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1.  Caspase-7: a protease involved in apoptosis and inflammation 
Caspase-7 was considered to be redundant with caspase-3 because these related cystein proteases share an optimal peptide recognition sequence and have several endogenous protein substrates in common. In addition, both caspases are proteolytically activated by the initiator caspases-8 and -9 during death receptor- and DNA-damage-induced apoptosis, respectively. However, a growing body of biochemical and physiological data indicate that caspase-7 also differs in significant ways from caspase-3. For instance, several substrates are specifically cleaved by caspase-7, but not caspase-3. Moreover, caspase-7 activation requires caspase-1 inflammasomes under inflammatory conditions, while caspase-3 processing proceeds independently of caspase-1. Finally, caspase-7 deficient mice are resistant to endotoxemia, whereas caspase-3 knockout mice are susceptible. These findings suggest that specifically interfering with caspase-7 activation may hold therapeutic value for the treatment of cancer and inflammatory ailments.
doi:10.1016/j.biocel.2009.09.013
PMCID: PMC2787741  PMID: 19782763
caspase-7; caspase-3; apoptosis; inflammation
2.  Effector Caspases and Leukemia 
Caspases, a family of aspartate-specific cysteine proteases, play a major role in apoptosis and a variety of physiological and pathological processes. Fourteen mammalian caspases have been identified and can be divided into two groups: inflammatory caspases and apoptotic caspases. Based on the structure and function, the apoptotic caspases are further grouped into initiator/apical caspases (caspase-2, -8, -9, and -10) and effector/executioner caspases (caspase-3, -6, and -7). In this paper, we discuss what we have learned about the role of individual effector caspase in mediating both apoptotic and nonapoptotic events, with special emphasis on leukemia-specific oncoproteins in relation to effector caspases.
doi:10.1155/2011/738301
PMCID: PMC3103908  PMID: 21647292
3.  Caspases: evolutionary aspects of their functions in vertebrates 
Journal of Fish Biology  2009;74(4):727-753.
Caspases (cysteine-dependent aspartyl-specific protease) belong to a family of cysteine proteases that mediate proteolytic events indispensable for biological phenomena such as cell death and inflammation. The first caspase was identified as an executioner of apoptotic cell death in the worm Caenorhabditis elegans. Additionally, a large number of caspases have been identified in various animals from sponges to vertebrates. Caspases are thought to play a pivotal role in apoptosis as an evolutionarily conserved function; however, the number of caspases that can be identified is distinct for each species. This indicates that species-specific functions or diversification of physiological roles has been cultivated through caspase evolution. Furthermore, recent studies suggest that caspases are also involved in inflammation and cellular differentiation in mammals. This review highlights vertebrate caspases in their universal and divergent functions and provides insight into the physiological roles of these molecules in animals.
doi:10.1111/j.1095-8649.2009.02184.x
PMCID: PMC2779465  PMID: 20735596
apoptosis; caspase; evolution; inflammation; terminal differentiation; vertebrates
4.  Caspase-1 Promiscuity Is Counterbalanced by Rapid Inactivation of Processed Enzyme* 
The Journal of Biological Chemistry  2011;286(37):32513-32524.
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.
doi:10.1074/jbc.M111.225862
PMCID: PMC3173193  PMID: 21757759
Apoptosis; Caspase; Enzyme Inactivation; Inflammation; Protease; Caspase Substrates; Caspase-1; Cell Death
5.  Comparison of activated caspase detection methods in the gentamicin-treated chick cochlea 
Hearing research  2008;240(1-2):1-11.
Aminoglycoside antibiotics induce caspase-dependent apoptotic death in cochlear hair cells. Apoptosis, a regulated form of cell death, can be induced by many stressors, which activate signaling pathways that result in the controlled dismantling of the affected cell. The caspase family of proteases is activated in the apoptotic signaling pathway and is responsible for cellular destruction. The initiator caspase-9 and the effector caspase-3 are both activated in chick cochlear hair cells following aminoglycoside exposure. We have analyzed caspase activation in the avian cochlea during gentamicin-induced hair cell death to compare two different methods of caspase detection: caspase antibodies and CaspaTag kits. Caspase antibodies bind to the cleaved activated form of caspase-9 or caspase-3 in specific locations in fixed tissue. CaspaTag is a fluorescent inhibitor that binds to a reactive cysteine residue on the large subunit of the caspase heterodimer in unfixed tissue.
