Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Review

Noncaspase proteases in apoptosis

Abstract

Biochemical and genetic analysis of apoptosis has determined that intracellular proteases are key effectors of cell death pathways. In particular, early studies have pointed to the primacy of caspase proteases as mediators of execution. More recently, however, evidence has accumulated that noncaspases, including cathepsins, calpains, granzymes, and the proteasome complex, also have roles in mediating and promoting cell death. An important goal is to understand the importance of distinct noncaspases in various forms of apoptosis, and to determine whether pathways mediated by noncaspase proteases intersect with those mediated by caspases. In this review the roles of noncaspase proteases in the biochemistry of apoptosis will be discussed. Leukemia (2000) 14, 1695–1703.

Introduction

The demise of cells via apoptosis hinges on the activation of cellular proteases. Elucidation of the specific proteases involved in apoptotic execution began in the early 1990s with genetic and biochemical studies of apoptotic cell death in the nematode C. elegans. These studies revealed that apoptosis in C. elegans was dependent on an intracellular protease bearing considerable homology to the human interleukin-1β converting enzyme, or ICE.123 Subsequent studies have led to the identification of 13 mammalian proteases that are related to ICE, and these proteases, along with ICE, have been termed caspase proteases (caspase-1 to caspase-13).45 The term caspase, or c-aspase, reflects the fact that these enzymes are cysteine proteases that cleave substrate proteins after aspartate residues. Caspases are initially synthesized in the cell as inactive zymogens. However, in response to an apoptotic stimulus, initiator caspases such as caspase-8 or -9 undergo processing to active forms.5 Active initiator caspases then cleave and activate downstream executioner caspases such as caspase-3 or -7, thereby initiating a cascade of caspase activation. Once activated, executioner caspases cleave cellular substrate proteins promoting the destruction of the cell.

The role of caspases in mammalian apoptosis has been intensively investigated using a variety of approaches: (1) pharmacologic inhibition of caspases using peptide inhibitors; (2) inhibition of caspases with endogenous or viral inhibitory proteins; and (3) targeted gene disruption. These studies have shown that inhibition of caspases significantly delays apoptosis induced by a wide variety of stimuli in a variety of different cell types. Moreover, gene knockout experiments have shown that different members of the caspase family appear to be important for apoptosis execution in different cell types.

While the importance of caspases in apoptosis is clearly established, recent studies have indicated that several other types of proteases also may play a role in the execution process. The involvement of noncaspase proteases is suggested by observations that inhibition of caspase proteases generally causes delays, but does not fully block, cell death resulting from most apoptotic stimuli. In addition, implicated noncaspases frequently are upregulated or activated during apoptosis, and their inhibition, as with caspase inhibition, can serve to delay apoptosis. The noncaspase proteases that have been most closely linked with apoptosis are cathepsins, calpains, granzymes, and the proteasome. This review will focus on the current state of knowledge regarding the involvement and role of cathepsins, calpains, and granzymes in the execution process. The role of the proteasome complex, which has been strongly implicated in apoptosis, has been recently reviewed elsewhere.6

Cathepsins and apoptosis

The cathepsin protease family consists of at least 12 known members.78 Cathepsins can be subdivided into three distinct groups, based on the amino acid that comprises the active site residue: (1) serine proteases (cathepsins A and G), (2) cysteine proteases (cathepsins B, C, H, K, L, S, and T), and (3) aspartate proteases (cathepsins D and E). Like the caspases, cathepsins are synthesized as inactive zymogens, and activation involves proteolytic processing.910111213 As discussed below, the involvement of cathepsins in apoptotic cell death has primarily been studied in nonhematopoietic systems. Evidence from these systems, however, should provide the basis for investigating the role of cathepsins in the death of both normal and malignant hematopoietic cells.14

The most extensive evidence linking cathepsins with apoptosis has come from studies of the cysteine protease, cathepsin B, and the aspartate protease, cathepsin D. Therefore, this review will focus on these two members of the cathepsin family. Both cathepsin B and cathepsin D are found primarily in lysosomes or endosomes. Historically, it has been presumed that these proteases are mainly involved in the terminal degradation of proteins within the lysosomal compartment. However, both proteases can also be secreted, and once secreted can degrade collagen, fibronectin, laminin, and proteoglycans.915 The degradation of extracellular matrix components by cathepsins B and D accounts for the ability of these proteases to promote cell migration or malignant invasion.16171819 Indeed, overexpression and secretion of cathepsin D, which can be induced by estrogen, closely correlates with high metastatic potential in breast cancer.20212223 Additional evidence (described below) suggests that cathepsins B and D are translocated to the cytoplasm during apoptosis. This is significant, since most of the known apoptosis execution pathways occur in the cytoplasm. Thus, the translocation of cathepsins to the cytoplasm may allow these proteases to intersect with and augment other apoptosis signaling mechanisms.

Early evidence suggesting a role for cathepsins B and D in apoptosis came from studies of apoptotic cell death in rat prostate and mammary tissues. Following androgen withdrawal in prostate, or following lactation in breast, massive apoptosis occurs in the epithelial cells of these tissues.2425 Concurrent with this apoptosis, there is upregulation of mRNA and protein for both cathepsin B and D.2627 While these initial findings suggested a role for cathepsins in regressing prostate and mammary gland, they did not directly demonstrate cathepsin involvement in the execution pathways. It remained possible that cathepsins were simply needed for ‘cleanup’ in the dying cells.

More definitive experiments showing a role for cathepsins in apoptotic execution have come from studies of bile salt-induced apoptosis in cultured hepatocytes. This in vitro model mimics apoptosis that occurs in human hepatocytes in diseases where normal bile flow is impaired. In cell culture, bile salt-induced apoptosis was found to be markedly inhibited by CA-074-Me, a specific inhibitor of cathepsin B, and by pepstatin A, an inhibitor of cathepsin D.2829 Antisense-mediated downregulation of cathepsin B also markedly inhibited hepatocyte apoptosis following treatment with bile salts.28 Together, these findings showed that, similar to caspases, cathepsins B and D, can be important components of apoptosis execution pathways. Interestingly, pepstatin A was found to inhibit bile salt-induced cathepsin B activation, while CA-074-Me failed to impact cathepsin D activation.29 This revealed that during bile salt-induced apoptosis, cathepsin D is activated upstream of cathepsin B. Thus, like the caspases, cathepsins may be activated in a cascade-like fashion during apoptosis. In support of this idea, cathepsin D has been shown to directly cleave and activate cathepsin B.3031

Cathepsins B and D exert opposing effects on apoptosis in serum- and neurotrophic factor-deprived neuronal cells. During apoptosis of serum-deprived rat PC12 cells, the levels of cathepsin B protein decline while those of cathepsin D increase.32 Interestingly, cathepsin D levels are also elevated in the pyramidal neurons of Alzheimers patients.33 In PC12 cells, inhibition of cathepsin B with CA-074, or downregulation of cathepsin B with antisense oligonucleotides resulted in enhanced apoptosis.34 By contrast, inhibition of cathepsin D with pepstatin A significantly inhibited PC12 cell apoptosis. Similar results were seen using dorsal root ganglion neurons deprived of nerve growth factor.34 It will be interesting to see whether these findings can be extended to hematopoietic cells. Many hematopoietic cells are dependent on specific cytokines for survival, and undergo apoptosis when deprived of these essential factors.3536 Future studies are needed to define the role and involvement of cathepsins during cytokine withdrawal-induced apoptosis.3738

The involvement of cathepsin D in the execution pathways triggered by a variety of other stimuli has been demonstrated by Deiss et al.39 This group isolated antisense cathepsin D cDNA while screening an antisense library for clones that could inhibit IFN-γ-induced apoptosis in HeLa cells. In addition to apoptosis caused by IFN-γ, antisense cathepsin D also markedly inhibited Fas-induced apoptosis. Moreover, pepstatin A was found to inhibit both forms of cell death, as well as apoptosis induced by TNF-α treatment of U937 histiocytic lymphoma cells. IFN-γ- and TNF-α-induced apoptosis were also associated with induction and processing of cathepsin D protein.

Cathepsin D also participates in the execution of cells treated with chemotherapy drugs or radiation. Wu et al,40 using a subtractive hybridization strategy, identified cathepsin D mRNA as an upregulated transcript in doxorubicin-treated cells. Induction of cathepsin D protein was observed in ML1 leukemic cells treated with doxorubicin, etoposide, or γ-irradiation. Upregulation of cathepsin D may be impacted by p53, as two p53 binding sites were found in the promoter region of cathepsin D. Wu et al40 further showed that drug-induced apoptosis was inhibited by pepstatin A. Moreover, fibroblasts derived from cathepsin D−/− gene knockout mice displayed enhanced resistance to adriamycin and etoposide relative to fibroblasts from wild-type mice.40 Similar studies by Roberts et al29 have suggested the involvement of both cathepsin B and cathepsin D in camptothecin-induced apoptosis of a hepatocyte cell line (Hep3B). Following treatment of Hep3B cells with camptothecin, an increase in the cellular activity of cathepsin B was observed, as measured by cleavage of z-ValLeuLys-7-amino-4-chloromethylcoumarin. Increased activity of cathepsin D, as measured with the substrate D-PheSerPhePheAlaAla-p-aminobenzoate, was also detected. Inhibition of cathepsins B and D with CA-074-Me and pepstatin A, respectively, markedly diminished camptothecin-induced apoptosis. Also, as observed in bile salt-induced apoptosis of hepatocytes, cathepsin D is upstream of cathepsin B in the camptothecin-induced cell death pathway.

