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.     

Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells.

The activity of ICE-like proteases or caspases is essential for apoptosis. Multiple caspases participate in apoptosis in mammalian cells but how many caspases are involved and what is their relative contribution to cell death is poorly understood. To identify caspases activated in apoptotic cells, we developed an approach to simultaneously detect multiple active caspases. Using tumor cells as a model, we have found that CPP32 (caspase 3) and Mch2 (caspase 6) are the major active caspases in apoptotic cells, and are activated in response to distinct apoptosis-inducing stimuli and in all cell lines analyzed. Both CPP32 and Mch2 are present in apoptotic cells as multiple active species. In a given cell line these species remained the same irrespective of the apoptotic stimulus used. However, the species of CPP32 and Mch2 detected varied between cell lines, indicating differences in caspase processing. The strategy described here is widely applicable to identify active caspases involved in apoptosis.     

Expression and biological activity of X-linked inhibitor of apoptosis (XIAP) in human malignant glioma.

The inhibitor-of-apoptosis (IAP) proteins are a novel family of antiapoptotic proteins that are thought to inhibit cell death via direct inhibition of caspases. Here, we report that human malignant glioma cell lines express XIAP, HIAP-1 and HIAP-2 mRNA and proteins. NAIP was not expressed. IAP proteins were not cleaved during CD95 ligand (CD95L)-induced apoptosis, and loss of IAP protein expression was not responsible for the potentiation of CD95L-induced apoptosis when protein synthesis was inhibited. LN-18 cells are highly sensitive to CD95-mediated apoptosis, whereas LN-229 cells require co-exposure to CD95L and a protein synthesis inhibitor, CHX, to acquire sensitivity to apoptosis. Adenoviral XIAP gene transfer blocked caspase 8 and 3 processing in both cell lines in the absence of CHX. Apoptosis was blocked in the absence and in the presence of CHX. However, XIAP failed to block caspase 8 processing in LN-229 cells in the presence of CHX. There was considerable overlap of the effects of XIAP on caspase processing with those of BCL-2 and the viral caspase inhibitor crm-A. These data define complex regulatory mechanisms for CD95-mediated apoptosis in glioma cells and indicate that there may be a distinct pathway of death receptor-mediated apoptosis that is readily activated when protein synthesis is inhibited. The constitutive expression of natural caspase inhibitors may play a role in the resistance of these cells to apoptotic stimuli that directly target caspases, including radiochemotherapy and immune-mediated tumor cell lysis.     

Serial killers: ordering caspase activation events in apoptosis

Caspases participate in the molecular control of apoptosis in several guises; as triggers of the death machinery, as regulatory elements within it, and ultimately as a subset of the effector elements of the machinery itself. The mammalian caspase family is steadily growing and currently contains 14 members. At present, it is unclear whether all of these proteases participate in apoptosis. Thus, current research in this area is focused upon establishing the repertoire and order of caspase activation events that occur during the signalling and demolition phases of cell death. Evidence is accumulating to suggest that proximal caspase activation events are typically initiated by molecules that promote caspase aggregation. As expected, distal caspase activation events are likely to be controlled by caspases activated earlier in the cascade. However, recent data has cast doubt upon the functional demarcation of caspases into signalling (upstream) and effector (downstream) roles based upon their prodomain lengths. In particular, caspase-3 may perform an important role in propagating the caspase cascade, in addition to its role as an effector caspase within the death programme. Here, we discuss the apoptosis-associated caspase cascade and the hierarchy of caspase activation events within it.     

Mechanisms mediating caspase activation in cell death

The initial activation of a caspase in a caspase cascade is a crucial event that determines whether a cell will ultimately undergo cell death. Although each cell contains a number of different caspases, only a small subset may be required for apoptosis in response to a specific stimulus. It now seems that each caspase cascade has two types of caspases involved, the upstream or class I caspases, and the downstream or class II caspases. Class I caspases are characterised by long amino-terminal prodomains that carry specific protein - protein interaction domains which mediate oligomerisation of caspases, often assisted by specific adaptor molecules. Oligomerisation appears to be sufficient for autocatalytic activation of class I caspases. Once the first caspase in the pathway has been activated, it processes downstream caspases initiating a cascade of amplifying events that lead to the apoptotic death of a cell. This article reviews our current understanding of mechanisms that mediate the activation of caspases.     