To induce cochlear hair cell loss, 1-2 week-old chickens received a single injection of gentamicin (300 mg/kg). Chicks were sacrificed 24, 30, 42, 48, 72, or 96 h after injection. Cochleae were dissected and labeled for activated caspase-9 or caspase-3 using either caspase-directed antibodies or CaspaTag kits. Ears were co-labeled with either phalloidin or myosin VI to visualize hair cells and to determine the progression of cochlear damage. The timing of caspase activation was similar for both assays; however, caspase-9 and caspase-3 antibodies labeled only those cells currently undergoing apoptotic cell death. Conversely, CaspaTag-labeled all the cells that have undergone apoptotic cell death and ejection from the sensory epithelium, in addition to those that are currently in the cell death process. This makes CaspaTag ideal for showing an overall pattern or level of cell death over a period of time, while caspase antibodies provide a snapshot of cell death at a specific time point.
doi:10.1016/j.heares.2008.03.003
PMCID: PMC2430666  PMID: 18487027
Apoptosis; Caspase; Avian; Aminoglycoside; Cochlea; Gentamicin; Hair cells
6.  Dual Role of Caspase-11 in Mediating Activation of Caspase-1 and Caspase-3 under Pathological Conditions 
The Journal of Cell Biology  2000;149(3):613-622.
Caspase-11, a member of the murine caspase family, has been shown to be an upstream activator of caspase-1 in regulating cytokine maturation. We demonstrate here that in addition to its defect in cytokine maturation, caspase-11–deficient mice have a reduced number of apoptotic cells and a defect in caspase-3 activation after middle cerebral artery occlusion (MCAO), a mouse model of stroke. Recombinant procaspase-11 can autoprocess itself in vitro. Purified active recombinant caspase-11 cleaves and activates procaspase-3 very efficiently. Using a positional scanning combinatorial library method, we found that the optimal cleavage site of caspase-11 was (I/L/V/P)EHD, similar to that of upstream caspases such as caspase-8 and -9. Our results suggest that caspase-11 is a critical initiator caspase responsible for the activation of caspase-3, as well as caspase-1 under certain pathological conditions.
PMCID: PMC2174843  PMID: 10791975
caspase-11; initiator caspase; stroke; middle cerebral artery occlusion; apoptosis
7.  A caspase-like decoy molecule enhances the activity of a paralogous caspase in the yellow fever mosquito, Aedes aegypti 
Caspases are cysteine proteases that play critical roles in apoptosis and other key cellular processes. A mechanism of caspase regulation that has been described in mammals and nematodes involves caspase-like decoy molecules, enzymatically inactive caspase homologs that have arisen by gene duplication and acquired the ability to regulate other caspases. Caspase-like decoy molecules are not found in Drosophila melanogaster, raising the question of whether this type of caspase regulation exists in insects. Phylogenomic analysis of caspase genes from twelve Drosophila and three mosquito species revealed several examples of duplicated caspase homologs lacking critical catalytic residues, making them candidate caspase-like decoy molecules. One of these, CASPS18 from the mosquito Aedes aegypti, is a homolog of the D. melanogaster caspase Decay and contains substitutions in two critical amino acid positions, including the catalytic cysteine residue. As expected, CASPS18 lacked caspase activity, but co-expression of CASPS18 with a paralogous caspase, CASPS19, in mosquito cells or co-incubation of CASPS18 and CASPS19 recombinant proteins resulted in greatly enhanced CASPS19 activity. The discovery of potential caspase-like decoy molecules in several insect species opens new avenues for investigating caspase regulation in insects, particularly in disease vectors such as mosquitoes.
doi:10.1016/j.ibmb.2010.04.011
PMCID: PMC2902568  PMID: 20417712
apoptosis; Drosophila; insect; phylogenetic analysis
8.  Regulation of the Drosophila Apoptosome through Feedback Inhibition 
Nature cell biology  2008;10(12):1440-1446.
Apoptosis is induced by caspases, which are members of the cysteine protease family 1. Caspases are synthesized as inactive zymogens and initiator caspases first gain activity by associating with an oligomeric complex of their adaptor proteins, such as the apoptosome 2,3. Activated initiator caspases subsequently cleave and activate effector caspases. While such a proteolytic cascade would predict that a small number of active caspases could irreversibly amplify caspase activity and trigger apoptosis, many cells can maintain moderate levels of caspase activity to perform non-apoptotic roles in cellular differentiation, shape change and migration 4. Here we show that the Drosophila apoptosome engages in a feedback inhibitory loop, thereby moderating its activation level in vivo. Specifically, the adaptor protein Apaf-1 lowers the level of its associated initiator caspase, Dronc, without triggering apoptosis. Conversely, Dronc lowers Apaf-1 protein levels. This mutual suppression depends upon Dronc’s catalytic site and a caspase cleavage site within Apaf-1. Moreover, the Drosophila Inhibitor of Apoptosis Protein 1 (Diap1) is required for this process. We speculate that this feedback inhibition allows cells to regulate the degree of caspase activation for apoptotic and non-apoptotic purposes.
doi:10.1038/ncb1803
PMCID: PMC2617731  PMID: 19011620
9.  Individual caspase-10 isoforms play distinct and opposing roles in the initiation of death receptor-mediated tumour cell apoptosis 
Cell Death & Disease  2011;2(3):e125-.
