Activation of apoptosis pathways by granzyme B

Cytotoxic lymphocytes induce apoptosis of target cells by degranulating and releasing the serine protease granzyme B and the pore forming protein perforin. Granzyme B is an aspartic acid protease similar to members of the interleukin 1beta converting enzyme (ICE) family. We review the evidence for the participation members of the ICE family of proteases and cdc2 kinase in granzyme B-induced apoptosis.     

Role of ICE-related and other proteases in Fas-mediated apoptosis

Interleukin-1beta-converting enzyme (ICE)-like proteases comprise a novel family of unusual cysteine proteases which have been implicated in programmed cell death in both invertebrates and mammals. Current available evidence indicates a role of ICE proteases as central executioners of apoptosis triggered by the cell surface receptor Fas (APO-l). The presence of multiple mammalian ICE proteases with partially overlapping but distinct activities suggests a complex proteolytic cascade which is induced upon Fas ligation. The precise role of single members of the ICE family in Fas-mediated apoptosis, however, is still unclear. Here, we summarize the present knowledge about the relevance of ICE proteases, their potential targets, and interaction with unrelated proteases in cell death mediated by Fas and other apoptotic stimuli.     

The cytotoxic cell protease granzyme B initiates apoptosis in a cell-free system by proteolytic processing and activation of the ICE/CED-3 family protease, CPP32, via a novel two-step mechanism.

The major mechanism of cytotoxic lymphocyte killing involves the directed release of granules containing perforin and a number of proteases onto the target cell membrane. One of these proteases, granzyme B, has an unusual substrate site preference for Asp residues, a property that it shares with members of the emerging interleukin-1beta-converting enzyme (ICE)/CED-3 family of proteases. Here we show that granzyme B is sufficient to reproduce rapidly all of the key features of apoptosis, including the degradation of several protein substrates, when introduced into Jurkat cell-free extracts. Granzyme B-induced apoptosis was neutralized by a tetrapeptide inhibitor of the ICE/CED-3 family protease, CPP32, whereas a similar inhibitor of ICE had no effect. Granzyme B was found to convert CPP32, but not ICE, to its active form by cleaving between the large and small subunits of the CPP32 proenzyme, resulting in removal of the prodomain via an autocatalytic step. The cowpox virus protein CrmA, a known inhibitor of ICE family proteases as well as granzyme B, inhibited granzyme B-mediated CPP32 processing and apoptosis. These data demonstrate that CPP32 activation is a key event during apoptosis initiated by granzyme B.     

Caspase-3/CPP32-like activity is not sufficient to mediate apoptosis in an IL-2 dependent T cell line

CTLL cells undergo apoptosis when cultured in the absence of IL-2. The IL-1-converting-enzyme (ICE)/ caspase family has been implicated as an integral component of some forms of apoptosis. Numerous members of the caspase family have been identified, and it appears as if caspase-3/CPP32 plays a critical role. Previously we demonstrated that ICE/caspase-1 expression increases in CTLL cells during apoptosis; however, inhibition of ICE activity did not abrogate apoptotic death. The purpose of this report is to determine if other members of the caspase family are involved in T cell apoptosis induced by growth factor starvation. We show that cytosolic CPP32-like activity, as measured by the cleavage of DEVD-pNA and poly(ADP-ribose) polymerase (PARP), increases during apoptosis following growth factor deprivation. Cytosolic CPP32-like activity is inhibited in cells treated with the broad spectrum ICE family inhibitor boc-aspartyl(OMe)-fluoromethylketone (D-FMK) and by VAD-FMK and DEVD-FMK which have greater specificity for CPP32-like ICE homologs; however, only the broad spectrum ICE inhibitor D-FMK inhibited apoptosis. Our results suggest that apoptosis induced by growth factor deprivation involves the caspase family, but increased CPP32-like activity is not sufficient to mediate apoptosis induced by IL-2 starvation.     

Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases.

