The ICE family of cysteine proteases as effectors of cell death

Programmed cellular suicide follows a set of distinct morphological events involving profound cytoplasmic and nuclear changes. The recent discovery of a family of mammalian homologues of the Caenorhabditis elegans cell death protein CED-3 is now providing insight into how these events might be brought about. These mammalian proteins encode cysteine proteases with homology to the interleukin-1beta converting enzyme (ICE). CED-3 and seven of its currently known mammalian homologues cleave their substrates after an aspartate residue, a property shared only by the cytotoxic T cell (CTL) protease granzyme B which is necessary for the CTL-mediated killing of target cells. A number of proteins previously known to be cleaved in cells undergoing apoptosis have now been shown to be targeted by ICE-like proteases. Although many questions remain, it is becoming increasingly clear that this unique group of proteases play a central effector role in the process of physiological cell death. This article reviews various aspects of the ICE family of proteases.     

Proteolytic mechanisms in necrotic cell death and neurodegeneration

Programmed neuronal cell death is required during development to achieve the accurate wiring of the nervous system. However, genetic or accidental factors can lead to the premature, non-programmed death of neurons during adult life. Inappropriate death of cells in the nervous system is the cause of multiple neurodegenerative disorders. Pathological neuronal death can occur by apoptosis, by necrosis or by a combination of both. Necrotic cell death underlies the pathology of devastating neurological diseases such as neurodegenerative disorders, stroke or trauma. However, little is known about the molecular mechanisms that bring about necrotic cell death. Proteases play crucial roles in neuron degeneration by exerting both regulatory and catabolic functions. Elevated intracellular calcium is the most ubiquitous feature of neuronal death with the concomitant activation of cysteine calcium-dependent proteases, calpains. Calpains and lysosomal, catabolic aspartyl proteases, play key roles in the necrotic death of neurons. In this review, we survey the recent literature on the role of cysteine and aspartyl proteases in necrosis and neurodegeneration, aiming to delineate common proteolytic mechanisms mediating cellular destruction.     

Caspase-dependent inactivation of proteasome function during programmed cell death in Drosophila and man

The caspase family of cysteine proteases plays a conserved role in the coordinate demolition of cellular structures during programmed cell death from nematodes to man. Because cells undergoing programmed cell death in nematodes, flies, and mammals all share common features, this suggests that caspases target a common set of cellular structures in each of these organisms. However, although many substrates for mammalian caspases have been identified, few substrates for these proteases have been identified in invertebrates. To search for similarities between the repertoires of proteins targeted for proteolysis by caspases in flies and mammals, we have performed proteomics-based screens in Drosophila and human cell lines undergoing apoptosis. Here we show that several subunits of the proteasome undergo caspase-dependent proteolysis in both organisms and that this results in diminished activity of this multicatalytic protease complex. These data suggest that caspase-dependent proteolysis decreases protein turnover by the proteasome and that this is a conserved event in programmed cell death from Drosophila to mammals.     

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.     

Caspase-independent programmed cell death with necrotic morphology.

Cell death is generally classified into two large categories: apoptosis represents active, programmed cell death, while necrosis represents passive cell death without underlying regulatory mechanisms. Recent progress revealed that caspases, a family of cysteine proteases, play a central role in the regulation of apoptosis. Unexpectedly, however, caspase inhibition occasionally turns the morphology of programmed cell death from apoptotic into necrotic without inhibiting death itself. In this article, we review different models of caspase-independent programmed cell death showing necrotic-like morphology, including our Ras-mediated caspase-independent cell death. Based on these findings, we suggest the existence of a necrotic-like cell death regulated by cellular intrinsic death programs distinct from that of apoptosis. Even though type 2 physiological cell death, or autophagic degeneration, has been recognized as a necrotic-like programmed cell death for a long time, the underlying molecular mechanisms have not been identified despite its physiological significance. This has been in part due to the previous absence of adequate caspase-independent cellular models to study, recent efforts may now help to elucidate these mechanisms.     

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.     

Calpain and cell death

Neither the early nor the late steps in apoptosis have been defined biochemically. Several different signalling pathways have been implicated, and these are familiar from other signalling paradigms. In what way could they lead to cell death, when under the usual conditions they are involved in reversible activation events? A possible role for proteolysis is suggested, because the cleavage of a peptide bond is one of the few irreversible processes in cellular metabolism, and death, after all, is an irreversible outcome. In this review we discuss the calcium-dependent neutral protease calpain, a member of the papain family of cysteine proteases quite distinct from the ICE family. Calpain has been shown to play an essential role in several important examples of physiologic apoptosis. It seems to play its part after the various 'private' pathways have been invoked, but before the final common pathway.     