The cysteine protease caspase-8 is an essential executioner of the death receptor (DR) apoptotic pathway. The physiological function of its homologue caspase-10 remains poorly understood, and the ability of caspase-10 to substitute for caspase-8 in the DR apoptotic pathway is still controversial. Here, we analysed the particular contribution of caspase-10 isoforms to DR-mediated apoptosis in neuroblastoma (NB) cells characterised by their resistance to DR signalling. Silencing of caspase-8 in tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive NB cells resulted in complete resistance to TRAIL, which could be reverted by overexpression of caspase-10A or -10D. Overexpression experiments in various caspase-8-expressing tumour cells also demonstrated that caspase-10A and -10D isoforms strongly increased TRAIL and FasL sensitivity, whereas caspase-10B or -10G had no effect or were weakly anti-apoptotic. Further investigations revealed that the unique C-terminal end of caspase-10B was responsible for its degradation by the ubiquitin–proteasome pathway and for its lack of pro-apoptotic activity compared with caspase-10A and -10D. These data highlight in several tumour cell types, a differential pro- or anti-apoptotic role for the distinct caspase-10 isoforms in DR signalling, which may be relevant for fine tuning of apoptosis initiation.
doi:10.1038/cddis.2011.8
PMCID: PMC3101821  PMID: 21368896
caspase-10; caspase-8; apoptosis; TRAIL; neuroblastoma
10.  Caspase-8 as a Therapeutic Target in Cancer 
Cancer letters  2010;332(2):133-140.
Caspase-8 is an apical caspase which initiates programmed cell death following death receptor ligation. This central role in apoptosis has prompted significant clinical interest in regulating caspase-8 expression and proteolytic activity. However, caspase-8 has also been found to play a number of non-apoptotic roles in cells, such as promoting activation NFκB signaling, regulating autophagy and altering endosomal trafficking, and enhancing cellular adhesion and migration. Therefore, depending upon the specific cellular context, caspase-8 may either potentiate or suppress tumor malignancy. Accordingly, a marked heterogeneity exists in the expression patterns of caspase-8 among different tumor types. Therapeutics have been developed which can increase caspase-8 expression, yet it remains unclear whether this approach will be beneficial in all cases. Care is warranted, and the role of caspase-8 should be addressed on a case by case basis.
doi:10.1016/j.canlet.2010.07.022
PMCID: PMC3049203  PMID: 20817393
11.  Both the Caspase CSP-1 and a Caspase-Independent Pathway Promote Programmed Cell Death in Parallel to the Canonical Pathway for Apoptosis in Caenorhabditis elegans 
PLoS Genetics  2013;9(3):e1003341.
Caspases are cysteine proteases that can drive apoptosis in metazoans and have critical functions in the elimination of cells during development, the maintenance of tissue homeostasis, and responses to cellular damage. Although a growing body of research suggests that programmed cell death can occur in the absence of caspases, mammalian studies of caspase-independent apoptosis are confounded by the existence of at least seven caspase homologs that can function redundantly to promote cell death. Caspase-independent programmed cell death is also thought to occur in the invertebrate nematode Caenorhabditis elegans. The C. elegans genome contains four caspase genes (ced-3, csp-1, csp-2, and csp-3), of which only ced-3 has been demonstrated to promote apoptosis. Here, we show that CSP-1 is a pro-apoptotic caspase that promotes programmed cell death in a subset of cells fated to die during C. elegans embryogenesis. csp-1 is expressed robustly in late pachytene nuclei of the germline and is required maternally for its role in embryonic programmed cell deaths. Unlike CED-3, CSP-1 is not regulated by the APAF-1 homolog CED-4 or the BCL-2 homolog CED-9, revealing that csp-1 functions independently of the canonical genetic pathway for apoptosis. Previously we demonstrated that embryos lacking all four caspases can eliminate cells through an extrusion mechanism and that these cells are apoptotic. Extruded cells differ from cells that normally undergo programmed cell death not only by being extruded but also by not being engulfed by neighboring cells. In this study, we identify in csp-3; csp-1; csp-2 ced-3 quadruple mutants apoptotic cell corpses that fully resemble wild-type cell corpses: these caspase-deficient cell corpses are morphologically apoptotic, are not extruded, and are internalized by engulfing cells. We conclude that both caspase-dependent and caspase-independent pathways promote apoptotic programmed cell death and the phagocytosis of cell corpses in parallel to the canonical apoptosis pathway involving CED-3 activation.