The Fas receptor mediates a signalling cascade resulting in programmed cell death (apoptosis) within hours of receptor cross-linking. In this study Fas activated the stress-responsive mitogen-activated protein kinases, p38 and JNK, within 2 h in Jurkat T lymphocytes but not the mitogen-responsive kinase ERK1 or pp70S6k. Fas activation of p38 correlated temporally with the onset of apoptosis, and transfection of constitutively active MKK3 (glu), an upstream regulator of p38, potentiated Fas-induced cell death, suggesting a potential involvement of the MKK3/p38 activation pathway in Fas-mediated apoptosis. Fas has been shown to require ICE (interleukin-1 beta-converting enzyme) family proteases to induce apoptosis from studies utilizing the cowpox ICE inhibitor protein CrmA, the synthetic tetrapeptide ICE inhibitor YVAD-CMK, and the tripeptide pan-ICE inhibitor Z-VAD-FMK. In this study, crmA antagonized, and YVAD-CMK and Z-VAD-FMK completely inhibited, Fas activation of p38 kinase activity, demonstrating that Fas-dependent activation of p38 requires ICE/CED-3 family members and conversely that the MKK3/p38 activation cascade represents a downstream target for the ICE/CED-3 family proteases. Intriguingly, p38 activation by sorbitol and etoposide was resistant to YVAD-CMK and Z-VAD-FMK, suggesting the existence of an additional mechanism(s) of p38 regulation. The ICE/CED-3 family-p38 regulatory relationship described in the current work indicates that in addition to the previously described destructive cleavage of substrates such as poly(ADP ribose) polymerase, lamins, and topoisomerase, the apoptotic cysteine proteases also function to regulate stress kinase signalling cascades.     

Targeted expression of ced-3 and Ice induces programmed cell death in Drosophila

CED-3 is a cysteine protease required for programmed cell death in the nematode, Caenorhabditis elegans, and shares a sequence similarity with mammalian ICE (interleukin-1beta converting enzyme) family proteases. Both CED-3 and ICE family proteases can induce programmed cell death in mammalian cells. Structural and functional similarities between CED-3 and ICE family proteases indicate that the mechanism of cell death is evolutionarily conserved, suggesting the presence of a similar mechanism involving CED-3/ICE-like proteases in Drosophila. Here we determined whether CED-3 or ICE functions to induce programmed cell death in Drosophila. We have generated transformant lines in which ced-3 or Ice is ectopically expressed using the GAL4-UAS system. Expression of CED-3 and ICE can elicit cell death in Drosophila and the cell death was blocked by coexpressing the p35 gene which encodes a viral inhibitor of CED-3/ICE proteases. Results support the idea that the mechanism of programmed cell death controlled by CED-3/ICE is conserved among widely divergent animal species including Drosophila, and the system described provides a tool to dissect cell death mechanism downstream of CED-3/ICE proteases.     

The role of interleukin-1 in the pathogenesis of human Intervertebral disc degeneration

In this study, we investigated the hypotheses that in human intervertebral disc (IVD) degeneration there is local production of the cytokine IL-1, and that this locally produced cytokine can induce the cellular and matrix changes of IVD degeneration. Immunohistochemistry was used to localize five members of the IL-1 family (IL-1α, IL-1β, IL-1Ra (IL-1 receptor antagonist), IL-1RI (IL-1 receptor, type I), and ICE (IL-1β-converting enzyme)) in non-degenerate and degenerate human IVDs. In addition, cells derived from non-degenerate and degenerate human IVDs were challenged with IL-1 agonists and the response was investigated using real-time PCR for a number of matrix-degrading enzymes, matrix proteins, and members of the IL-1 family.     

Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death

In the accompanying paper by Weil et al. (1996) we show that staurosporine (STS), in the presence of cycloheximide (CHX) to inhibit protein synthesis, induces apoptotic cell death in a large variety of nucleated mammalian cell types, suggesting that all nucleated mammalian cells constitutively express all of the proteins required to undergo programmed cell death (PCD). The reliability of that conclusion depends on the evidence that STS-induced, and (STS + CHS)-induced, cell deaths are bona fide examples of PCD. There is rapidly accumulating evidence that some members of the Ced-3/Interleukin-1 beta converting enzyme (ICE) family of cysteine proteases are part of the basic machinery of PCD. Here we show that Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a cell-permeable, irreversible, tripeptide inhibitor of some of these proteases, suppresses STS-induced and (STS + CHX)-induced cell death in a wide variety of mammalian cell types, including anucleate cytoplasts, providing strong evidence that these are all bona fide examples of PCD. We show that the Ced-3/ICE family member CPP32 becomes activated in STS- induced PCD, and that Bcl-2 inhibits this activation. Most important, we show that, in some cells at least, one or more CPP32-family members, but not ICE itself, is required for STS-induced PCD. Finally, we show that zVAD-fmk suppresses PCD in the interdigital webs in developing mouse paws and blocks the removal of web tissue during digit development, suggesting that this inhibition will be a useful tool for investigating the roles of PCD in various developmental processes.     

Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons.

Sympathetic neurons undergo programmed cell death (PCD) upon deprivation of nerve growth factor (NGF). PCD of neurons is blocked by inhibitors of the interleukin-1beta converting enzyme (ICE)/Ced-3-like cysteine protease, indicating involvement of this class of proteases in the cell death programme. Here we demonstrate that the proteolytic activities of the proteasome are also essential in PCD of neurons. Nanomolar concentrations of several proteasome inhibitors, including the highly selective inhibitor lactacystin, not only prolonged survival of NGF-deprived neurons but also prevented processing of poly(ADP-ribose) polymerase which is known to be cleaved by an ICE/Ced-3 family member during PCD. These results demonstrate that the proteasome is a key regulator of neuronal PCD and that, within this process, it is involved upstream of proteases of the ICE/Ced-3 family. This order of events was confirmed in macrophages where lactacystin inhibited the proteolytic activation of precursor ICE and the subsequent generation of active interleukin-1beta.     

ICE/CED-3 proteasesin apoptosis

An expanding family of cysteine proteases, of which interleukin-1beta-converting enzyme (ICE) is the prototype, has been shown to play a key role in mammalian cell apoptosis. ICE is both a structural and functional homologue of the nematode 'death gene' ced-3. Here, Moira Whyte discusses how functional characterization of these ICE-like proteases and identification of their substrates is helping to elucidate the biochemical processes underlying the stereotyped morphology of apoptosis and to identify potential targets for therapy.     

Inflammatory caspases and inflammasomes: master switches of inflammation

Fifteen years have passed since the cloning and characterization of the interleukin-1beta-converting enzyme (ICE/caspase-1), the first identified member of a family of proteases currently known as caspases. Caspase-1 is the prototypical member of a subclass of caspases involved in cytokine maturation termed inflammatory caspases that also include caspase-4 caspase -5, caspase -11 and caspase -12. Efforts to elucidate the molecular mechanisms involved in the activation of these proteases have uncovered an important role for the NLR family members, NALPs, NAIP and IPAF. These proteins promote the assembly of multiprotein complexes termed inflammasomes, which are required for activation of inflammatory caspases. This article will review some evolutionary aspects, biochemical evidences and genetic studies, underlining the role of inflammasomes and inflammatory caspases in innate immunity against pathogens, autoinflammatory syndromes and in the biology of reproduction.     

人肺癌细胞CPP32基因的克隆及表达

蛋白酶尤其是ＩＣＥ家族的蛋白酶是细胞死亡机制的核心成分．ＩＣＥ蛋白酶家族中,ＣＰＰ３２（又称Ｙａｍａ,ａｐｏｐａｉｎ）在不同形式的凋亡途径中起核心作用．为深入研究ＣＰＰ３２的结构与功能,克隆了ＣＰＰ３２基因,并在大肠杆菌中进行了表达．采用ＲＴ－ＰＣＲ技术从人肺癌细胞株中获得了ＣＰＰ３２蛋白酶基因．ＤＮＡ序列分析表明,该基因由已报道的编码ＣＰＰ３２αｐ２０亚单位和ＣＰＰ３２βｐ１０亚单位的核苷酸组成,提示ＩＣＥ家族蛋白酶寡聚化可能受ＤＮＡ水平调控．将获得的ＣＰＰ３２基因分别重组到ｐＢＶ３２１和ｐＥＸ３１Ｂ载体上,并分别转化到大肠杆菌中,均获得了ＣＰＰ３２基因的较高表达,表达产物主要以包涵体形式存在．     

A novel human protease similar to the interleukin-1 beta converting enzyme induces apoptosis in transfected cells.

We have identified a novel cDNA encoding a protein (named TX) with > 50% overall sequence identity with the interleukin-1 beta converting enzyme (ICE) and approximately 30% sequence identity with the ICE homologs NEDD-2/ICH-1L and CED-3. A computer homology model of TX was constructed based on the X-ray coordinates of the ICE crystal recently published. This model suggests that TX is a cysteine protease, with the P1 aspartic acid substrate specificity retained. Transfection experiments demonstrate that TX is a protease which is able to cleave itself and the p30 ICE precursor, but not to generate mature IL-1 beta from pro-IL-1 beta. In addition, this protein induces apoptosis in transfected COS cells. TX therefore delineates a new member of the growing Ice/ced-3 gene family coding for proteases with cytokine processing activity or involved in programmed cell death.     