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 role of cytosolic Ca2+ in cell injury, necrosis and apoptosis.

Increases in cytosolic Ca2+ are believed to be a pivotal signal in the regulation of cell injury, cell death, cell proliferation, cellular differentiation and cellular aging. Changes in the concentration of cytosolic Ca2+ are involved in both acute and chronic cell injury, as well as in accidental or programmed cell death. Signalling in all of these phenomena is dependent on mediated activities of a number of intracellular factors, including phospholipases, proteases and endonucleases. The coordinate regulation of these factors, as well as of oncogene activation, seems to play a role both in the processes of cell injury and cell death, and in the recovery from injury in sublethally injured cells.     

Pathways to caspase activation

Apoptosis or programmed cell death is an active form of cell death which is essential for tissue homeostasis. Many proteins are involved in the molecular signal transduction of apoptosis. The caspase enzymes, a family of specific cysteine proteases, play a central role in cell death machinery. In this review, we mainly discuss the current understanding of several pathways to activate caspases and some key proteins related to these pathways.     

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.     

Apoptosis: controlled demolition at the cellular level

Apoptosis is characterized by a series of dramatic perturbations to the cellular architecture that contribute not only to cell death, but also prepare cells for removal by phagocytes and prevent unwanted immune responses. Much of what happens during the demolition phase of apoptosis is orchestrated by members of the caspase family of cysteine proteases. These proteases target several hundred proteins for restricted proteolysis in a controlled manner that minimizes damage and disruption to neighbouring cells and avoids the release of immunostimulatory molecules.     

Understanding IAP function and regulation: a view from Drosophila

Apoptosis is an active form of cell suicide that results in the orderly death and phagocytosis of cells during normal development and in the adult. Many death signals lead to the activation of members of a family of cysteine proteases known as caspases. These proteins act to transduce death signals from different cellular compartments and they cleave a number of cellular proteins, leading ultimately to many of the biochemical and morphological events associated with death. Many mechanisms act to inhibit cell death upstream of caspase activation. However, only one family of cellular proteins, the inhibitors of apoptosis (IAPs), has been identified that inhibit caspase activation and/or activity. The observations that IAP function is essential for cell survival in Drosophila, and that IAP expression is deregulated in many forms of cancer in humans, argue that IAPs are important cell death inhibitors and that deregulation of their function is likely to be important in human disease. Here we review IAP function, with particular reference to insights that study of the Drosophila IAPs has provided. We also discuss some directions for future study.     

Fas Induces Cytoplasmic Apoptotic Responses and Activation of the MKK7-JNK/SAPK and MKK6-p38 Pathways Independent of CPP32-like Proteases

IL-1β converting enzyme (ICE) family cysteine proteases are subdivided into three groups; ICE-, CPP32-, and Ich-1–like proteases. In Fas-induced apoptosis, activation of ICE-like proteases is followed by activation of CPP32-like proteases which is thought to be essential for execution of the cell death. It was recently reported that two subfamily members of the mitogen-activated protein kinase superfamily, JNK/SAPK and p38, are activated during Fas-induced apoptosis. Here, we have shown that MKK7, but not SEK1/ MKK4, is activated by Fas as an activator for JNK/ SAPK and that MKK6 is a major activator for p38 in Fas signaling. Then, to dissect various cellular responses induced by Fas, we used several peptide inhibitors for ICE family proteases in Fas-treated Jurkat cells and KB cells. While Z-VAD-FK which inhibited almost all the Fas-induced cellular responses blocked the activation of JNK/SAPK and p38, Ac-DEVD-CHO and Z-DEVD-FK, specific inhibitors for CPP32-like proteases, which inhibited the Fas-induced chromatin condensation and DNA fragmentation did not block the activation of JNK/SAPK and p38. Interestingly, these DEVD-type inhibitors did not block the Fas-induced morphological changes (cell shrinkage and surface blebbing), induction of Apo2.7 antigen, or the cell death (as assessed by the dye exclusion ability). These results suggest that the Fas-induced activation of the JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events (such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the cytoplasm or the cell death itself. Thus, the Fas signaling pathway diverges into multiple, separate processes, each of which may be responsible for part of the apoptotic cellular responses.     

