Activation of the c-Jun N-terminal kinase/stress-activated protein kinase pathway by overexpression of caspase-8 and its homologs.

Caspase-8 is the most proximal caspase in the caspase cascade and possesses a prodomain consisting of two homologous death effector domains (DEDs). We have discovered that caspase-8 and its homologs can physically interact with tumor necrosis factor receptor-associated factor family members and activate the c-Jun N-terminal kinase (JNK, or stress-activated protein kinase) pathway. This ability resides in the DED-containing prodomain of these proteins and is independent of their role as cell death proteases. A point mutant in the first DED of caspase-8 can block JNK activation induced by several death domain receptors. Inhibition of JNK activation blocks apoptosis mediated by caspase-10, Mach-related inducer of toxicity/cFLIP, and Fas/CD95, thereby suggesting a cooperative role of this pathway in the mediation of caspase-induced apoptosis.     

Portrait of a killer: the mitochondrial apoptosome emerges from the shadows

Apoptosis (programmed cell death) is a physiological process used to eliminate superfluous, damaged, infected, or aged cells in multicellular organisms. During apoptosis the cellular architecture is dismantled from within in a highly controlled fashion. Members of the caspase family of cysteine proteases are responsible for the destructive phase of apoptosis. One major pathway to caspase activation involves the formation of a multisubunit protease activation complex called the apoptosome. The apoptosome is assembled in response to signals that provoke mitochondrial outer membrane permeabilization and the release of cytochrome c into the cytosol. Recent studies indicate that the apoptosome is a wheel-like structure consisting of seven molecules of Apaf-1 and a similar number of caspase-9 dimers. Knowledge of the structure of the apoptosome will likely lead to the design of therapeutic modulators of apoptosis.     

Ars moriendi: Proteases as sculptors of cellular suicide

The Ars moriendi, which translates to “The Art of Dying,” encompasses two Latin texts that gave advice on how to die well and without fear according to the Christian precepts of the late Middle Ages. Given that ten to hundred billion cells die in our bodies every day, it is obvious that the concept of a well and orderly (“regulated”) death is also paramount at the cellular level. In apoptosis, as the most well-studied form of regulated cell death, proteases of the caspase family are the central mediators. However, caspases are not the only proteases that act as sculptors of cellular suicide, and therefore, we here provide an overview of the impact of proteases in apoptosis and other forms of regulated cell death.     

Manganese induces apoptosis of human B cells: caspase-dependent cell death blocked by bcl-2.

Manganese ions block apoptosis of phagocytes induced by various agents. The prevention of apoptosis was attributed to the activation of manganous superoxide dismutase (Mn-SOD) and to the antioxidant function of free Mn2+ cations. However, the effect of Mn2+ on B cell apoptosis is not documented. In this study, we investigated the effects of Mn2+ on the apoptotic process in human B cells. We observed that Mn2+ but not Mg2+ or Ca2+, inhibited cell growth and induced apoptosis of activated tonsilar B cells, Epstein Barr virus (EBV)-negative Burkitt's lymphoma cell lines (BL-CL) and EBV-transformed B cell lines (EBV-BCL). In the same conditions, no apoptosis was observed in U937, a monoblastic cell line. Induction of B cell apoptosis by Mn2+ was time- and dose-dependent. The cell permeable tripeptide inhibitor of ICE family cysteine proteases, zVAD-fmk, suppressed Mn2+-induced apoptosis. Furthermore, Mn2+ triggered the activation of interleukin-1beta converting enzyme (ICE/caspase 1), followed by the activation of CPP32/Yama/Apopain/caspase-3. In addition, poly-(ADP-ribose) polymerase (PARP), a cellular substrate for CPP32 protease was degraded to generate apoptotic fragments in Mn2+-treated B cell lines. The inhibitor, zVAD-fmk suppressed Mn2+-triggered CPP32 activation and PARP cleavage and apoptosis. These results indicate that the activation of caspase family proteases is required for the apoptotic process induced by Mn2+ treatment of B cells. While the caspase-1 inhibitor YVAD was unable to block apoptosis, the caspase-3 specific inhibitor DEVD-cmk, partially inhibited Mn2+-induced CPP32 activation, PARP cleavage and apoptosis of cells. Moreover, Bcl-2 overexpression in BL-CL effectively protected cells from apoptosis and cell death induced by manganese. This is the first report showing the involvement of Mn2+ in the regulation of B lymphocyte death presumably via a caspase-dependent process with a death-protective effect of Bcl-2.     