Author Summary
Caspases are cysteine proteases that in many cases drive apoptosis, an evolutionarily conserved and highly stereotyped form of cellular suicide with functions in animal development and tissue maintenance. The dysregulation of apoptosis can contribute to diseases as diverse as cancer, autoimmunity, and neurodegeneration. Caspases are often thought to be required for, or even to define, apoptosis. Although there is evidence that apoptosis can occur in the absence of caspase activity, caspase-independence can be difficult to prove, as most animals have multiple caspases. The nematode Caenorhabditis elegans has four caspases, CED-3, CSP-1, CSP-2, and CSP-3. CED-3 has a well-established role in apoptosis, but less is known about the functions of the CSP caspases. In this study, we show that CSP-1 promotes apoptosis in the developing C. elegans embryo and that CSP-1 is regulated differently than its homolog CED-3. Furthermore, we show that apoptosis and the engulfment of dying cells can occur in mutants lacking all four caspases, proving that neither apoptosis nor cell-corpse engulfment require caspase function and that caspase-independent activities can contribute to apoptosis of some cells during animal development.
doi:10.1371/journal.pgen.1003341
PMCID: PMC3591282  PMID: 23505386
12.  Lack of involvement of mitochondrial factors in caspase activation in a Drosophila cell-free system 
Cell death and differentiation  2005;13(7):1222-1234.
Although mitochondrial proteins play well-defined roles in caspase activation in mammalian cells, the role of mitochondrial factors in caspase activation in Drosophila is unclear. Using cell-free extracts, we demonstrate that mitochondrial factors play no apparent role in Drosophila caspase activation. Cytosolic extract from apoptotic S2 cells, in which caspases were inhibited, induced caspase activation in cytosolic extract from normal S2 cells. Mitochondrial extract did not activate caspases, nor did it influence caspase activation by cytosolic extract. Silencing of Hid, Reaper, or Grim reduced caspase activation by apoptotic cell extract. Furthermore, a peptide representing the amino terminus of Hid was sufficient to activate caspases in cytosolic extract, and this activity was not enhanced by addition of mitochondria or mitochondrial lysate. The Hid peptide also induced apoptosis when introduced into S2 cells. These results suggest that caspase activation in Drosophila is regulated solely by cytoplasmic factors and does not involve any mitochondrial factors.
doi:10.1038/sj.cdd.4401821
PMCID: PMC2575646  PMID: 16322754
apoptosis; mitochondria; cytochrome c; Drosophila; caspase; Hid; Reaper; Grim; DIAP1
13.  Hair Cell Death in the Avian Basilar Papilla: Characterization of the in vitro Model and Caspase Activation  
Caspases are a family of proteases that have been implicated as key mediators of cell death. Although nonspecific inhibition of caspase activation has been reported to prevent mammalian sensory hair cell death, the exact roles of individual caspases during hair cell death are unclear. In other systems, the activation of initiator caspases, such as caspase-8 and caspase-9, can lead to the activation of the effector caspase-3. We have begun to systematically characterize hair cell death in an in vitro system by examining the activation of these specific caspases in degenerating hair cells after acutely damaging the whole avian basilar papilla with gentamicin. Basilar papillae (BP) displayed a dose-dependent hair cell loss after a 24-h treatment with gentamicin at concentrations of 0.1, 0.5, and 2.0 mM. When treated with 0.5 mM gentamicin for 6, 12, or 24 h, hair cells first began to degenerate in the basal third of the BP and damage progressed apically. Supplementation of z-VAD-fmk, a general caspase inhibitor, provided short-term protection against gentamicin-induced hair cell death. Treatment with gentamicin for 6 or 12 h promoted the expression of active caspase-3 and active caspase-9 in many hair cells along the BP as shown by immunohistochemistry. At these time-points, specific fluorescent-labeled peptide substrates detected more active caspase-3, caspase-8, and caspase-9 in gentamicin-treated hair cells relative to controls. Our data indicate that auditory hair cells degenerate as a result of gentamicin exposure in a caspase-dependent manner. Specifically, the upstream caspases, caspase-8 and caspase-9, and the downstream caspase-3 are activated in aminoglycoside-damaged hair cells.
doi:10.1007/s10162-002-3016-8
PMCID: PMC3202452  PMID: 12417974
14.  Identification and functional characterization of AMVp33, a novel homolog of the baculovirus caspase inhibitor p35 found in Amsacta moorei entomopoxvirus 
Virology  2006;358(2):436-447.