The Drosophila caspase Ice is important for many apoptotic cell deaths and for spermatid individualization, a nonapoptotic process

Caspase family proteases play important roles in the regulation of apoptotic cell death. Initiator caspases are activated in response to death stimuli, and they transduce and amplify these signals by cleaving and thereby activating effector caspases. In Drosophila, the initiator caspase Nc (previously Dronc) cleaves and activates two short-prodomain caspases, Dcp-1 and Ice (previously Drice), suggesting these as candidate effectors of Nc killing activity. dcp-1-null mutants are healthy and possess few defects in normally occurring cell death. To explore roles for Ice in cell death, we generated and characterized an Ice null mutant. Animals lacking Ice show a number of defects in cell death, including those that occur during embryonic development, as well as during formation of adult eyes, arista and wings. Ice mutants exhibit subtle defects in the destruction of larval tissues, and do not prevent destruction of salivary glands during metamorphosis. Cells from Ice animals are also markedly resistant to several stresses, including X-irradiation and inhibition of protein synthesis. Mutations in Ice also suppress cell death that is induced by expression of Rpr, Wrinkled (previously Hid) and Grim. These observations demonstrate that Ice plays an important non-redundant role as a cell death effector. Finally, we demonstrate that Ice participates in, but is not absolutely required for, the non-apoptotic process of spermatid differentiation.     

Death without caspases, caspases without death

Apoptosis is a conserved cell-death process displaying characteristic morphological and molecular changes including activation of caspase proteases. Recent work challenges the accepted roles of these proteases. New investigations in mice and the nematode Caenorhabditis elegans suggest that there could be caspase-independent pathways leading to cell death. In addition, another type of cell death displaying autophagic features might depend on caspases. Recent studies also indicate that caspase activation does not always lead to cell death and, instead, might be important for cell differentiation. Here, we review recent evidence for both the expanded roles of caspases and the existence of caspase-independent cell-death processes. We suggest that cellular context plays an important role in defining the consequences of caspase activation.     

Imaging Proteolytic Activity in Live Cells and Animal Models

In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors); their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulated activation of apoptosis by the caspase family of proteases has been linked to diseases such as cancer, autoimmunity and inflammation. In an effort to better understand the role of proteases in health and disease, a luciferase biosensor is described which can quantitatively report proteolytic activity in live cells and mouse models. The biosensor, hereafter referred to as GloSensor Caspase 3/7 has a robust signal to noise (50–100 fold) and dynamic range such that it can be used to screen for pharmacologically active compounds in high throughput campaigns as well as to study cell signaling in rare cell populations such as isolated cancer stem cells. The biosensor can also be used in the context of genetically engineered mouse models of human disease wherein conditional expression using the Cre/loxP technology can be implemented to investigate the role of a specific protease in living subjects. While the regulation of apoptosis by caspase''s was used as an example in these studies, biosensors to study additional proteases involved in the regulation of normal and pathological cellular processes can be designed using the concepts presented herein.     

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.     

Inhibitor of apoptosis proteins in Drosophila: gatekeepers of death

Regulation of apoptosis is crucial to ensure cellular viability, and failure to do so is linked to several human pathologies. The apoptotic cell death programme culminates in the activation of caspases, a family of highly specific cysteine proteases essential for the destruction of the cell. Although best known for their role in executing apoptosis, caspases also play important signalling roles in non-apoptotic processes, such as regulation of actin dynamics, innate immunity, cell proliferation, differentiation and survival. Under such conditions, caspases are activated without killing the cell. Caspase activation and activity is subject to complex regulation, and various cellular and viral inhibitors have been identified that control the activity of caspases in their apoptotic and non-apoptotic roles. Members of the Inhibitor of APoptosis (IAP) protein family ensure cell viability in Drosophila by directly binding to caspases and regulating their activities in a ubiquitin-dependent manner. The observation that IAPs are essential for cell survival in Drosophila, and are frequently deregulated in human cancer, contributing to tumourigenesis, chemoresistance, disease progression and poor patient survival, highlights the importance of this family of caspase regulators in health and disease. Here we summarise recent advances from Drosophila that start to elucidate how the cellular response to caspase activation is modulated by IAPs and their regulators.