Caspases: evolutionary aspects of their functions in vertebrates

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.     

Apoptotic pathways: involvement of lysosomal proteases

Apoptosis or programmed cell death is the major mechanism used by multicellular organisms to remove infected, excessive and potentially dangerous cells. Cysteine proteases from the caspase family play a crucial role in the process. However, there is increasing evidence that lysosomal proteases are also involved in apoptosis. In this review various lysosomal proteases and their potential contribution to propagation of apoptosis are discussed.     

The CASBAH: a searchable database of caspase substrates

Apoptosis is coordinated by members of the caspase family of aspartic acid-specific proteases. Other members of this protease family also play essential roles in inflammation where they participate in the maturation of pro-inflammatory cytokines. To date, almost 400 substrates for the apoptosis-associated caspases have been reported and there are likely to be hundreds more yet to be discovered. Thus, the fraction of the proteome that is degraded (the degradome) by caspases during the demolition phase of apoptosis appears to be quite substantial. Despite this, we still know surprisingly little concerning how caspases provoke some of the signature events in apoptosis, such as membrane phosphatidylserine externalization, cellular retraction, chromatin condensation and apoptotic body production. The inflammatory caspases appear to be much more specific proteases than those involved in apoptosis and only two confirmed substrates for these proteases have been described to date. Here, we have compiled a comprehensive list of caspase substrates and describe a searchable web resource (The Casbah; www.casbah.ie) which contains information pertaining to all currently known caspase substrates. We also discuss some of the unresolved issues relating to caspase-dependent events in apoptosis and inflammation.     

Destabilizing influences in apoptosis: sowing the seeds of IAP destruction

Inhibitor of apoptosis proteins (IAPs) can block apoptosis through interactions with members of the caspase family of cysteine proteases. Recent developments suggest that ubiquitin-proteasome mediated destruction of the Drosophila IAP, DIAP1, is a key event during the initiation of programmed cell death in the fly.     

Caspases at the crossroads of immune-cell life and death

Caspases are responsible for crucial aspects of inflammation and immune-cell death that are disrupted in a number of genetic autoimmune and autoinflammatory diseases. The caspase family of proteases can be divided into pro-apoptotic and pro-inflammatory members based on their substrate specificity and participation in separate signalling cascades. However, as discussed here, evidence has emerged over the past few years that a number of the caspases thought to be involved solely in apoptosis also contribute to specific aspects of immune-cell development, activation and differentiation, and can even protect cells from some forms of cell death.     

Distinct cleavage products of nuclear proteins in apoptosis and necrosis revealed by autoantibody probes

A central mechanism in apoptosis is the activation of proteases of the caspase (cysteine aspartases) family. Protease activation has also been implicated in necrosis, but its role in this cell death process and the identity of the proteases involved and their substrates, are unknown. Using human autoantibodies to well characterized cellular proteins as detecting probes in immunoblotting, we observed that a defined and somewhat similar set of nuclear proteins, including poly (ADP-ribose) polymerase (PARP) and DNA topoisomerase I (Topo I), were selectively cleaved during both apoptosis and necrosis of cultured cells induced by various stimuli. The resulting cleavage products were distinctively different in the two cell death pathways. In contrast to apoptosis, the cleavages of PARP and Topo I during necrosis were not blocked by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk). These findings suggest that different proteases act in apoptosis and necrosis, and that although both cell death processes result in selective cleavage of almost identical cellular proteins, they can be distinguished immunochemically on the basis of their cleavage products.     