Members of the baculovirus p35 gene family encode proteins that specifically inhibit caspases, cysteine proteases that are involved in apoptosis. To date, p35 homologs have only been found in baculoviruses. We have identified AMVp33, a gene from Amsacta moorei entomopoxvirus with low but significant homology to baculovirus p35 genes. Expression of AMVp33 blocked apoptosis in several different insect and human cell lines. Purified recombinant P33 protein was an efficient inhibitor of insect and human effector caspases, but not initiator caspases. P33 was cleaved by effector caspases, and the resulting cleavage fragments stably associated with the caspases. Mutation of the predicted caspase cleavage site in P33 eliminated cleavage, caspase inhibition, and anti-apoptotic function. Thus, AMVp33 encodes a caspase inhibitor similar to baculovirus P35 with a preference for effector caspases. This is the first report of a p35 homolog from any viral or cellular genome outside of the baculovirus family.
doi:10.1016/j.virol.2006.08.043
PMCID: PMC2582192  PMID: 17010407
15.  The Enigmatic Roles of Caspases in Tumor Development 
Cancers  2010;2(4):1952-1979.
One function ascribed to apoptosis is the suicidal destruction of potentially harmful cells, such as cancerous cells. Hence, their growth depends on evasion of apoptosis, which is considered as one of the hallmarks of cancer. Apoptosis is ultimately carried out by the sequential activation of initiator and executioner caspases, which constitute a family of intracellular proteases involved in dismantling the cell in an ordered fashion. In cancer, therefore, one would anticipate caspases to be frequently rendered inactive, either by gene silencing or by somatic mutations. From clinical data, however, there is little evidence that caspase genes are impaired in cancer. Executioner caspases have only rarely been found mutated or silenced, and also initiator caspases are only affected in particular types of cancer. There is experimental evidence from transgenic mice that certain initiator caspases, such as caspase-8 and -2, might act as tumor suppressors. Loss of the initiator caspase of the intrinsic apoptotic pathway, caspase-9, however, did not promote cellular transformation. These data seem to question a general tumor-suppressive role of caspases. We discuss several possible ways how tumor cells might evade the need for alterations of caspase genes. First, alternative splicing in tumor cells might generate caspase variants that counteract apoptosis. Second, in tumor cells caspases might be kept in check by cellular caspase inhibitors such as c-FLIP or XIAP. Third, pathways upstream of caspase activation might be disrupted in tumor cells. Finally, caspase-independent cell death mechanisms might abrogate the selection pressure for caspase inactivation during tumor development. These scenarios, however, are hardly compatible with the considerable frequency of spontaneous apoptosis occurring in several cancer types. Therefore, alternative concepts might come into play, such as compensatory proliferation. Herein, apoptosis and/or non-apoptotic functions of caspases may even promote tumor development. Moreover, experimental evidence suggests that caspases might play non-apoptotic roles in processes that are crucial for tumorigenesis, such as cell proliferation, migration, or invasion. We thus propose a model wherein caspases are preserved in tumor cells due to their functional contributions to development and progression of tumors.
doi:10.3390/cancers2041952
PMCID: PMC3840446  PMID: 24281211
caspase; cancer; apoptosis; tumor suppressor; non-apoptotic functions; mutations; LOH
16.  Caspase-2 Maintains Bone Homeostasis by Inducing Apoptosis of Oxidatively-Damaged Osteoclasts 
PLoS ONE  2014;9(4):e93696.
Osteoporosis is a silent disease, characterized by a porous bone micro-structure that enhances risk for fractures and associated disabilities. Senile, or age-related osteoporosis (SO), affects both men and women, resulting in increased morbidity and mortality. However, cellular and molecular mechanisms underlying senile osteoporosis are not fully known. Recent studies implicate the accumulation of reactive oxygen species (ROS) and increased oxidative stress as key factors in SO. Herein, we show that loss of caspase-2, a cysteine aspartate protease involved in oxidative stress-induced apoptosis, results in total body and femoral bone loss in aged mice (20% decrease in bone mineral density), and an increase in bone fragility (30% decrease in fracture strength). Importantly, we demonstrate that genetic ablation or selective inhibition of caspase-2 using zVDVAD-fmk results in increased numbers of bone-resorbing osteoclasts and enhanced tartrate-resistant acid phosphatase (TRAP) activity. Conversely, transfection of osteoclast precursors with wild type caspase-2 but not an enzymatic mutant, results in a decrease in TRAP activity. We demonstrate that caspase-2 expression is induced in osteoclasts treated with oxidants such as hydrogen peroxide and that loss of caspase-2 enhances resistance to oxidants, as measured by TRAP activity, and decreases oxidative stress-induced apoptosis of osteoclasts. Moreover, oxidative stress, quantified by assessment of the lipid peroxidation marker, 4-HNE, is increased in Casp2-/- bone, perhaps due to a decrease in antioxidant enzymes such as SOD2. Taken together, our data point to a critical and novel role for caspase-2 in maintaining bone homeostasis by modulating ROS levels and osteoclast apoptosis during conditions of enhanced oxidative stress that occur during aging.