While inhibition of cathepsins clearly delays some forms of apoptosis, any discussion of the role of cathepsins in apoptotic execution should consider the issue of subcellular localization. Apoptosis signal transduction pathways have been found to occur in the cytoplasm, on the inner surface of the plasma membrane, in mitochondria, and in the nucleus.5414243444546 In particular, caspase-mediated signaling occurs predominantly in the cytoplasm.54748 By contrast, cathepsins B and D are localized in lysosomes, or are secreted by the cell. This raises the question as to how these enzymes can facilitate apoptosis signaling. An emerging line of evidence suggests that during apoptosis, cathepsins B and D are translocated from lysosomes to other subcellular localizations. Three studies have utilized gold-labeled antibodies followed by electron microscopic immunocytochemistry to examine cathepsin D localization during apoptosis.495051 Li et al50 found that treatment of the mouse macrophage cell line J-774 or human macrophages with apoptosis-inducing oxidized LDL changed the pattern of cathepsin D staining from granular lysosomal to diffuse cytoplasmic. Similar results were seen in rat myocytes treated with the quinone naphthazarin,51 and in cells treated with hydrogen peroxide.49 In the case of bile salt-induced apoptosis of hepatocytes, Roberts et al28 performed subcellular fractionation, followed by Western blotting, and found that preexisting cathepsin B redistributed to the nucleus in treated cells. This group also expressed a cathepsin B-green fluorescent protein chimeric molecule, and observed translocation of the fluorescent signal to the hepatocyte nucleus following treatment with bile salts. Another group has examined the localization of cathepsin B in response to the agent atractyloside.52 Atractyloside is known to induce mitochondrial permeability transition, and subsequent apoptosis. Surprisingly, it was shown that atractyloside caused the release of cathepsin B from highly purified lysosomes. Thus, current evidence indicates that cathepsins may be released from lysosomal compartments during some forms of apoptosis. It remains unclear, however, whether this is a general apoptosis phenomenon, or is restricted to execution induced by only some apoptotic stimuli. In addition, it is unclear whether release of cathepsins from lysosomes is controlled by pores or translocators, or is simply the consequence of damage to lysosomal membranes during the apoptotic process.

To date, relatively little is known about potential intersections of cathepsin- and caspase-mediated pathways, although it seems likely that these pathways will be integrated in some fashion. One report has shown that cathepsin G, which is abundantly expressed in neutrophils, can cleave and activate procaspase-7 in vitro.53 Cathepsin G cleaves procaspase-7 between the large and small subunits in the zymogen molecule. Another report has demonstrated that cathepsin B can readily cleave caspase-1 and procaspase-11.52 In addition, cathepsin B showed weak cleavage activity towards procaspase-2, -6, -7 and -14. It was not determined, however, whether cathepsin B-mediated cleavage of procaspases resulted in the activation of these enzymes. Also, while cathepsin G and cathepsin B clearly can cleave certain procaspases in vitro, it is not known whether cathepsin-mediated cleavage of caspases occurs in whole cells. Thus, it is difficult to establish whether caspase activation may be upstream or downstream of cathepsin activation during apoptosis. Perhaps the use of highly selective inhibitors will help to address these issues. In this regard, however, a caveat should be raised. Peptides based on caspase cleavage sequences, and derivatized with fluoromethyl ketone or chloromethyl ketone have commonly been used as specific inhibitors of caspases in vitro and in whole cells. Strikingly, a recent study found that z-VAD-FMK, z-DEVD-FMK, and Ac-YVAD-CMK potently inhibited cathepsin B activity both in vitro and in cells.54 Therefore, the impact of these inhibitors on biochemical events may be due to inhibition of either caspases or cathepsins, or both.

Calpains and apoptosis

Calpains comprise a family of cytoplasmic neutral cysteine proteases.5556575859 Both ubiquitously expressed and tissue-specific calpains have been identified.586061 The most ubiquitous calpains are μ-calpain (or calpain I) and m-calpain (calpain II). The μ- and m-calpains have been the focus of most studies involving calpains, and this review will focus exclusively on these two isoforms. It should be kept in mind, however, that tissue-specific calpains may have essential in vivo roles in apoptosis execution. In addition, calpain homologs have been identified in organisms such as C. elegans, where genetic analyses may provide clues regarding the importance of these proteases.62

Both μ-calpain and m-calpain are composed of two subunits, a large catalytic subunit of approximately 80 kDa, and a smaller subunit of approximately 30 kDa.5863 The 80 kDa subunits specific for μ-calpain and m-calpain are encoded by distinct genes, while the 30 kDa subunit is shared between the two isoforms. Both enzymes bind and require Ca++ for optimal activity.555657596465 Although μ- and m-calpains appear to have the same substrate specificity, they are functionally distinquished on the basis of their Ca++ sensitivity. μ-Calpain requires micromolar concentrations of Ca++ for optimal activity, while m-calpain requires millimolar concentrations.

The activation and activity of calpains is influenced by several factors. As mentioned, calpains must bind Ca++ in order to be active, and sequences resembling EF-hand Ca++ binding motifs are found in both the 80 and 30 kDa subunits.66 Another interesting feature of the calpains is their ability to associate with membrane phospholipids. Membrane association occurs in response to certain cellular stimuli, and this association may lower the intrinsic Ca++ requirements of these enzymes.67 A third factor influencing calpain activity may be autolysis. Following the binding of Ca++, calpains undergo autolysis, with cleavages occurring near the amino terminii of the large and small subunits.686970717273 Autolysis appears to increase activity and lower the requirement for Ca++. Although it remains controversial as to whether autolysis is absolutely required for activation, Western blotting, looking for conversion of calpains to lower molecular weight forms, is a convenient and frequently used means of assessing calpain activation in cells. Finally, a fourth factor influencing calpain activity is the presence of calpastatin, a 110 kDa endogenous protein inhibitor.7475 Calpastatin is commonly expressed and is highly specific for calpains; it is not known to inhibit any other proteases. The expression and function of calpastatin may be regulated during apoptosis by other proteases, including caspases (discussed below).

Calpains have been implicated in apoptosis based on two types of observations: (1) the activation of calpains during cell death and (2) the inhibition of apoptotic execution by various calpain inhibitors. Calpain activation has been assessed using several different assays including, Western blotting to detect calpain autolysis, Western blotting to detect cleavage of known calpain substrate proteins, and in vitro and whole cell assays to detect cleavage of the fluorogenic calpain substrate N-succinyl-leu-leu-val-tyr-7-amido-4-methylcourmarin (N-s-LLVY-AMC). Using these and a few less common assays, calpain activation has been detected in response to a variety of apoptotic stimuli, in a variety of different cell types. For example, in murine thymocytes treated with dexamethasone, calpain activity increases rapidly, peaking within 1 h of treatment.76 Also, in freshly isolated, apoptosis-prone, human neutrophils, calpain enzymes are found to be constitutively active.77 Treatment of BL30A Burkitt’s lymphoma cells with radiation induces rapid calpain activation (15 min), as measured by cleavage of the calpain substrate fodrin.78 By contrast, incubation of HL-60 cells with 9-amino-20(S)-camptothecin causes slow autolysis/activation of calpain (8–10 h) compared with rapid activation of caspase-3 (2 h;79). Activation of calpains has also been observed in nonhematopoietic cells. In neuronally differentiated PC12 cells, induction of apoptosis by ceramide analogs stimulates calpain-mediated cleavage of N-s-LLVY-AMC.80 Similar activation of N-s-LLVY-AMC cleavage activites are seen during apoptosis caused by reovirus infection of cultured fibroblasts (81) or in vivo ischemia-reperfusion injury in rat hepatocytes.82

In addition to observations of calpain activation in experimental apoptosis, activation or overexpression of calpains has also been seen in human diseases marked by excessive cell death. For example, considerable activation of calpains is seen in brain tissue from patients with Alzheimer’s disease when compared with normal brain tissue.8384 Moreover, Alzheimer’s brains also exhibit significantly reduced expression of the calpain inhibitor calpastatin.84 In Parkinson’s disease, elevated expression of m-calpain is found in the mesencephalon region of the brain.85 Thus, it is possible that overzealous expression and/or activation of calpains may contribute to inappropriate cell loss in some important pathological conditions.

While the activation of calpains during apoptosis suggests an involvement of these enzymes in the execution process, it does not directly demonstrate an essential role. An understanding of the importance of calpains in apoptotic execution has come from studies using calpain inhibitors. A number of different calpain inhibitors have been developed. Unfortunately, many of the earlier inhibitors are somewhat nonspecific. Thus, experiments using these inhibitors must be interpreted with caution; it is possible that actions ascribed to calpains may actually be due to other cross-inhibited proteases (eg the proteasome or cysteine proteases in the cathepsin family). The first generation of calpain inhibitors included the active site inhibitors leupeptin and E64 (E64d is the cell permeable derivative of E64).86878889 These inhibitors also exhibit considerable activity against the proteasome and lysosomal cysteine proteases. More recent active site inhibitors include calpain inhibitor I (N-acetyl-leu-leu-norleucinal (aLLnL)), calpain inhibitor II (N-acetyl-leu-leu-methioninal (aLLM)), and calpeptin (benzyloxycarbonyl-leu-norleucinal).888990 Although more specific than leupeptin and E64, these inhibitors also inhibit to some degree lysosomal cysteine proteases and the proteasome. An even more specific calpain inhibitor is the compound PD150606 [3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid], which interacts with Ca++ binding sites in the calpain enzymes.91 Finally, the most specific calpain inhibitor known to exist is the protein calpastatin, which directly binds to calpains and potently inhibits their activities.7475 To date, however, relatively few studies have employed calpastatin to investigate the role of calpains in apoptotic cell death.