Oxidative stress induces caspase-independent retinal apoptosis in vitro

Apoptosis is the mode of cell death in retinitis pigmentosa (RP), a heterogeneous group of retinal degenerations. The activation of the caspase proteases forms a pivotal step in the initiation and execution phase of apoptosis in many cells. Inhibition of caspases has been reported to prevent apoptosis in many model systems. However, we demonstrate the absence of caspase activation during retinal cell apoptosis in vitro which involves phosphatidylserine (PS) externalisation, DNA nicking and cell shrinkage. In addition, zVAD-fmk, DEVD-CHO and BD-fmk, inhibitors of the caspases, were unable to alter the characteristics or kinetics of apoptosis, implying that retinal cell death in vitro follows a caspase-independent pathway. We have previously demonstrated the ability of reactive oxygen species (ROS) to act as mediators of retinal cell apoptosis in vitro as well as the ability of antioxidants to prevent retinal cell apoptosis. Here we demonstrate the oxidative inactivation of caspases in this model of retinal apoptosis and provide evidence for an oxidative stress driven cell death pathway that does not involve caspase activity and which retains key features of apoptotic cell death. Furthermore, our data indicates that apoptotic events such as PS exposure, DNA nicking and cell shrinkage may occur independently of caspase activity.     

A role for caspases in the differentiation of erythroid cells and macrophages

Several cysteine proteases of the caspase family play a central role in many forms of cell death by apoptosis. Other enzymes of the family are involved in cytokine maturation along inflammatory response. In recent years, several caspases involved in cell death were shown to play a role in other cellular processes such as proliferation and differentiation. In the present review, we summarize the current knowledge of the role of caspases in the differentiation of erythroid cells and macrophages. Based on these two examples, we show that the nature of involved enzymes, the pathways leading to their activation in response to specific growth factors, and the specificity of the target proteins that are cleaved by the activated enzymes strongly differ from one cell type to another. Deregulation of these pathways is thought to play a role in the pathophysiology of low-grade myelodysplastic syndromes, characterized by excessive activation of caspases and erythroid precursor apoptosis, and that of chronic myelomonocytic leukemia, characterized by a defective activation of caspases in monocytes exposed to M-CSF, which blocks their differentiation.     

Regulation of caspase activity in apoptosis

Intracellular cysteine aspartate-specific proteases (caspases) play both signaling and effector roles in realizing the program of cell death. Caspases function as proteolytic cascades unique for each cell type and signal triggering apoptosis. All parts of the proteolytic cascades are duplicated and controlled by feedback signals. Amplification cycles between pairs of caspases (the third and the sixth, the ninth and the third, the twelfth and the sixth, and others) help multiply the initial apoptotic signal. The presence of physiological inhibitors of apoptosis that directly interact with caspases creates a multilevel regulatory network of apoptosis in cell. The caspase proteolytic cascades are also regulated by sphingolipid secondary messengers, among them ceramide, sphingosine, and their phosphates. Moreover, an association of the caspase signaling with ubiquitin-dependent proteolysis is shown in cells. In particular, the use of extracellular activators and inhibitors of caspases allows irreversible activation of apoptosis in tumor cells or the prevention of neuron death in neurodegenerative diseases.     

Cell death in pancreatitis: caspases protect from necrotizing pancreatitis

Mechanisms of cell death in pancreatitis remain unknown. Parenchymal necrosis is a major complication of pancreatitis; also, the severity of experimental pancreatitis correlates directly with necrosis and inversely with apoptosis. Thus, shifting death responses from necrosis to apoptosis may have a therapeutic value. To determine cell death pathways in pancreatitis and the possibility of necrosis/apoptosis switch, we utilized the differences between the rat model of cerulein pancreatitis, with relatively high apoptosis and low necrosis, and the mouse model, with little apoptosis and high necrosis. We found that caspases were greatly activated during cerulein pancreatitis in the rat but not mouse. Endogenous caspase inhibitor X-linked inhibitor of apoptosis protein (XIAP) underwent complete degradation in the rat but remained intact in the mouse model. Furthermore, XIAP inhibition with embelin triggered caspase activation in the mouse model, implicating XIAP in caspase blockade in pancreatitis. Caspase inhibitors decreased apoptosis and markedly stimulated necrosis in the rat model, worsening pancreatitis parameters. Conversely, caspase induction with embelin stimulated apoptosis and decreased necrosis in mouse model. Thus, caspases not only mediate apoptosis but also protect from necrosis in pancreatitis. One protective mechanism is through degradation of receptor-interacting protein (RIP), a key mediator of "programmed" necrosis. We found that RIP was cleaved (i.e. inactivated) in the rat but not the mouse model. Caspase inhibition restored RIP levels; conversely, caspase induction with embelin triggered RIP cleavage. Our results indicate key roles for caspases, XIAP, and RIP in the regulation of cell death in pancreatitis. Manipulating these signals to change the pattern of death responses presents a therapeutic strategy for treatment of pancreatitis.     