doi:10.1371/journal.pone.0093696
PMCID: PMC3972236  PMID: 24691516
17.  Inflammasome-Dependent Release of the Alarmin HMGB1 in Endotoxemia 
Endotoxin administration recapitulates many of the host responses to sepsis. Inhibitors of the cysteine protease caspase 1 have long been sought as a therapeutic because mice lacking caspase 1 are resistant to LPS-induced endotoxic shock. According to current thinking, caspase 1-mediated shock requires the proinflammatory caspase 1 substrates IL-1β and IL-18. We show, however, that mice lacking both IL-1β and IL-18 are normally susceptible to LPS-induced splenocyte apoptosis and endotoxic shock. This finding indicates the existence of another caspase 1-dependent mediator of endotoxemia. Reduced serum high mobility group box 1 (HMGB1) levels in caspase 1-deficient mice correlated with their resistance to LPS. A critical role for HMGB1 in endotoxemia was confirmed when mice deficient for IL-1β and IL-18 were protected from a lethal dose of LPS by pretreatment with HMGB1-neutralizing Abs. We found that HMGB1 secretion from LPS-primed macrophages required the inflammasome components apoptotic speck protein containing a caspase activation and recruitment domain (ASC), caspase 1 and Nalp3, whereas HMGB1 secretion from macrophages infected in vitro with Salmonella typhimurium was dependent on caspase 1 and Ipaf. Thus, HMGB1 secretion, which is critical for endotoxemia, occurs downstream of inflammasome assembly and caspase 1 activation.
doi:10.4049/jimmunol.1000803
PMCID: PMC3428148  PMID: 20802146
18.  Caspase-2 Is Localized at the Golgi Complex and Cleaves Golgin-160 during Apoptosis 
The Journal of Cell Biology  2000;149(3):603-612.
Caspases are an extended family of cysteine proteases that play critical roles in apoptosis. Animals deficient in caspases-2 or -3, which share very similar tetrapeptide cleavage specificities, exhibit very different phenotypes, suggesting that the unique features of individual caspases may account for distinct regulation and specialized functions. Recent studies demonstrate that unique apoptotic stimuli are transduced by distinct proteolytic pathways, with multiple components of the proteolytic machinery clustering at distinct subcellular sites. We demonstrate here that, in addition to its nuclear distribution, caspase-2 is localized to the Golgi complex, where it cleaves golgin-160 at a unique site not susceptible to cleavage by other caspases with very similar tetrapeptide specificities. Early cleavage at this site precedes cleavage at distal sites by other caspases. Prevention of cleavage at the unique caspase-2 site delays disintegration of the Golgi complex after delivery of a pro-apoptotic signal. We propose that the Golgi complex, like mitochondria, senses and integrates unique local conditions, and transduces pro-apoptotic signals through local caspases, which regulate local effectors.
PMCID: PMC2174848  PMID: 10791974
signaling; subcellular; substrate; coiled coil; protease
19.  FLIPL induces caspase-8 activity in the absence of interdomain caspase-8 cleavage and alters substrate specificity 
The Biochemical journal  2011;433(3):447-457.
Caspase-8 is an initiator caspase that is activated by death receptors to initiate the extrinsic pathway of apoptosis. Caspase-8 activation involves dimerization and subsequent interdomain autoprocessing of caspase-8 zymogens, and recently published work has established that elimination of the autoprocessing site of caspase-8 abrogates its pro-apoptotic function while leaving its proliferative function intact. The observation that the developmental abnormalities of caspase-8 deficient mice are shared by mice lacking the dimerization adapter FADD or the caspase paralog FLIPL has led to the hypothesis that FADD-dependent formation of heterodimers between caspase-8 and FLIPL could mediate the developmental role of caspase-8. Using an inducible dimerization system we demonstrate that cleavage of the catalytic domain of caspase-8 is crucial for its activity in the context of activation by homodimerization. However, we find that use of FLIPL as a partner for caspase-8 in dimerization-induced activation rescues the requirement for intersubunit linker proteolysis in both protomers. Moreover, before processing, caspase-8 in complex with FLIPL does not generate a fully active enzyme, but an attenuated species able to process only select natural substrates. Based on these results we propose a mechanism of caspase-8 activation by dimerization in the presence of FLIPL, as well as a mechanism of caspase-8 functional divergence in apoptotic and non-apoptotic pathways.