Ionizing radiation-induced, Bax-mediated cell death is dependent on activation of cysteine and serine proteases.

Bcl-2 family proteins and interleukin-1-beta converting enzyme/Caenorhabditis elegans cell death gene-3 (ICE/CED-3) family proteases (caspases) represent the basic regulators of apoptosis. However, the precise mechanism by which they interact is unclear. In this study, we found that gamma-radiation-induced apoptosis of leukemia cells was associated with activation of multiple caspases and bax up-regulation. Membrane changes and caspase activities were suppressed by specific caspase inhibitors. Similarly, the serine protease inhibitors z-Ala-Ala-Asp-cmk (AAD) and tosyl-lysine chloromethyl ketone (TLCK) also prevented caspase activation and poly(ADP-ribose) polymerase cleavage in vivo but had no effect on caspase activity in vitro. TLCK also prevented bax up-regulation as a result of its inhibitory effect on p53 function. Inhibitors of caspases and serine proteases partially prevented cell death, suggesting a caspase involvement in Bax-mediated cell death. We propose an ordering of signaling events in Bax-mediated cell death, including steps upstream and downstream of p53 and bax up-regulation.     

Caspases: potential targets for regulating cell death

While in multicellular organisms all cells inexorably die, there are several different ways provided for the realization of cell death. One of them, apoptosis, represents a universal energy-dependent and tightly regulated physiologic process of cell death in both normal and pathologic tissues. The execution of apoptosis appears to be uniformly mediated through consecutive activation of the members of a caspase family. This review briefly summarizes current knowledge on the molecular mechanisms of caspase activation and the inhibitory components of caspase cascades. The suitability of caspases as a new potential therapeutic target is discussed next. Particular attention is focused on two broad categories of caspase-directed compounds: highly specific caspase inhibitors that distinctly block the progress of apoptosis and caspase activators that selectively induce cell death in a variety of in vitro and in vivo systems. These agents promise to be useful clinically, either alone or in combination with more conventional therapeutics.     

Death by proteases in plants: whodunit

Several studies have shown that protease inhibitors can suppress programmed cell death in various plant species and plant tissues. This is especially true of caspase inhibitors that can block programmed cell death and its marker DNA laddering. There are up to six different caspase-like activities that can be measured in plant extracts, the most prominent being caspase1-like and caspase3-like. These activities can be located in vacuoles and also in the nucleus or the cytoplasm. This represents a striking apparent similarity with animal programmed cell death. Because there are no caspase orthologue in plant genomes, a major challenge is to identify these proteases. Recently two proteases with caspase-like activities have been recognized as belonging to two different protease families that are not closely related to animal caspases. Various other protease families have been implicated and this suggests that complex protease networks have been recruited for the plant cell demise.     

Identification of an inhibitor of caspase activation from heart extracts; ATP blocks apoptosome formation

By revealing the biochemistry of apoptosis it is expected we will both improve our understanding of diseases where apoptosis plays an important role and aid the development of therapies for these disorders. Caspases are a family of proteases whose activity is required for apoptosis. In this study, a cell-free system was used to investigate the mechanism of caspase-9 activation in extracts from heart cells. Unlike extracts from other cell types, heart extracts were found to activate caspases poorly. This could be explained by the low levels of Apaf-1 in heart cells. However, subsequent testing showed that heart extracts contained an inhibitor of caspase activation that could block caspase activation in extracts from different cell types. Subsequent purification of the inhibitor of caspase activation from these extracts identified ATP. Caspase-9 is activated by recruitment into a multi-protein complex, the apoptosome, which then activates downstream caspases that kill the cell. Importantly, size exclusion chromatography showed that ATP inhibits apoptosome formation at physiologically relevant concentrations. Together these data support the hypothesis that intracellular ATP concentration is a critical factor in determining whether an apoptotic stimulus can induce apoptosome formation. Thus, the well described fall in intracellular ATP apoptosis is not an epiphenomenon but may be a pro-apoptotic event contributing to cell death.     