As perhaps may have been expected, numerous studies have shown that calpain inhibitors inhibit apoptosis in response to some, but not all, apoptotic stimuli. Studies by Squier et al7692 showed that dexamethasone-induced apoptosis of mouse thymocytes was inhibited by E64d and calpain inhibitor I. It was also found that calpain inhibitor I could inhibit thymocyte apoptosis caused by low-dose radiation, A23187 ionophore, ionomycin, and forskolin; each of these forms of apoptosis requires new RNA synthesis in the thymocyte.92 By contrast, calpain inhibitor I could not prevent apoptosis that is not dependent on RNA synthesis, namely apoptosis caused by heat shock or valinomycin. In human neutrophils, calpain inhibitor I and PD150606 were shown to inhibit apoptosis caused by cycloheximide treatment, but not apoptosis resulting from Fas stimulation.93 During spontaneous neutrophil apoptosis, inhibition of calpains by calpeptin alone, or the proteasome-specific inhibitor, lactacystin, alone had no effect on the rate of cell death.77 However, when used in combination, these inhibitors displayed a synergistic inhibitory effect, indicating synergy between calpains and the proteasome in spontaneous neutrophil apoptosis. Related studies have revealed that antisense-mediated downregulation of calpastatin inhibitor protein significantly accelerates neutrophil apoptosis.93 Studies with HL-60 cells have shown that calpain inhibition by calpeptin fails to block DNA fragmentation and loss of viability caused by treatment with camptothecin.79 However, calpeptin was able to block TNF-induced U937 cell apoptosis, while calpain inhibitor I delayed U937 apoptosis resulting from calphostin treatment.9495 In human platelets, calpeptin blocked A23187-induced caspase fragmentation, cleavage of apoptosis substrate proteins, and microvesiculation.96 Studies with nonhematopoietic cells have shown that calpain inhibitor I and PD150606 blocked reovirus-induced apoptosis in murine fibroblasts, and calpain inhibitors I and II blocked TGF-β-induced DNA fragmentation and loss of viability in rat hepatocytes.8197 In addition, calpain inhibitor I blocked apoptosis in chick ciliary neurons deprived of CNTF, and the calpain inhibitor MDL 28,170 inhibited the death of rat hippocampal pyramidal neurons treated with β-amyloid peptide or staurosporin.9899 Taken together, the studies described above show that calpains can play important roles in the apoptotic execution of both hematopoietic and nonhematopoietic cell types.

The ability of calpains to promote apoptosis in a variety of systems raises questions regarding the molecular mechanism(s) of calpain action. Undoubtedly, the cleavage of specific substrate proteins accounts for the proapoptotic activity of calpain, since the catalytic activity of the enzyme is required. A large variety of proteins have been shown to be calpain substrates, including actin, alpha-actinin, talin, filamin,77100 fodrin,94101 gelsolin,96 FAK,102 integrin β3,103 PKC α/β/γ,104 calcium/calmodulin-dependent kinase IV,105 c-mos,106 c-fos, c-jun,107108 cyclin D1,109 p53,110 procaspase-3 and -9,96 and Bax.111 This group of substrate proteins is clearly very diverse. However, it should be noted that several of these proteins are cytoskeletal proteins or proteins that can associate with cell membranes. This has led to speculation that calpains may be particularly important in destruction of cellular architecture during apoptosis. The impact of calpain-mediated cleavage of the apoptosis regulatory proteins p53, Bax, and procaspase-3 and -9 is presently unclear. In the case of caspase-3 and -9, calpain cleavage neither activates nor inactivates the caspase enzyme.96 Unfortunately, many of the proteins identified as calpain substrates have only been shown to be cleaved using in vitro experiments. Thus, it remains unclear how many of these proteins are directly cleaved by calpains in vivo. Also, it is currently unknown which substrates may be priority substrates, whose cleavage is critical to the proapoptotic action of calpain.

A number of studies have indicated cross-talk between calpain-mediated pathways and caspase-mediated pathways. In some cases, caspase activation has been reported to be upstream of calpain activation, while in other cases the opposite has been found. Wood and Newcomb79 determined that calpain activation in camptothecin-treated HL-60 cells can be blocked by the pancaspase inhibitor z-VAD-FMK. By contrast, the calpain inhibitor calpeptin failed to block activation of caspases. Interestingly, two groups have reported that activated caspases cleave and inactivate calpastatin inhibitor.112113 Caspase-mediated calpastatin cleavage was seen in Jurkat and U937 cells following stimulation of Fas or TNF receptors, or following treatment with staurosporin. It remains undetermined, however, whether cleavage of calpastatin inhibitor is entirely responsible for the calpain activation in these cells. An opposite relationship between calpains and caspases has been reported during radiation-induced apoptosis. Waterhouse et al78 observed that following treatment of BL30A lymphoma cells with radiation, calpain was activated early, and caspases were activated significantly later. Moreover, inhibition of calpain activity abrogated downstream activation of caspase-3. In yet another study, calpain activation was found to occur independently of caspase activation during activation of platelets.96 Given these different findings, it seems likely that the relationship between caspases and calpains may vary depending on the cell type and apoptotic stimulus.

Granzyme B and apoptosis

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are important for the host defense against viruses, parasitic agents, and transformed cells.114115 CTLs and NK cells induce apoptosis in target cells using at least two distinct mechanisms. One mechanism involves stimulation of cell surface death receptors (such as Fas) on the target cells by death ligands expressed on the surface of the effector cell.116117 This leads to activation of caspase cascades in the target cell. Another mechanism, termed ‘granule exocytosis’, involves the vectoral transfer of the contents of effector cell cytoplasmic granules into the target cell.118119120 Key components of these granules are perforin and the granzyme family of serine proteases.

Perforin is a 70 kDa protein that binds to membrane phosphorylcholine groups in a calcium-dependent manner.121122123 Following binding, perforin inserts into the membrane and oligomerizes, forming pores. This permeabilization of the membrane may facilitate the entry of other molecules, including granzymes, into the target cell.

Within the granules of CTLs and NK cells, granzymes A and B are particularly abundant.124 Granzyme B (also called fragmentin or cytotoxic T cell protease (CCP)) shares with caspases the unique characteristic of cleaving substrate proteins after aspartate residues.125126127128129 An important role for granzyme B in the induction of target cell apoptosis has been demonstrated using gene knockout mice. CTLs and NK cells derived from granzyme B−/− mice exhibit greatly reduced capacity to induce apoptotic DNA fragmentation in target cells.119130 Earlier complementary studies showed that purified granzyme B alone did not promote apoptosis when added to target cells. However, cotreatment with purified granzyme B and perforin proteins induced marked DNA fragmentation and apoptotic features in four lymphoma target cell lines.129 Based on the ability of perforin to form membrane pores, it has been the general consensus that granzyme B gains entry into target cells through perforin pores. This mode of entry, however, is currently being called into question. Several studies have shown that granzyme B is internalized by target cells in the absence of added perforin.131132133134135 The internalized granzyme B has been reported to reside in the cytoplasm,132133 or in a novel vesicular compartment.134 The triggering of apoptosis in cells that have internalized granzyme B requires further addition of perforin to the cells.131132133134135 It may be that perforin is required for the release of granzyme B from internal vesicles in the target cell. Other studies have indicated that perforin facilitates translocation of granzyme B to the nucleus, and that nuclear localization is critical to the ability of granzyme B to cause apoptosis.132133134135136137

While the importance of granzyme B subcellular localization remains controversial, it is quite clear that granzyme B has the ability to impact the caspase pathway of apoptosis. Studies done in vitro, have shown that granzyme B is capable of cleaving procaspase-3, -6, -7, -8, -9 and -10.138139140141142143144145146147148149150151152153 In the case of procaspases-3, -7 and -9, granzyme B-mediated processing has been shown to generate active caspase enzymes.138141146149 More importantly, studies with whole cells have shown that caspases are activated in target cells following coincubation with granzyme B and perforin.139153154 It remains to be determined, however, which caspases are the preferred in vivo substrates for granzyme B. In any event, it is reasonable to propose that granzyme B may promote apoptosis simply by cleaving and activating endogenous caspases in the target cell.

In cells undergoing granzyme B/perforin-mediated apoptosis, cleavage of the caspase substrate proteins PARP, lamin B, and U1–70 kDa is also observed.151153154155 These cleavage events appear to be due to caspases activated by granzyme B, and not the result of direct granzyme B cleavage, since cleavage of all three proteins is inhibited by 100 μM DEVD- or VAD-containing peptides (100 μM DEVD-CHO or VAD peptides inhibit caspases, but not granzyme B).139151153154155 Two additional caspase substrate proteins, DNA-PKcs and NuMA, are also cleaved in granzyme B/perforin-treated cells, but cleavage of these proteins is insensitive to DEVD or VAD peptide inhibitors.155 Moreover, the sizes of the DNA-PKcs and NuMA proteolytic fragments generated by granzyme B differ from those resulting from caspase cleavage. This indicates that during granzyme B-mediated apoptosis, important cellular substrates are cleaved in a caspase-independent fashion. The importance of these caspase-independent cleavage events remains to be determined. However, the fact that granzyme B/perforin-mediated DNA fragmentation and apoptotic death is significantly delayed by 100 μM DEVD/VAD,139153154 underscores the necessity for caspase activation during this form of apoptosis.

