanism able to restrict bacterial replication in vitro and control IFN-γ levels in vivo in Casp1(KO) mice. This work underscores the crosstalk between inflammasome components and the apoptotic machinery and highlights the versatility of the pathway, which can switch from pyroptosis to apoptosis.