Apoptosis: checkpoint at the mitochondrial frontier.

Apoptosis, an evolutionarily conserved form of cell death, requires a regulated program. Central to the apoptotic program is a family of cysteine proteases, known as caspases, that cleave a subset of cellular proteins, resulting in the stereotypic morphological changes of apoptotic cell death. In living cells caspases are present as inactive zymogens and become activated in response to pro-apoptotic stimuli. Mitochondria participate in the activation of caspases by releasing cytochrome c into the cytosol where it binds to the adaptor molecule Apaf-1 (apoptotic protease activating factor 1) and causes its oligomerization. This renders Apaf-1 competent to recruit and activate the cell death initiator caspase, pro-caspase-9. Once caspase-9 is activated, it cleaves and activates downstream cell death effector caspases. Bcl-2, an apoptosis inhibitor localized to mitochondrial outer membranes, prevents cytochrome c release, caspase activation and cell death. This review discusses recent advances on the role of mitochondria and cytochrome c in the central pathway leading to apoptotic cell death.     

The N-terminal internal region of BLM is required for the formation of dots/rod-like structures which are associated with SUMO-1

Chemotherapeutic drug-induced apoptosis of human malignant glioma cells involves the death receptor-independent activation of caspases other than caspases 3 or 8 (Glaser et al., Oncogene 18, 5044-5053, 1999). Here, we report that caspases 1, 2, 3, 7, 8, and 9 are constitutively expressed in most human malignant glioma cell lines. Cytotoxic drug-induced apoptosisinvolves delayed activation of caspases 2, 7, and 9, but not 8 and 3, and is blocked by a broad spectrum caspase inhibitor, zVAD-fmk. Cytochrome c release from mitochondria precedes caspase activation during drug-induced apoptosis and is unaffected by zVAD-fmk or ectopic expression of the viral caspase inhibitor, crm-A. In contrast, ectopic expression of BCL-X(L) prevents drug-induced cytochrome c release, caspase activation and cell death. Thus, cancer chemotherapy targets the mitochondrial, caspase-dependent death pathway in human malignant glioma cells.     

Drosophila caspase DRONC is required for specific developmental cell death pathways and stress-induced apoptosis

Proteases of the caspase family play key roles in the execution of apoptosis. In Drosophila there are seven caspases, but their roles in cell death have not been studied in detail due to a lack of availability of specific mutants. Here, we describe the generation of a specific mutant of the Drosophila gene encoding DRONC, the only caspase recruitment domain (CARD) containing apical caspase in the fly. dronc mutants are pupal lethal and our studies show that DRONC is required for many forms of developmental cell deaths and apoptosis induced by DNA damage. Furthermore, we demonstrate that DRONC is required for the autophagic death of larval salivary glands during metamorphosis, but not for histolysis of larval midguts. Our results indicate that DRONC is involved in specific developmental cell death pathways and that in some tissues, effector caspase activation and cell death can occur independently of DRONC.     

Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.     

Caspase knockouts: matters of life and death

Apoptosis, the seemingly counter-intuitive act of physiological cell suicide, is accomplished by an evolutionarily conserved death program that is centered on the activation of a group of intracellular cysteine proteases known as caspases. It is now clear that both extra- and intra-cellular stimuli induce apoptosis by triggering the activation of these otherwise latent proteases in a process that culminates in caspase-mediated disintegration of cellular contents and their subsequent absorption by neighboring cells. While many elegant in vitro studies have demonstrated the requirement of caspase activities for the execution of most, if not all, apoptosis, the precise contribution of individual caspases in vivo and how they functionally relate to each other remain poorly elucidated. Fortunately, the generation of various caspase deficient mice through gene targeting has provided a unique window of opportunity to definitely examine the physiological function of these caspases in vivo. As the list of caspase knockouts grows, we considered it was time to review what we have been learned, from these studies about the exact role of individual caspases in mediating apoptotic events. We will also provide our prediction on the direction of future studies in this ever-growing field of caspases.     

Proteases for cell suicide: functions and regulation of caspases.