Granzyme A and apoptosis

Granzyme A is the most abundant protease found in the granules of CTL cells. The mature granzyme A enzyme is a disulphide cross-linked homodimer of 50 kDa that cleaves substrate proteins following lysine or arginine residues.127156157 Although granzyme A is capable of inducing apoptosis after loading into target cells, the mechanism of action of this protease differs significantly from that of granzyme B. In addition, based on the experimental systems that have been employed thus far, it appears that the role of granzyme A in CTL-induced apoptosis is far more subtle than that of granzyme B. Mice which are deficient in granzyme A expression (granzyme A−/− mice), exhibit relatively normal CTL-mediated cytotoxicity.158 The only reported defect for granzyme A−/− mice is an inability to clear the mouse pox virus Ectromelia.159 By contrast, CTLs from granzyme B−/− mice are capable of inducing target cell death only after prolonged coincubation.130 Thus, granzyme B is critically important for rapid CTL killing. The possibility that granzyme A does have some role in CTL-mediated killing, has been suggested by recent experiments using mice that are deficient in both granzyme A and granzyme B. CTLs from granzyme A−/−/granzyme B−/− mice are unable to induce target cell DNA fragmentation, even after prolonged coincubation.160 This indicates that granzyme A activity accounts for the ability of granzyme B−/− CTLs to induce target cell apoptosis after prolonged exposure. Therefore, granzyme A may allow CTLs to kill target cells under conditions where granzyme B activity is inhibited (eg target cells that express granzyme B inhibitors).

Studies using recombinant proteins have shown that coincubation of granzyme A and perforin with target cells leads to rapid (within 2 h) accumulation of DNA single-strand breaks.161162 This contrasts with the rapid degradation of DNA to oligonucleosomal-length fragments seen in cells treated with granzyme B and perforin. Granzyme A/perforin treatment also leads to nuclear condensation.162 The DNA single-strand breakage and nuclear condensation that occurs in response to granzyme A is insensitive to caspase inhibitors, indicating that these actions of granzyme A are caspase-independent.162 Consistent with this, granzyme A/perforin treatment does not result in processing/activation of procaspase-3 or cleavage of the caspase substrate proteins PARP, lamin B, or rho-GTPase.162 By contrast, granzyme B-induced DNA fragmentation is strictly dependent on the activation of caspases. Both granzyme A and granzyme B (in conjunction with perforin) also induce target cell cytolysis, and in both cases this is a caspase-independent event. Taken together, current evidence indicates that granzyme B is the primary CTL mediator of target cell DNA fragmentation and apoptotic death, and that the apoptotic effects of this protease are mediated primarily through the activation of caspases. Granzyme A, on the other hand, may be more of a default or specialized mediator of target cell apoptosis, with the pathways initiated by granzyme A being distinctly different from those initiated by granzyme B.

References

  1. 1

    Yuan J, Shaham S, Ledoux S, Ellis HM, Horvitz HR . The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1β-converting enzyme Cell 1993 75: 641–652

    CAS  Article  Google Scholar 

  2. 2

    Cerretti DP, Kozlosky CJ, Mosley B, Nelson N, Van Ness K, Greenstreet TA, March CJ, Kronheim SR, Druck T, Cannizzaro LA, Huebner K, Black RA . Molecular cloning of the interleukin-1β converting enzyme Science 1992 256: 97–100

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Thornberry NA, Bull HG, Calaycay JR, Chapman KT, Howard AD, Kostura MJ, Miller DK, Mokineaux SM, Weidner JR, Aunins J, Elliston KO, Ayala JM, Casano FJ, Chin J, Ding GJ-F, Egger LA, Gaffney EP, Limjuco G, Palyha OC, Raju SM, Rolando AM, Salley JP, Yamin T-T, Lee TD, Shively JE, MacCross M, Mumford RA, Schmidt JA, Tocci MJ . A novel heterodimeric cysteine protease is required for interleukin-1β processing in monocytes Nature 1992 356: 768–774

    CAS  Google Scholar 

  4. 4

    Alnemri ES, Livingston DJ, Nicholson DW, Salvesen G, Thornberry NA, Wong WW, Yuan J . Human ICE/CED-3 protease nomenclature Cell 1996 87: 171

    CAS  Article  Google Scholar 

  5. 5

    Thornberry NA, Lazebnik Y . Caspases: enemies within Science 1998 281: 1312–1316

    CAS  Google Scholar 

  6. 6

    Orlowski RZ . The role of the ubiquitin-proteasome pathway in apoptosis Cell Death Differ 1999 6: 303–313

    CAS  Google Scholar 

  7. 7

    Schwartz MK . Tissue cathepsins as tumor markers Clin Chem Acta 1995 237: 67–78

    CAS  Google Scholar 

  8. 8

    Chapman HA, Riese RJ, Shi GP . Emerging roles for cysteine proteases in human biology Ann Rev Physiol 1997 59: 63–88

    CAS  Google Scholar 

  9. 9

    Westley B, Rochefort H . A secreted glycoprotein induced by estrogen in human breast cancer cell lines Cell 1980 20: 353–362

    CAS  Google Scholar 

  10. 10

    Capony F, Rougeot C, Montcourrier P, Cavailles V, Salazar G, Rochefort H . Increased secretion, altered processing and glycosylation of procathepsin D in mammary cancer cells Cancer Res 1989 49: 3904–3909

    CAS  PubMed  Google Scholar 

  11. 11

    Erickson AH . Biosynthesis of lysosomal endopeptidases J Cell Biochem 1989 40: 31–41

    CAS  PubMed  Google Scholar 

  12. 12

    Fujita H, Tanaka Y, Noguchi Y, Kono A, Himeno M, Kato K . Isolation and sequencing of a cDNA clone encoding rat liver lysosomal cathepsin D and the structure of three forms of mature enzymes Biochem Biophys Res Commun 1991 179: 190–196

    CAS  PubMed  Google Scholar 

  13. 13

    Godbold GD, Ahn K, Yeyeodu S, Lee LF, Ting JP, Erickson AH . Biosynthesis and intracellular targeting of the lysosomal aspartic proteinase cathepsin D Adv Exp Med Biol 1998 436: 153–162

    CAS  PubMed  Google Scholar 

  14. 14

    Kornblau SM . The role of apoptosis in the pathogenesis, prognosis, and therapy of hematologic malignancies Leukemia 1998 12: (Suppl1) 41–46

    Google Scholar 

  15. 15

    Mort JS, Recklies AD . Interrelationship of active and latent secreted human cathepsin B precursors Biochem J 1986 233: 57–63

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Sloane BF, Honn KV . Cysteine proteinases and metastasis Cancer Metast Rev 1984 3: 249–263

    CAS  Google Scholar 

  17. 17

    Briozzo P, Morisset M, Capony F, Rougeot C, Rochefort H . In vitro degradation of extracellular matrix with Mr 52,000 cathepsin D secreted by breast cancer cells Cancer Res 1988 48: 3688–3692

    CAS  PubMed  Google Scholar 

  18. 18

    Leto G, Gebbia N, Rausa L, Tumminello FM . Cathepsin D in the malignant progression of neoplastic disease Anticancer Res 1992 12: 235–240

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Mignatti P, Rifkin DB . Biology and biochemistry of proteinases in tumor invasion Physiol Rev 1993 73: 161–195

    CAS  PubMed  Google Scholar 

  20. 20

    Thorpe SM, Rochefort H, Garcia M, Freiss G, Christensen IJ, Khalaf S, Paolucci F, Pau B, Rasmussen BB, Rose C . Association between high concentration of 52-kDa cathepsin D and poor prognosis in primary human breast cancer Cancer Res 1989 49: 6008–6014

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Tandon AK, Clark GM, Chamness GC, Chirgwin JM, McGuire WL . Cathepsin D and prognosis in breast cancer New Engl J Med 1990 322: 297–302

    CAS  PubMed  Google Scholar 

  22. 22

    Kute TE, Shao ZM, Sugg NK, Long RT, Russell GB, Case D . Cathepsin D as a prognostic indicator of node-negative breastcancer patients using both immunoassays and enzymatic assays Cancer Res 1992 52: 198–203

    Google Scholar 

  23. 23

    Garcia M, Platet N, Liaudet E, Laurent V, Derocq D, Brouillet J-P, Rochefort H . Biological and clinical significance of cathepsin D in breast cancer metastasis Stem Cells 1996 14: 642–650

    CAS  PubMed  Google Scholar 

  24. 24

    English HF, Kyprianou N, Isaacs JT . Relationship between DNA fragmentation and apoptosis in programmed cell death in the rat prostate following castration Prostate 1989 15: 233–250

    CAS  PubMed  Google Scholar 

  25. 25

    Walker NI, Bennett RE, Kerr JF . Cell death by apoptosis during involution of the lactating breast in mice and rats Am J Anat 1989 185: 19–32

    CAS  PubMed  Google Scholar 

  26. 26

    Guenette RS, Mooibroek M, Wong K, Wong P, Tenniswood M . Cathepsin B, a cysteine protease implicated in metastatic progression, is also expressed during regression of the rat prostate and mammary glands Eur J Biochem 1994 226: 311–321

    CAS  PubMed  Google Scholar 

  27. 27

    Sensibar JA, Liu XX, Patai B, Alger B, Lee C . Characterization of castration-induced cell death in the rat prostate by immunohistochemical localization of cathepsin D Prostate 1990 16: 263–276