Proteolytic specificity of caspases is required to signal the appearance of apoptotic morphology

The caspase family of cysteine proteases is essential for implementation of physiological cell death. Since a wide variety of cellular proteins is cleaved by caspases during apoptosis, it has been predicted that digestion of proteins crucial to maintaining the life of a cell is central to apoptosis. To assess the role of the proteolytic destruction during apoptosis, we introduced the non-specific protease proteinase K into intact cells. This introduction led to extensive digestion of cellular proteins, including physiological caspase-substrates. Caspase-3-like activity was induced rapidly, followed by morphological signs of apoptosis such as membrane blebbing and nuclear condensation. The caspase inhibitor Z-VAD-fmk inhibited the appearance of these morphological changes without reducing the extent of intracellular proteolysis by proteinase K. Loss of integrity of the cell membrane, however, was not blocked by Z-VAD-fmk. This system thus generated conditions of extensive destruction of caspase substrates by proteinase K in the absence of apoptotic morphology. Taken together, these experiments suggest that caspases implement cell death not by protein destruction but by proteolytic activation of specific downstream effector molecules.     

Serine proteases mediate apoptosis-like cell death and phagocytosis under caspase-inhibiting conditions

Effective execution of apoptosis requires the activation of caspases. However, in many cases, broad-range caspase inhibitors such as Z-VAD.fmk do not inhibit cell death because death signaling continues via basal caspase activities or caspase-independent processes. Although death mediators acting under caspase-inhibiting conditions have been identified, it remains unknown whether they trigger a physiologically relevant cell death that shows typical signs of apoptosis, including phosphatidylserine (PS) exposure and the removal of apoptotic cells by phagocytosis. Here we show that cells treated with ER stress drugs or deprived of IL-3 still show hallmarks of apoptosis such as cell shrinkage, membrane blebbing, mitochondrial release of cytochrome c, PS exposure and phagocytosis in the presence of Z-VAD.fmk. Cotreatment of the stressed cells with Z-VAD.fmk and the serine protease inhibitor Pefabloc (AEBSF) inhibited all these events, indicating that serine proteases mediated the apoptosis-like cell death and phagocytosis under these conditions. The serine proteases were found to act upstream of an increase in mitochondrial membrane permeability as opposed to the serine protease Omi/HtrA2 which is released from mitochondria at a later stage. Thus, despite caspase inhibition or basal caspase activities, cells can still be phagocytosed and killed in an apoptosis-like fashion by a serine protease-mediated mechanism that damages the mitochondrial membrane.     

Lysosomal protease pathways to apoptosis. Cleavage of bid, not pro-caspases, is the most likely route

We investigated the mechanism of lysosome-mediated cell death using purified recombinant pro-apoptotic proteins, and cell-free extracts from the human neuronal progenitor cell line NT2. Potential effectors were either isolated lysosomes or purified lysosomal proteases. Purified lysosomal cathepsins B, H, K, L, S, and X or an extract of mouse lysosomes did not directly activate either recombinant caspase zymogens or caspase zymogens present in an NT2 cytosolic extract to any significant extent. In contrast, a cathepsin L-related protease from the protozoan parasite Trypanosoma cruzi, cruzipain, showed a measurable caspase activation rate. This demonstrated that members of the papain family can directly activate caspases but that mammalian lysosomal members of this family may have been negatively selected for caspase activation to prevent inappropriate induction of apoptosis. Given the lack of evidence for a direct role in caspase activation by lysosomal proteases, we hypothesized that an indirect mode of caspase activation may involve the Bcl-2 family member Bid. In support of this, Bid was cleaved in the presence of lysosomal extracts, at a site six residues downstream from that seen for pathways involving capase 8. Incubation of mitochondria with Bid that had been cleaved by lysosomal extracts resulted in cytochrome c release. Thus, cleavage of Bid may represent a mechanism by which proteases that have leaked from the lysosomes can precipitate cytochrome c release and subsequent caspase activation. This is supported by the finding that cytosolic extracts from mice ablated in the bid gene are impaired in the ability to release cytochrome c in response to lysosome extracts. Together these data suggest that Bid represents a sensor that allows cells to initiate apoptosis in response to widespread adventitious proteolysis.     