Caspases are a large family of evolutionarily conserved proteases found from Caenorhabditis elegans to humans. Although the first caspase was identified as a processing enzyme for interleukin-1beta, genetic and biochemical data have converged to reveal that many caspases are key mediators of apoptosis, the intrinsic cell suicide program essential for development and tissue homeostasis. Each caspase is a cysteine aspartase; it employs a nucleophilic cysteine in its active site to cleave aspartic acid peptide bonds within proteins. Caspases are synthesized as inactive precursors termed procaspases; proteolytic processing of procaspase generates the tetrameric active caspase enzyme, composed of two repeating heterotypic subunits. Based on kinetic data, substrate specificity, and procaspase structure, caspases have been conceptually divided into initiators and effectors. Initiator caspases activate effector caspases in response to specific cell death signals, and effector caspases cleave various cellular proteins to trigger apoptosis. Adapter protein-mediated oligomerization of procaspases is now recognized as a universal mechanism of initiator caspase activation and underlies the control of both cell surface death receptor and mitochondrial cytochrome c-Apaf-1 apoptosis pathways. Caspase substrates have bene identified that induce each of the classic features of apoptosis, including membrane blebbing, cell body shrinkage, and DNA fragmentation. Mice deficient for caspase genes have highlighted tissue- and signal-specific pathways for apoptosis and demonstrated an independent function for caspase-1 and -11 in cytokine processing. Dysregulation of caspases features prominently in many human diseases, including cancer, autoimmunity, and neurodegenerative disorders, and increasing evidence shows that altering caspase activity can confer therapeutic benefits.     

Cell survival and proliferation in Drosophila S2 cells following apoptotic stress in the absence of the APAF-1 homolog, ARK, or downstream caspases

In Drosophila, the APAF-1 homolog ARK is required for the activation of the initiator caspase DRONC, which in turn cleaves the effector caspases DRICE and DCP-1. While the function of ARK is important in stress-induced apoptosis in Drosophila S2 cells, as its removal completely suppresses cell death, the decision to undergo apoptosis appears to be regulated at the level of caspase activation, which is controlled by the IAP proteins, particularly DIAP1. Here, we further dissect the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. We found that the induction of apoptosis was dependent in each case on expression of ARK and DRONC and surviving cells continued to proliferate. We noted a difference in the effects of silencing the executioner caspases DCP-1 and DRICE; knock-down of either or both of these had dramatic effects to sustain cell survival following depletion of DIAP1, but had only minor effects following cellular stress. Our results suggest that the executioner caspases are essential for death following DIAP1 knock-down, indicating that the initiator caspase DRONC may lack executioner functions. The apparent absence of mitochondrial outer membrane permeabilization (MOMP) in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted.     

Many cuts to ruin: a comprehensive update of caspase substrates

Apoptotic cell death is executed by the caspase-mediated cleavage of various vital proteins. Elucidating the consequences of this endoproteolytic cleavage is crucial for our understanding of cell death and other biological processes. Many caspase substrates are just cleaved as bystanders, because they happen to contain a caspase cleavage site in their sequence. Several targets, however, have a discrete function in propagation of the cell death process. Many structural and regulatory proteins are inactivated by caspases, while other substrates can be activated. In most cases, the consequences of this gain-of-function are poorly understood. Caspase substrates can regulate the key morphological changes in apoptosis. Several caspase substrates also act as transducers and amplifiers that determine the apoptotic threshold and cell fate. This review summarizes the known caspase substrates comprising a bewildering list of more than 280 different proteins. We highlight some recent aspects inferred by the cleavage of certain proteins in apoptosis. We also discuss emerging themes of caspase cleavage in other forms of cell death and, in particular, in apparently unrelated processes, such as cell cycle regulation and cellular differentiation.     

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.     

Death receptor-induced activation of initiator caspase 8 is antagonized by serine/threonine kinase PAK4

Normal cell growth requires a precisely controlled balance between cell death and survival. This involves activation of different types of intracellular signaling cascades within the cell. While some types of signaling proteins regulate apoptosis, or programmed cell death, other proteins within the cell can promote survival. The serine/threonine kinase PAK4 can protect cells from apoptosis in response to several different types of stimuli. As is the case for other members of the p21-activated kinase (PAK) family, one way that PAK4 may promote cell survival is by phosphorylating and thereby inhibiting the proapoptotic protein Bad. This leads in turn to the inhibition of effector caspases such as caspase 3. Here we show that in response to cytokines which activate death domain-containing receptors, such as the tumor necrosis factor and Fas receptors, PAK4 can inhibit the death signal by a different mechanism. Under these conditions, PAK4 inhibits apoptosis early in the caspase cascade, antagonizing the activation of initiator caspase 8. This inhibition, which does not require PAK4's kinase activity, may involve inhibition of caspase 8 recruitment to the death domain receptors. This role in regulating initiator caspases is an entirely novel role for the PAK proteins and suggests a new mechanism by which these proteins promote cell survival.