    CAS  PubMed  Google Scholar 

  28. 28

    Roberts LR, Kurosawa H, Bronk SF, Fesmier PJ, Agellon LB, Leung W-Y, Mao F, Gores GJ . Cathepsin B contributes to bile salt-induced apoptosis of rat hepatocytes Gastroenterology 1997 113: 1714–1726

    CAS  PubMed  Google Scholar 

  29. 29

    Roberts LR, Adjei PN, Gores GJ . Cathepsins as effector proteases in hepatocyte apoptosis Cell Biochem Biophys 1999 30: 71–88

    CAS  Article  Google Scholar 

  30. 30

    Nishimura Y, Kawabata T, Kato K . Identification of latent cathepsins B and L in microsomal lumen: characterization of enzymatic activation and proteolytic processing in vitro Arch Biochem Biophys 1988 261: 64–71

    CAS  Google Scholar 

  31. 31

    Rowan AD, Mason P, Mach L, Mort JS . Rat procathepsin B: Proteolytic processing to the mature form in vitro J Biol Chem 1992 267: 15993–15999

    CAS  PubMed  Google Scholar 

  32. 32

    Shibata M, Kanamori S, Isahara K, Ohsawa Y, Konishi A, Kametaka S, Watanabe T, Ebisu S, Ishido K, Kominami E, Uchiyama Y . Participation of cathepsins B and D in apoptosis of PC12 cells following serum deprivation Biochem Biophys Res Commun 1998 251: 199–203

    CAS  PubMed  Google Scholar 

  33. 33

    Cataldo AM, Barnett JL, Berman SA, Li J, Quarless S, Bursztajn S, Lippa C, Nixon RA . Gene expression and cellular content of cathepsin D in Alzheimer’s disease brain: evidence for early up-regulation of the endosomal-lysosomal system Neuron 1995 14: 671–680

    CAS  Google Scholar 

  34. 34

    Isahara K, Ohsawa Y, Kanamori S, Shibata M, Waguri S, Sato N, Gotow T, Watanabe T, Momoi T, Urase K, Kominami E, Uchiyama Y . Regulation of a novel pathway for cell death by lysosomal aspartic and cysteine proteinases Neuroscience 1999 91: 233–249

    CAS  PubMed  Google Scholar 

  35. 35

    Lotem J, Sachs L . Control of apoptosis in hematopoiesis and leukemia by cytokines, tumor suppressor and oncogenes Leukemia 1996 10: 925–931

    CAS  PubMed  Google Scholar 

  36. 36

    Drexler HG, Zaborski M, Quentmeier H . Cytokine response profiles of human myeloid factor-dependent leukemia cell lines Leukemia 1997 11: 701–708

    CAS  PubMed  Google Scholar 

  37. 37

    Antoku K, Liu Z, Johnson DE . IL-3 withdrawal activates a CrmA-insensitive poly(ADP-ribose) polymerase cleavage enzyme in factor-dependent myeloid progenitor cells Leukemia 1998 12: 682–689

    CAS  PubMed  Google Scholar 

  38. 38

    Blalock WL, Weinstein-Oppenheimer C, Chang F, Hoyle PE, Wang XY, Algate PA, Franklin RA, Oberhaus SM, Steelman LS, McCubrey JA . Signal transduction, cell cycle regulatory, and anti-apoptotic pathways regulated by IL-3 in hematopoietic cells: possible sites for intervention with anti-neoplastic drugs Leukemia 1999 13: 1109–1166

    CAS  Google Scholar 

  39. 39

    Deiss LP, Galinka H, Berissi H, Cohen O, Kimchi A . Cathepsin D protease mediates programmed cell death induced by interferon-γ, Fas/APO-1 and TNF-α EMBO J 1996 15: 3861–3870

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Wu GS, Saftig P, Peters C, El-Deiry WS . Potential role for cathepsin D in p53-dependent tumor suppression and chemosensitivity Oncogene 1998 16: 2177–2183

    CAS  PubMed  Google Scholar 

  41. 41

    Luo X, Budihardjo I, Zou H, Slaughter C, Wang X . Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors Cell 1998 94: 481–490

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Li H, Zhu H, Xu C-J, Yuan J . Cleavage of BID by caspase 8 mediates the mitochondrial damage in Fas pathway of apoptosis Cell 1998 94: 491–501

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Friesen C, Fulda S, Debatin KM . Cytotoxic drugs and the CD95 pathway Leukemia 1999 13: 1854–1858

    CAS  Google Scholar 

  44. 44

    Adams JM, Cory S . The Bcl-2 protein family: arbiters of cell survival Science 1998 281: 1322–1326

    CAS  Google Scholar 

  45. 45

    Liu X, Zou H, Slaughter C, Wang X . DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis Cell 1997 89: 175–184

    CAS  Article  Google Scholar 

  46. 46

    Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S . A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD Nature 1998 391: 43–50

    CAS  Google Scholar 

  47. 47

    Rowan S, Fisher DE . Mechanisms of apoptotic cell death Leukemia 1997 11: 457–465

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Wolf BB, Green DR . Suicidal tendencies: apoptotic cell death by caspase family proteinases J Biol Chem 1999 274: 20049–20052

    CAS  PubMed  Google Scholar 

  49. 49

    Brunk UT, Zhang H, Roberg K, Ollinger K . Lethal hydrogen peroxide toxicity involves lysosomal iron-catalyzed reactions with membrane damage Redox Rep 1995 1: 267–277

    CAS  PubMed  Google Scholar 

  50. 50

    Li W, Yuan XM, Olsson AG, Brunk UT . Uptake of oxidized LDL by macrophages results in partial lysosomal enzyme inactivation and relocation Arterioscler Thromb Vasc Biol 1998 18: 177–184

    CAS  PubMed  Google Scholar 

  51. 51

    Roberg K, Ollinger K . Oxidative stress causes relocation of the lysosomal enzyme cathepsin D with ensuing apoptosis in neonatal rat cardiomyocytes Am J Pathol 1998 152: 1151–1156

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Vancompernolle K, Van Herreweghe F, Pynaert G, Van de Craen M, De Vos K, Totty N, Sterling A, Fiers W, Vandenabeele P, Grooten J . Atractyloside-induced release of cathepsin B, a protease with caspase-processing activity FEBS Lett 1998 438: 150–158

    CAS  PubMed  Google Scholar 

  53. 53

    Zhou Q, Salvesen GS . Activation of pro-caspase-7 by serine proteases includes a non-canonical specificity Biochem J 1997 324: 361–364

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Schotte P, Declercq W, Van Huffel S, Vandenabeele P, Beyaert R . Non-specific effects of methyl ketone peptide inhibitors of caspases FEBS Lett 1999 442: 117–121

    CAS  PubMed  Google Scholar 

  55. 55

    Guroff G . A neutral, calcium-activated proteinase from the soluble fraction of rat brain J Biol Chem 1964 239: 149–155

    CAS  PubMed  Google Scholar 

  56. 56

    Mellgren RL . Canine cardiac calcium-dependent proteases: resolution of two forms with different requirements for calcium FEBS Lett 1980 109: 129–133

    CAS  PubMed  Google Scholar 

  57. 57

    Murachi T, Tanaka K, Hatanaka M, Murakami T . Intracellular Ca2+-dependent protease (calpain) and its high-molecular-weight endogenous inhibitor (calpastatin) Adv Enzyme Reg 1981 19: 407–424

    CAS  Google Scholar 

  58. 58

    Sorimachi H, Saido TC, Suzuki K . New era of calpain research: discovery of tissue-specific calpains FEBS Lett 1994 341: 1–5

    Google Scholar 

  59. 59

    Carfoli E, Molinari M . Calpain: a protease in search of a function? Biochem Biophys Res Commun 1998 247: 193–203

    Google Scholar 

  60. 60

    Sorimachi H, Ishiura S, Suzuki K . A novel tissue-specific calpain species expressed predominantly in the stomach comprises two alternative splicing products with and without Ca(2+)-binding domain J Biol Chem 1993 268: 19476–19482

    CAS  PubMed  Google Scholar 

  61. 61

    Sorimachi H, Toyama-Sorimachi N, Saido TC, Kawasaki H, Sugita H, Miyasaka M, Arahata K, Ishiura S, Suzuki K . Muscle-specific calpain, p94, is degraded by autolysis immediately after translation, resulting in disappearance from muscle J Biol Chem 1993 268: 10593–10605

    CAS  PubMed  Google Scholar 

  62. 62

    Wilson R, Ainscough R, Anderson K, Baynes C, Berks M, Bonfield J, Burton J, Connell M, Copsey T, Cooper J, Coulson A, Craxton M, Dear S, Du Z, Durbin R, Favello A, Fraser A, Fulton L, Gardner A, Green P, Hawkins T, Hillier L, Jler M, Johnston L, Jones M, Kershaw J, Kirsten J, Laisster N, Latreille P, Lightning J, Lloyd C, Mortimore B, O’Callaghan M, Parsons J, Percy C, Rifken L, Roopra A, Saunders D, Shownkeen R, Sims M, Smaldon N, Smith A, Smith M, Sonnhammer E, Staden R, Sulston J, Thierry-Mieg J, Thomas K, Vaudin M, Vaughan K, Waterston R, Watson A, Weinstock L, Wilkinson-Sproat J, Wohldman P . 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans Nature 1994 368: 32–38