Prevention of apoptosis in CNTF-dependent neurons by a mutant ICE and by viral protein CrmA but not by proto-oncogene product Bcl-2

The interleukin-1beta converting enzyme (ICE) gene family, (homologues of C. elegans cell death gene product Ced-3) plays an important role in controlling programmed cell death. Nerve growth factor (NGF) promotes survival of cultured embryonic chicken dorsal root ganglion neurons. Ciliary ganglion neurons depend exclusively on ciliary neurotrophic factor (CNTF) for survival. Complete depletion of NGF or CNTF from culture medium induces apoptosis in both types of neurons. We can prevent apoptosis, due either to NGF or CNTF withdrawal and in either type of neuron, by overexpression of a mutant inactive ICE and an ICE inhibitor, the product of cowpox virus gene crmA. Bcl-2 does not prevent apoptosis in CNTF-dependent ciliary neurons or DRG neurons as it does in NGF-dependent neurons. These results suggest that neuronal cell death is mediated through a common effector mechanism involving the Ice family of genes, whereas different suppression mechanisms are engaged depending upon the specific neurotrophic factors present.     

The current ICE age: biology and evolution of SXT-related integrating conjugative elements

SXT is an integrating conjugative element (ICE) that was initially isolated from a 1992 Vibrio cholerae O139 clinical isolate from India. This approximately 100-kb ICE encodes resistance to multiple antibiotics. SXT or closely related ICEs are now present in most clinical and some environmental V. cholerae isolates from Asia and Africa. SXT-related ICEs are not limited to V. cholerae. It is now clear that so-called IncJ elements such as R391 are closely related to SXT. More than 25 members of the SXT/R391 family of ICEs have now been identified in environmental and clinical isolates of diverse species of gamma-proteobacteria worldwide. In this review, we discuss the diversity, evolution and biology of this family of ICEs.     

ICE processing and kinetic mechanism

Interleukin-1β converting enzyme (ICE) has been the focus of major scientific efforts to discover pharmaceutically effective inhibitors. Little is known about the rates of the individual steps in catalysis. We report here that the rates of the two individual chemical steps in catalysis (acylation and deacylation) are each partially rate-limiting. This keeps the overall rate of the reaction less than 3% of the rate of the reaction for papain with its optimized substrate. Eight human ICE-like proteases have been published to date. They have levels of sequence identity that range from around 30% to greater than 50% throughout the full lengths of the proteins. This degree of relatedness increases when only the active domains are compared. This indicates that the greatest variability between family members occurs in their N-terminal prodomains. We propose several possibilities for the role for these prodomains in the regulation of enzyme processing. J. Cell. Biochem. 64:11–18. © 1997 Wiley-Liss, Inc.     

In vitro and in vivo studies of ICE inhibitors

Interleukin-1β-converting enzyme (ICE) is a cysteine protease responsible for proteolytic activation of the biologically inactive interleukin-1β precursor to the proinflammatory cytokine. ICE and homologous proteases also appear to mediate intracellular protein degradation during programmed cell death. Inhibition of ICE is a new antiinflammatory strategy being explored by the design of both reversible inhibitors and irreversible inactivators of the enzyme. Such compounds are capable of blocking release of interleukin-1β from human monocytes. ICE inhibitors that cross react against multiple ICE homologs can also block apoptosis in diverse cell types. ICE inhibitors impart protection in vivo from endotoxin-induced sepsis and collagen-induced polyarthritis in rodent models. Further optimization of the current generation of peptidyl ICE inhibitors will be required to produce agents suitable for administration in chronic inflammatory and neurodegenerative diseases. J. Cell. Biochem. 64:19–26. © Wiley-Liss, Inc.     

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.     

Synthesis and evaluation of tricyclic pyrrolopyrimidinones as dipeptide mimetics: inhibition of interleukin-1beta-converting enzyme

The application of a tricyclic pyrrolopyrimidinone scaffold for the synthesis of peptidomimetic inhibitors of interleukin-1beta-converting enzyme (ICE) is reported. The synthesis of the tricyclic scaffold and conversion of it to a variety of target ICE inhibitors were accomplished in 4-5 steps. In vitro biological evaluation of the tricyclic pyrrolopyrimidinones revealed fair to good ICE inhibitors, with the most active compound exhibiting an IC50 of 14 nM in a caspase-1 enzyme binding assay.