Caspase-2 is resistant to inhibition by inhibitor of apoptosis proteins (IAPs) and can activate caspase-7

Caspases are a family of cysteine proteases with roles in cytokine maturation or apoptosis. Caspase-2 was the first pro-apoptotic caspase identified, but its functions in apoptotic signal transduction are still being elucidated. This study examined the regulation of the activity of caspase-2 using recombinant proteins and a yeast-based system. Our data suggest that for human caspase-2 to be active its large and small subunits must be separated. For maximal activity its prodomain must also be removed. Consistent with its proposed identity as an upstream caspase, caspase-2 could provoke the activation of caspase-7. Caspase-2 was not subject to inhibition by members of the IAP family of apoptosis inhibitors.     

Induction of necrotic-like cell death by tumor necrosis factor alpha and caspase inhibitors: novel mechanism for killing virus-infected cells

Induction of apoptotic cell death generally requires the participation of cysteine proteases belonging to the caspase family. However, and similar to most cell types, mouse fibroblasts are normally resistant to tumor necrosis factor alpha (TNF-alpha)-induced apoptosis. Surprisingly, TNF-alpha treatment of vaccinia virus-infected mouse fibroblasts resulted in necrotic-like cell death, which was significantly reduced in cells infected with a vaccinia virus mutant lacking the caspase inhibitor B13R. Furthermore, TNF-alpha also induced necrotic-like cell death of fibroblasts in the presence of peptidyl caspase inhibitors. In both cases, necrosis was accompanied by generation of superoxide species. Caspase inhibitors also sensitized fibroblasts to killing by double-stranded RNA and gamma interferon. In all cases, cell death was efficiently blocked by antioxidants or mitochondrial respiratory chain inhibitors. These results define a new mitochondrion-dependent mechanism which may be important in the killing of cells infected with viruses encoding caspase inhibitors.     

Baculovirus inhibitor of apoptosis functions at or upstream of the apoptotic suppressor P35 to prevent programmed cell death.

Members of the inhibitor of apoptosis (iap) gene family prevent programmed cell death induced by multiple signals in diverse organisms, suggesting that they act at a conserved step in the apoptotic pathway. To investigate the molecular mechanism of iap function, we expressed epitope-tagged Op-iap, the prototype viral iap from Orgyia pseudotsugata nuclear polyhedrosis virus, by using novel baculovirus recombinants and stably transfected insect cell lines. Epitope-tagged Op-iap blocked both virus- and UV radiation-induced apoptosis. With or without apoptotic stimuli, Op-IAP protein (31 kDa) cofractionated with cellular membranes and the cytosol, suggesting a cytoplasmic site of action. To identify the step(s) at which Op-iap blocks apoptosis, we monitored the effect of Op-iap expression on in vivo activation of the insect CED-3/ICE death proteases (caspases). Op-iap prevented in vivo caspase-mediated cleavage of the baculovirus substrate inhibitor P35 and blocked caspase activity upon viral infection or UV irradiation. However, unlike the stoichiometric inhibitor P35, Op-IAP failed to affect activated caspase as determined by in vitro protease assays. These findings provide the first biochemical evidence that Op-iap blocks activation of the host caspase or inhibits its activity by a mechanism distinct from P35. Moreover, as suggested by the capacity of Op-iap to block apoptosis induced by diverse signals, including virus infection and UV radiation, iap functions at a central point at or upstream from steps involving the death proteases.