    CAS  Google Scholar 

  63. 63

    Sasaki T, Yoshimura N, Kikuchi T, Hatanaka M, Kitahara A, Sakihama T, Murachi T . Similarity and dissimilarity in subunit structures of calpains I and II from various sources as demonstrated by immunological cross-reactivity J Biochem 1983 94: 2055–2061

    CAS  PubMed  Google Scholar 

  64. 64

    Murachi T . Intracellular regulatory system involving calpain and calpastatin Biochem Int 1989 18: 263–294

    CAS  PubMed  Google Scholar 

  65. 65

    Croall DE, DeMartino GN . Calcium-activated neutral protease (calpain) system: Structure, function, and regulation Physiol Rev 1991 71: 813–847

    CAS  PubMed  Google Scholar 

  66. 66

    Blanchard H, Grochulski P, Li Y, Arthur JS, Davies PL, Elce JS, Cygler M . Structure of a calpain Ca(2+)-binding domain reveals a novel EF-hand and Ca(2+)-induced conformational changes Nature Struct Biol 1997 4: 532–538

    CAS  PubMed  Google Scholar 

  67. 67

    Molinari M, Anagli J, Carafoli E . Ca(2+)-activated neutral protease is active in the erythrocyte membrane in its nonautolyzed 80-kDa form J Biol Chem 1994 269: 27992–27995

    CAS  PubMed  Google Scholar 

  68. 68

    Suzuki K, Tsuji S, Ishiura S, Kimura Y, Kubota S, Imahori K . Autolysis of calcium-activated neutral protease of chicken skeletal muscle J Biochem 1981 90: 1787–1793

    CAS  PubMed  Google Scholar 

  69. 69

    Mellgren RL, Repetti A, Muck TC, Easly J . Rabbit skeletal muscle calcium-dependent protease requiring millimolar CA2+. Purification, subunit structure, and Ca2+-dependent autoproteolysis J Biol Chem 1982 257: 7203–7209

    CAS  PubMed  Google Scholar 

  70. 70

    DeMartino GN, Huff CA, Croall DE . Autoproteolysis of the small subunit of calcium-dependent protease II activates and regulates protease activity J Biol Chem 1986 261: 12047–12052

    CAS  PubMed  Google Scholar 

  71. 71

    Imajoh S, Kawasaki H, Suzuki K . Limited autolysis of calcium-activated neutral protease (CANP): reduction of the Ca2+-requirement is due to the NH2-terminal processing of the large subunit J Biochem 1986 100: 633–642

    CAS  PubMed  Google Scholar 

  72. 72

    Inomata M, Kasai Y, Nakamura M, Kawashima S . Activation mechanism of calcium-activated neutral protease. Evidence for the existence of intramolecular and intermolecular autolyses J Biol Chem 1988 263: 19783–19787

    CAS  PubMed  Google Scholar 

  73. 73

    Elce JS, Hegadorn C, Arthur JSC . Autolysis, Ca2+ requirement, and heterodimer stability in m-calpain J Biol Chem 1997 272: 11268–11275

    CAS  PubMed  Google Scholar 

  74. 74

    Waxman L, Krebs EG . Identification of two protease inhibitors from bovine cardiac muscle J Biol Chem 1978 253: 5888–5891

    CAS  PubMed  Google Scholar 

  75. 75

    Emori Y, Kawasaki H, Imajoh S, Imahori K, Suzuki K . Endogenous inhibitor for calcium-dependent cysteine protease contains four internal repeats that could be responsible for its multiple reactive sites Proc Natl Acad Sci USA 1987 84: 3590–3594

    CAS  PubMed  Google Scholar 

  76. 76

    Squier MKT, Miller ACK, Malkinson AM, Cohen JJ . Calpain activation in apoptosis J Cell Physiol 1994 159: 229–237

    CAS  PubMed  Google Scholar 

  77. 77

    Knepper-Nicolai B, Savill J, Brown SB . Constitutive apoptosis in human neutrophils requires synergy between calpains and the proteosome downstream of caspases J Biol Chem 1998 273: 30530–30536

    CAS  PubMed  Google Scholar 

  78. 78

    Waterhouse NJ, Finucane DM, Green DR, Elce JS, Kumar S, Alnemri ES, Litwack G, Khanna KK, Lavin MF, Watters DJ . Calpain activation is upstream of caspases in radiation-induced apoptosis Cell Death Differ 1998 5: 1051–1061

    CAS  PubMed  Google Scholar 

  79. 79

    Wood DE, Newcomb EW . Caspase-dependent activation of calpain during drug-induced apoptosis J Biol Chem 1999 274: 8309–8315

    CAS  PubMed  Google Scholar 

  80. 80

    Xie H, Johnson GV . Ceramide selectively decreases tau levels in differentiated PC12 cells through modulation of calpain I J Neurochem 1997 69: 1020–1030

    CAS  PubMed  Google Scholar 

  81. 81

    Debiasi RL, Squier MKT, Pike B, Wynes M, Dermody TS, Cohen JJ, Tyler KL . Reovirus-induced apoptosis is preceded by increased cellular calpain activity and is blocked by calpain inhibitors J Virol 1999 73: 695–701

    CAS  PubMed  PubMed Central  Google Scholar 

  82. 82

    Kohli V, Madden JF, Bentley RC, Clavien P-A . Calpain mediates ishemic injury of the liver through modulation of apoptosis and necrosis Gastroenterol 1999 116: 168–178

    CAS  Google Scholar 

  83. 83

    Saito K, Elce JS, Hamos JE, Nixon RA . Widespread activation of calcium-activated neutral proteinase (calpain) in the brain in Alzheimer disease: a potential molecular basis for neuronal degeneration Proc Natl Acad Sci USA 1993 90: 2628–2632

    CAS  PubMed  Google Scholar 

  84. 84

    Nixon RA, Saito K-I, Grynspan F, Griffin WR, Katayama S, Honda T, Mohan PS, Shea TB, Beermann M . Calcium-activated neutral proteinase (calpain) system in aging and Alzheimer’s disease Ann NY Acad Sci 1994 747: 77–91

    CAS  PubMed  Google Scholar 

  85. 85

    Mouatt-Prigent A, Karlsson JO, Agid Y, Hirsch EC . Increased M-calpain expression in the mesencephalon of patients with Parkinson's disease but not in other neurodegenerative disorders involving the mesencephalon: a role in nerve cell death? Neurosci 1996 73: 979–987

    CAS  Google Scholar 

  86. 86

    Aoyagi T, Takeuchi T, Matsuzaki A, Kawamura K, Kondo S . Leupeptins, new protease inhibitors from Actinomycetes J Antibiotics 1969 22: 283–286

    CAS  Google Scholar 

  87. 87

    Barrett AJ, Kembhavi AA, Hanada K . E-64 [L-trans-epoxysuccinyl-leucyl-amido(4-guanidino)butane] and related epoxides as inhibitors of cysteine proteinases Acta Biol Med Germ 1981 40: 1513–1517

    CAS  PubMed  Google Scholar 

  88. 88

    Wang KK . Developing selective inhibitors of calpain Trends Pharm Sci 1990 11: 139–142

    CAS  PubMed  Google Scholar 

  89. 89

    Mehdi S . Cell-penetrating inhibitors of calpain Trends Biochem Sci 1991 16: 150–153

    CAS  PubMed  Google Scholar 

  90. 90

    Tsujinaka T, Kajiwara Y, Kambayashi J, Sakon M, Higuchi N, Tanaka T, Mori T . Synthesis of a new cell penetrating inhibitor (calpeptin) Biochem Biophys Res Commun 1988 153: 1201–1208

    CAS  PubMed  Google Scholar 

  91. 91

    Wang KK, Nath R, Posner A, Raser KJ, Buroker-Kilgore M, Hajimohammadreza I, Probert W, Marcoux FW, Ye Q, Takano E, Hatanaka M, Maki M, Caner H, Collins JL, Fergus A, Lee KS, Lunney EA, Hays SJ, Yuen P . An alpha-mercaptoacrylic acid derivative is a selective non-peptide cell-permeable calpain inhibitor and is neuroprotective Proc Natl Acad Sci USA 1996 93: 6687–6692

    CAS  PubMed  Google Scholar 

  92. 92

    Squier MKT, Cohen JJ . Calpain, an upstream regulator of thymocyte apoptosis J Immunol 1997 158: 3690–3697

    CAS  PubMed  Google Scholar 

  93. 93

    Squier MKT, Sehnert AJ, Sellins KS, Malkinson AM, Takano E, Cohen JJ . Calpain and calpastatin regulate neutrophil apoptosis J Cell Physiol 1999 178: 311–319

    CAS  PubMed  Google Scholar 

  94. 94

    Vanags DM, Porn-Ares I, Coppola S, Burgess DH, Orrenius S . Protease involvement in fodrin cleavage and phosphatidylserine exposure in apoptosis J Biol Chem 1996 271: 31075–31085

    CAS  PubMed  Google Scholar 

  95. 95

    Spinedi A, Oliverio S, Di Sano F, Piacentini M . Calpain involvement in calphostin C-induced apoptosis Biochem Pharm 1998 56: 1489–1492

    CAS  PubMed  Google Scholar 

  96. 96

    Wolf BB, Goldstein JC, Stennicke HR, Beere H, Amarante-Mendes GP, Salvesen GS, Green DR . Calpain functions in a caspase-independent manner to promote apoptosis-like events during platelet activation Blood 1999 94: 1683–1692