doi:10.1042/BJ20101738
PMCID: PMC4024219  PMID: 21235526
apoptosis; activation mechanism; protein dimerization
20.  Asc-Dependent and Independent Mechanisms Contribute to Restriction of Legionella Pneumophila Infection in Murine Macrophages 
The apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc) is an adaptor molecule that mediates inflammatory and apoptotic signals. Legionella pneumophila is an intracellular bacterium and the causative agent of Legionnaire's pneumonia. L. pneumophila is able to cause pneumonia in immuno-compromised humans but not in most inbred mice. Murine macrophages that lack the ability to activate caspase-1, such as caspase-1−/− and Nlrc4−/− allow L. pneumophila infection. This permissiveness is attributed mainly to the lack of active caspase-1 and the absence of its down stream substrates such as caspase-7. However, the role of Asc in control of L. pneumophila infection in mice is unclear. Here we show that caspase-1 is moderately activated in Asc−/− macrophages and that this limited activation is required and sufficient to restrict L. pneumophila growth. Moreover, Asc-independent activation of caspase-1 requires bacterial flagellin and is mainly detected in cellular extracts but not in culture supernatants. We also demonstrate that the depletion of Asc from permissive macrophages enhances bacterial growth by promoting L. pneumophila-mediated activation of the NF-κB pathway and decreasing caspase-3 activation. Taken together, our data demonstrate that L. pneumophila infection in murine macrophages is controlled by several mechanisms: Asc-independent activation of caspase-1 and Asc-dependent regulation of NF-κB and caspase-3 activation.
doi:10.3389/fmicb.2011.00018
PMCID: PMC3112328  PMID: 21713115
inflammasome; caspase-1; Legionella pneumophila; Asc
21.  Salmonella Exploits Caspase-1 to Colonize Peyer's Patches in a Murine Typhoid Model 
Salmonella typhimurium invades host macrophages and induces apoptosis and the release of mature proinflammatory cytokines. SipB, a protein translocated by Salmonella into the cytoplasm of macrophages, is required for activation of Caspase-1 (Casp-1, an interleukin [IL]-1β–converting enzyme), which is a member of a family of cysteine proteases that induce apoptosis in mammalian cells. Casp-1 is unique among caspases because it also directly cleaves the proinflammatory cytokines IL-1β and IL-18 to produce bioactive cytokines. We show here that mice lacking Casp-1 (casp-1−/− mice) had an oral S. typhimurium 50% lethal dose (LD50) that was 1,000-fold higher than that of wild-type mice. Salmonella breached the M cell barrier of casp-1−/− mice efficiently; however, there was a decrease in the number of apoptotic cells, intracellular bacteria, and the recruitment of polymorphonuclear lymphocytes in the Peyer's patches (PP) as compared with wild-type mice. Furthermore, Salmonella did not disseminate systemically in the majority of casp-1−/− mice, as demonstrated by significantly less colonization in the PP, mesenteric lymph nodes, and spleens of casp-1−/− mice after an oral dose of S. typhimurium that was 100-fold higher than the LD50. The increased resistance in casp-1−/− animals appears specific for Salmonella infection since these mice were susceptible to colonization by another enteric pathogen, Yersinia pseudotuberculosis, which normally invades the PP. These results show that Casp-1, which is both proapoptotic and proinflammatory, is essential for S. typhimurium to efficiently colonize the cecum and PP and subsequently cause systemic typhoid-like disease in mice.
PMCID: PMC2193260  PMID: 10899911
apoptosis; pathogenesis; intestine; inflammation; macrophages
22.  TAp73alpha protects small cell lung carcinoma cells from caspase-2 induced mitochondrial mediated apoptotic cell death 
Oncotarget  2011;2(12):1145-1154.
Caspase-2 is ubiquitously expressed and the most evolutionarily conserved mammalian caspase. It can be activated by a range of death stimuli prior to Bax activation and the occurrence of apoptotic mitochondrial dysfunctions. Caspase-2 has also been reported to exert tumour suppressor function in vivo. The full length TAp73alpha isoform is found up-regulated in various tumour types, and is reported in a cell-type specific manner to repress drug-induced apoptosis. Here, we report that TAp73alpha represses caspase-2 enzymatic activity and by this means reduce caspase-2 induced Bax activation, loss of mitochondrial transmembrane potential and resulting apoptosis. The inhibitory effect on caspase-2 requires the presence of the DNA binding domain and SAM domain region of TAp73alpha. In conclusion, the ability of TAp73alpha to act as an inhibitor of caspase-2-induced cell death together with its up-regulation in certain tumour types strengthen the potential oncogenic activities for this protein.