    CAS  Google Scholar 

  97. 97

    Gressner AM, Lahme B, Roth S . Attenuation of TGF-beta-induced apoptosis in primary cultures of hepatocytes by calpain inhibitors Biochem Biophys Res Commun 1997 231: 457–462

    CAS  PubMed  Google Scholar 

  98. 98

    Villa PG, Henzel WJ, Sensenbrenner M, Henderson CE, Pettmann B . Calpain inhibitors, but not caspase inhibitors, prevent actin proteolysis and DNA fragmentation during apoptosis J Cell Sci 1998 111: 713–722

    CAS  PubMed  Google Scholar 

  99. 99

    Jordan J, Galindo MF, Miller RJ . Role of calpain- and interleukin-1β converting enzyme-like proteases in the β-amyloid-induced death of rat hippocampal neurons in culture J Neurochem 1997 68: 1612–1621

    CAS  PubMed  Google Scholar 

  100. 100

    Yuan Y, Dopheide SM, Ivanidis C, Salem HH, Jackson SP . Calpain regulation of cytoskeletal signaling complexes in von Willebrand factor-stimulated platelets. Distinct roles for glycoprotein Ib-V-IX and glycoprotein IIb-IIIa (integrin alphaIIbbeta3) in von Willebrand factor-induced signal transduction J Biol Chem 1997 272: 21847–21854

    CAS  PubMed  Google Scholar 

  101. 101

    Martin SJ, O’Brien GA, Nishioka WK, McGahon AJ, Mahboubi A, Saido TC, Green DR . Proteolysis of fodrin (non-erythroid spectrin) during apoptosis J Biol Chem 1995 270: 6425–6428

    CAS  PubMed  Google Scholar 

  102. 102

    Cooray P, Yuan Y, Schoenwaelder SM, Mitchell CA, Salem HH, Jackson SP . Focal adhesion kinase (pp125FAK) cleavage and regulation by calpain Biochem J 1996 318: 41–47

    CAS  PubMed  PubMed Central  Google Scholar 

  103. 103

    Meredith J Jr, Mu Z, Saido T, Du X . Cleavage of the cytoplasmic domain of integrin β3 subunit during endothelial cell apoptosis J Biol Chem 1998 273: 19525–19531

    PubMed  Google Scholar 

  104. 104

    Kishimoto A, Mikawa K, Hashimotos K, Yasuda I, Tanaka S, Tominaga MT, Kuroda T, Nishizuka Y . Limited proteolysis of protein kinase C subspecies by calcium-dependent neutral protease (calpain) J Biol Chem 1989 264: 4088–4092

    CAS  PubMed  Google Scholar 

  105. 105

    McGinnis KM, Whitton MM, Gnegy ME, Wang KKW . Calcium/calmodulin-dependent protein kinase IV is cleaved by caspase-3 and calpain in SH-SY5Y human neuroblastoma cells undergoing apoptosis J Biol Chem 1998 273: 19993–20000

    CAS  PubMed  Google Scholar 

  106. 106

    Watanabe N, Vande Woude GF, Ikawa Y, Sagata N . Specific proteolysis of the c-mos proto-oncogene product by calpain on fertilization of Xenopus eggs Nature 1989 342: 505–511

    CAS  PubMed  Google Scholar 

  107. 107

    Hirai S, Kawasaki H, Yaniv M, Suzuki K . Degradation of transcription factors, c-Jun and c-Fos, by calpain FEBS Lett 1991 287: 57–61

    CAS  PubMed  Google Scholar 

  108. 108

    Watt F, Molloy PL . Specific cleavage of transcription factors by the thiol protease, m-calpain Nucleic Acids Res 1993 21: 5092–5100

    CAS  PubMed  PubMed Central  Google Scholar 

  109. 109

    Choi YH, Lee SJ, Nguyen P, Jang JS, Lee J, Wu ML, Takano E, Maki M, Henkart PA, Trepel JB . Regulation of cyclin D1 by calpain protease J Biol Chem 1997 272: 28479–28484

    CAS  PubMed  Google Scholar 

  110. 110

    Kubbutat MHG, Vousden KH . Proteolytic cleavage of human p53 by calpain: a potential regulator of protein stability Mol Cell Biol 1997 17: 460–468

    CAS  PubMed  PubMed Central  Google Scholar 

  111. 111

    Wood DE, Thomas A, Devi LA, Berman Y, Beavis RC, Reed JC, Newcomb EW . Bax cleavage is mediated by calpain during drug-induced apoptosis Oncogene 1998 17: 1069–1078

    CAS  Google Scholar 

  112. 112

    Porn-Ares MI, Samali A, Orrenius S . Cleavage of the calpain inhibitor, calpastatin, during apoptosis Cell Death Differ 1998 5: 1028–1033

    CAS  PubMed  Google Scholar 

  113. 113

    Wang KKW, Posmantur R, Nadimpalli R, Nath R, Mohan P, Nixon RA, Talanian RV, Keegan M, Herzog L, Allen H . Caspase-mediated fragmentation of calpain inhibitor protein calpastatin during apoptosis Arch Biochem Biophys 1998 356: 187–196

    CAS  PubMed  Google Scholar 

  114. 114

    Berke G . The CTL’s kiss of death Cell 1995 81: 9–12

    CAS  PubMed  Google Scholar 

  115. 115

    Kagi D, Lederman B, Burki K, Zinkernagel RM, Hengartner H . Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo Ann Rev Immunol 1996 14: 207–232

    CAS  Google Scholar 

  116. 116

    Nagata S, Golstein P . The Fas death factor Science 1995 267: 1449–1456

    CAS  Google Scholar 

  117. 117

    Ashkenazi A, Dixit VM . Death receptors: signaling and modulation Science 1998 281: 1305–1308

    CAS  PubMed  PubMed Central  Google Scholar 

  118. 118

    Doherty PC . Cell-mediated cytotoxicity Cell 1993 75: 607–612

    CAS  Google Scholar 

  119. 119

    Shresta S, MacIvor DM, Heusel JW, Russell JH, Ley TJ . Natural killer and lymphokine-activated killer cells require granzyme B for the rapid induction of apoptosis in susceptible target cells Proc Natl Acad Sci USA 1995 92: 5679–5683

    CAS  PubMed  Google Scholar 

  120. 120

    Shresta S, Heusel JW, Macivor DM, Wesselschmidt RL, Russell JH, Ley TJ . Granzyme B plays a critical role in cytotoxic lymphocyte-induced apoptosis Immunol Rev 1995 146: 211–221

    CAS  PubMed  Google Scholar 

  121. 121

    Masson D, Tschopp J . Isolation of a lytic pore-forming protein (perforin) from cytolytic T lymphocytes J Biol Chem 1985 260: 9069–9072

    CAS  PubMed  Google Scholar 

  122. 122

    Young JD-E, Hengartner H, Podack ER, Cohn ZA . Purification and characterization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer activity Cell 1986 44: 849–859

    CAS  PubMed  Google Scholar 

  123. 123

    Tschopp J, Schafer S, Masson D, Peitsch MC, Heusser C . Phosphorylcholine acts as a calcium dependent receptor molecule for lymphocyte perforin Nature 1989 337: 272–274

    CAS  PubMed  Google Scholar 

  124. 124

    Smyth MJ, O’Connor MD, Trapani JA . Granzymes: A variety of serine protease specificities encoded by genetically distinct subfamilies J Leuk Biol 1996 60: 555–562

    CAS  Google Scholar 

  125. 125

    Zunino SJ, Bleackley RC, Martinez J, Hudig D . RNKP-1, a novel natural killer cell-associated serine protease gene cloned from RNK-16 cytotoxic lymphocytes J Immunol 1990 144: 2001–2009

    CAS  PubMed  Google Scholar 

  126. 126

    Lobe CG, Havele C, Bleackley RC . Cloning of two genes that are specifically expressed in activated cytotoxic T lymphocytes Proc Natl Acad Sci USA 1986 83: 1448–1452

    CAS  PubMed  Google Scholar 

  127. 127

    Odake S, Kam CM, Narasimhan L, Poe M, Blake JT, Krahenbuhl O, Tschopp J, Powers JC . Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins Biochem 1991 30: 2217–2227

    CAS  Google Scholar 

  128. 128

    Poe M, Blake JT, Boulton DA, Gammon M, Sigal NH, Wu JK, Zweerink HJ . Human cytotoxic lymphocyte granzyme B: its purification from granules and the characterization of substrate and inhibitor specificity J Biol Chem 1991 266: 98–103

    CAS  PubMed  Google Scholar 

  129. 129

    Shi L, Kraut RP, Aebersold R, Greenberg AH . A natural killer cell granule protein that induces DNA fragmentation and apoptosis J Exp Med 1992 175: 553–566

    CAS  PubMed  Google Scholar 

  130. 130

    Heusel JW, Wesselschmidt RL, Shresta S, Russell JH, Ley TJ . Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells Cell 1994 76: 977–987

    CAS  Google Scholar 

  131. 131

    Froelich CJ, Orth K, Turbov J, Seth P, Gottlieb R, Babior B, Shah GM, Bleackley RC, Dixit VM, Hanna W . New paradigm for lymphocyte granule-mediated cytotoxicity. Target cells bind and internalize granzyme B, but an endosomolytic agent is necessary for cytosolic delivery and subsequent apoptosis J Biol Chem 1996 271: 29073–29079

    CAS  Google Scholar 

  132. 132

    Jans DA, Jans P, Briggs LJ, Sutton V, Trapani JA . Nuclear transport of granzyme B (fragmentin-2) J Biol Chem 1996 271: 30781–30789

    CAS  PubMed  Google Scholar 

  133. 133

    Shi L, Mai S, Israels S, Browne K, Trapani JA, Greenberg AH . Granzyme B (GraB) autonomously crosses the cell membrane and perforin initiates apoptosis and GraB nuclear localization J Exp Med 1997 185: 853–866

    Google Scholar 

  134. 134

    Pinkoski MJ, Hobman M, Heibein JA, Tomaselli K, Li F, Seth P, Froelich CJ, Bleackley RC . Entry and trafficking of granzyme B in target cells during granzyme B-perforin-mediated apoptosis Blood 1998 92: 1044–1054

    CAS  Google Scholar 

  135. 135

    Pinkoski MJ, Heibein JA, Barry M, Bleackley RC . Nuclear translocation of granzyme B in target cell apoptosis Cell Death Differ 2000 7: 17–24

    CAS  PubMed  PubMed Central  Google Scholar 

  136. 136

    Pinkoski MJ, Winkler U, Hudig D, Bleackley RC . Binding of granzyme B in the nucleus of target cells. Recognition of an 80-kilodalton protein J Biol Chem 1996 271: 10225–10229

    CAS  Google Scholar 

  137. 137

    Trapani JA, Browne KA, Smyth MJ, Jans DA . Localization of granzyme B in the nucleus. A putative role in the mechanism of cytotoxic lymphocyte-mediated apoptosis J Biol Chem 1996 271: 4127–4133

    CAS  PubMed  Google Scholar 

  138. 138

    Darmon AJ, Nicholson DW, Bleackley RC . Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B Nature 1995 377: 446–448

    CAS  PubMed  Google Scholar 

  139. 139

    Darmon AJ, Ley TJ, Nicholson DW, Bleackley RC . Cleavage of CPP32 by granzyme B represents a critical role for granzyme B in the induction of target cell DNA fragmentation J Biol Chem 1996 271: 21709–21712

    CAS  PubMed  PubMed Central  Google Scholar 

  140. 140

    Martin SJ, Amarante-Mendes GP, Shi LF, Chuang TH, Casiano CA, O’Brien GA, Fitzgerald P, Tan EM, Bokoch GM, Greenberg AH, Green DR . 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 EMBO J 1996 15: 2407–2416

    CAS  PubMed  PubMed Central  Google Scholar 

  141. 141

    Quan LT, Tewari M, O’Rourke K, Dixit VM, Snipas SJ, Poirier GG, Ray C, Pickup DJ, Salvesen GS . Proteolytic activation of the cell death protease Yama/CPP32 by granzyme B Proc Natl Acad Sci USA 1996 93: 1972–1976

    CAS  PubMed  Google Scholar 

  142. 142

    Fernandes-Alnemri T, Litwack G, Alnemri ES . Mch2, a new member of the apoptotic Ced-3/Ice cysteine protease gene family Cancer Res 1995 55: 2737–2742

    CAS  PubMed  PubMed Central  Google Scholar 

  143. 143

    Orth K, Chinnaiyan AM, Garg M, Froelich CJ, Dixit VM . The CED-3/ICE-like protease Mch2 is activated during apoptosis and cleaves the death substrate lamin A J Biol Chem 1996 271: 16443–16446

    CAS  PubMed  PubMed Central  Google Scholar 

  144. 144

    Fernandes-Alnemri T, Takahashi A, Armstrong R, Krebs J, Fritz L, Tomaselli KJ, Wang L, Yu Z, Croce CM, Salvesen G, Earnshaw WC, Litwack G, Alnemri ES . Mch3, a novel human apoptotic cysteine protease highly related to CPP32 Cancer Res 1995 55: 6045–6052

    CAS  PubMed  Google Scholar 

  145. 145

    Chinnnaiyan AM, Orth K, Hanna WL, Duan HJ, Poirier GG, Froelich CJ, Dixit VM . Cytotoxic T cell-derived granzyme B activates the apoptotic protease ICE-LAP3 Curr Biol 1996 6: 897–899

    Google Scholar 

  146. 146

    Gu Y, Sarnecki C, Fleming MA, Lippke JA, Bleackley RC, Su MS-S . Processing and activation of CMH-1 by granzyme B J Biol Chem 1996 271: 10816–10820

    CAS  PubMed  Google Scholar 

  147. 147

    Boldin MP, Goncharov TM, Goltsev YV, Wallach D . Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death Cell 1996 85: 803–815

    CAS  PubMed  PubMed Central  Google Scholar 

  148. 148

    Muzio M, Chinnaiyan AM, Kischkel FC, O’Rourke K, Shevchenko A, Ni J, Scaffidi C, Bretz JD, Zhang M, Gentz R, Mann M, Krammer PH, Peter ME, Dixit VM . FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex Cell 1996 85: 817–827

    CAS  PubMed  PubMed Central  Google Scholar 

  149. 149

    Duan H, Orth K, Chinnaiyan AM, Poirier GG, Froelich CJ, He W-W, Dixit VM . ICE-LAP6, a novel member of the ICE/Ced-3 gene family, is activated by the cytotoxic T cell protease granzyme B J Biol Chem 1996 271: 16720–16724

    CAS  PubMed  Google Scholar 

  150. 150

    Fernandes-Alnemri T, Armstrong RC, Krebs J, Srinivasula SM, Wang L, Bullrich F, Fritz LC, Trapani JA, Tomaselli KJ, Litwack G, Alnemri ES . In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains Proc Natl Acad Sci USA 1996 93: 7464–7469

    CAS  Google Scholar 

  151. 151

    Medema JP, Toes REM, Scaffidi C, Zheng TS, Flavell RA, Melief CJM, Peter ME, Offringa R, Krammer PH . Cleavage of FLICE (caspase-8) by granzyme B during cytotoxic T lymphocyte-induced apoptosis Eur J Immunol 1997 27: 3492–3498

    CAS  PubMed  Google Scholar 

  152. 152

    Van de Craen M, Van den brande I, Declercq W, Irmler M, Beyaert R, Tschopp J, Fiers W, Vandenabeele P . Cleavage of caspase family members by granzyme B: a comparative study in vitro Eur J Immunol 1997 27: 1296–1299

    CAS  PubMed  Google Scholar 

  153. 153

    Talanian RV, Yang XH, Turbov J, Seth P, Ghayur T, Casiano CA, Orth K, Froelich CJ . Granule-mediated killing: pathways for granzyme B-initiated apoptosis J Exp Med 1997 186: 1323–1331

    CAS  PubMed  PubMed Central  Google Scholar 

  154. 154

    Shi L, Chen G, MacDonald G, Bergeron L, Li H, Miura M, Rotello RJ, Miller DK, Li P, Seshadri T, Yuan J, Greenberg AH . Activation of an interleukin 1 converting enzyme-dependent apoptosis pathway by granzyme B Proc Natl Acad Sci USA 1996 93: 11002–11007

    CAS  PubMed  Google Scholar 

  155. 155

    Andrade F, Roy S, Nicholson D, Thornberry N, Rosen A, Casciola-Rosen L . Granzyme B directly and efficiently cleaves several downstream caspase substrates: implications for CTL-induced apoptosis Immunity 1998 8: 451–460

    CAS  Google Scholar 

  156. 156

    Gershenfeld HK, Weissman IL . Cloning of a cDNA for a T cell-specific serine protease from a cytotoxic T lymphocyte Science 1986 232: 854–858

    CAS  PubMed  Google Scholar 

  157. 157

    Masson D, Zamai M, Tschopp J . Identification of granzyme A isolated from cytotoxic T-lymphocyte-granules as one of the proteases encoded by CTL-specific genes FEBS Lett 1986 208: 84–88

    CAS  PubMed  Google Scholar 

  158. 158

    Ebnet K, Hausmann M, Lehmann-Grube F, Mullbacher A, Kopf M, Lamers M, Simon MM . Granzyme A-deficient mice retain potent cell-mediated cytotoxicity EMBO J 1995 14: 4230–4239

    CAS  PubMed  PubMed Central  Google Scholar 

  159. 159

    Mullbacher A, Ebnet K, Blanden RV, Hla RT, Stehle T, Museteanu C, Simon MM . Granzyme A is critical for recovery of mice from infection with the natural cytopathic viral pathogen, ectromelia Proc Natl Acad Sci USA 1996 93: 5783–5787

    CAS  PubMed  Google Scholar 

  160. 160

    Shresta S, Graubert TA, Thomas DA, Raptis SZ, Ley TJ . Granzyme A initiates an alternative pathway for granule-mediated apoptosis Immunity 1999 10: 595–605

    CAS  Google Scholar 

  161. 161

    Hayes MP, Berrebi GA, Henkart PA . Induction of target cell DNA release by the cytotoxic T lymphocyte granule protease granzyme A J Exp Med 1989 170: 933–946

    CAS  Google Scholar 

  162. 162

    Beresford PJ, Xia Z, Greenberg AH, Lieberman J . Granzyme A loading induces rapid cytolysis and a novel form of DNA damage independently of caspase activation Immunity 1999 10: 585–594

    CAS  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to DE Johnson.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Johnson, D. Noncaspase proteases in apoptosis. Leukemia 14, 1695–1703 (2000). https://doi.org/10.1038/sj.leu.2401879

Download citation

Keywords

  • apoptosis
  • cathepsin B
  • cathepsin D
  • calpain
  • granzyme A
  • granzyme B

Further reading

Search

Quick links