PMCID: PMC3282073  PMID: 22201672
p73; caspase-2; apoptosis; cancer
23.  The Drosophila melanogaster Apaf-1 homologue ARK is required for most, but not all, programmed cell death 
The Journal of Cell Biology  2006;172(6):809-815.
The Apaf-1 protein is essential for cytochrome c–mediated caspase-9 activation in the intrinsic mammalian pathway of apoptosis. Although Apaf-1 is the only known mammalian homologue of the Caenorhabditis elegans CED-4 protein, the deficiency of apaf-1 in cells or in mice results in a limited cell survival phenotype, suggesting that alternative mechanisms of caspase activation and apoptosis exist in mammals. In Drosophila melanogaster, the only Apaf-1/CED-4 homologue, ARK, is required for the activation of the caspase-9/CED-3–like caspase DRONC. Using specific mutants that are deficient for ark function, we demonstrate that ARK is essential for most programmed cell death (PCD) during D. melanogaster development, as well as for radiation-induced apoptosis. ark mutant embryos have extra cells, and tissues such as brain lobes and wing discs are enlarged. These tissues from ark mutant larvae lack detectable PCD. During metamorphosis, larval salivary gland removal was severely delayed in ark mutants. However, PCD occurred normally in the larval midgut, suggesting that ARK-independent cell death pathways also exist in D. melanogaster.
doi:10.1083/jcb.200512126
PMCID: PMC2063725  PMID: 16533943
24.  Cleavage and Inactivation of ATM during Apoptosis 
Molecular and Cellular Biology  1999;19(9):6076-6084.
The activation of the cysteine proteases with aspartate specificity, termed caspases, is of fundamental importance for the execution of programmed cell death. These proteases are highly specific in their action and activate or inhibit a variety of key protein molecules in the cell. Here, we study the effect of apoptosis on the integrity of two proteins that have critical roles in DNA damage signalling, cell cycle checkpoint controls, and genome maintenance—the product of the gene defective in ataxia telangiectasia, ATM, and the related protein ATR. We find that ATM but not ATR is specifically cleaved in cells induced to undergo apoptosis by a variety of stimuli. We establish that ATM cleavage in vivo is dependent on caspases, reveal that ATM is an efficient substrate for caspase 3 but not caspase 6 in vitro, and show that the in vitro caspase 3 cleavage pattern mirrors that in cells undergoing apoptosis. Strikingly, apoptotic cleavage of ATM in vivo abrogates its protein kinase activity against p53 but has no apparent effect on the DNA binding properties of ATM. These data suggest that the cleavage of ATM during apoptosis generates a kinase-inactive protein that acts, through its DNA binding ability, in a trans-dominant-negative fashion to prevent DNA repair and DNA damage signalling.
PMCID: PMC84521  PMID: 10454555
25.  Deletion of growth hormone receptor gene but not visceral fat removal decreases expression of apoptosis-related genes in the kidney—potential mechanism of lifespan extension 
Age  2011;34(2):295-304.
Mice homozygous for the targeted disruption of the growth hormone (GH) receptor (Ghr) gene (GH receptor knockout; GHRKO; KO) are hypoinsulinemic, highly insulin sensitive, normoglycemic, and long-lived. Visceral fat removal (VFR) is a surgical intervention which improves insulin signaling in normal (N) mice and rats and extends longevity in rats. We have previously demonstrated decreased expression level of certain pro-apoptotic genes in skeletal muscles and suggested that this may contribute to the regulation of longevity in GHRKO mice. Alterations in apoptosis-related genes expression in the kidneys also may potentially lead to lifespan extension. In this context, we decided to examine the renal expression of the following genes: caspase-3, caspase-9, caspase-8, bax, bad, bcl-2, Smac/DIABLO, Apaf-1, p53, and cytochrome c1 (cyc1) in male GHRKO and N mice subjected to VFR or sham surgery, at approximately 6 months of age. The kidneys were collected 2 months after VFR. As a result, caspase-3, caspase-9, and bax expressions were decreased in KO mice as compared to N animals. Expressions of Smac/DIABLO, caspase-8, bcl-2, bad, and p53 did not differ between KOs and N mice. VFR did not change the expression of the examined genes in KO or N mice. In conclusion, endocrine abnormalities in GHRKO mice result in decreased expression of pro-apoptotic genes and VFR did not alter the examined genes expression in N and KO mice. These data are consistent with a model in which alterations of GH signaling and/or insulin sensitivity lead to increased lifespan mediated by decreased renal expression of pro-apoptotic genes.
doi:10.1007/s11357-011-9232-6
PMCID: PMC3312636  PMID: 21431351
Apoptosis; GHRKO mice; Kidney; Gene expression; Caspases; Visceral fat removal

Results 1-25 (701726)