Interleukin-1β Converting Enzyme Family Research Articles

Differential Toxicity of Protease Inhibitors in Cultures of Cerebellar Granule Neurons

Involvement of proteases has been postulated in several neurodegenerative processes. Accordingly, protease inhibition has been proposed as a potential therapeutic tool to limit damage in some neuropathological states. The timed turn-over of proteins is, however, an essential biochemical process and its prolonged block may be dangerous to the cell. We report here data on toxicity consequent to 24-h exposure of cerebellar granule neurons in culture to inhibitors of different classes of proteases. Inhibition of calpains (calcium-activated cysteine proteases) resulted in dose-dependent neuronal death which largely occurred through apoptotic process. Leupeptin, an inhibitor acting on a broad spectrum of cellular serine proteases, was less toxic but resulted in definite morphological alteration of the cells. On the contrary, inhibitors of caspases, proteases belonging to the ICE (interleukin 1-β converting enzyme) family, did not apparently damage granule neurons upon exposure for 24 h to high concentrations (up to 200 μM) of two inhibitors specific for ICE (Ac-YAVD-CHO) and CPP-32 (Ac-DEVD-CHO), respectively. These results suggest that inhibition of proteases that are activated by stressful stimuli but are not essential for the normal functioning of healthy cells, as it is likely the case for caspases, may not be harmful to neurons. Instead, the potential risks and side effects of prolonged inhibition of proteases such as calpains, that regulate the disposal and the turn-over of key cellular proteins, should be carefully tested in the assessment of possible neuroprotective roles.

Distinct interleukin-1β-converting enzyme family proteases mediate cisplatin- and staurosporine-induced apoptosis of mouse proximal tubule cells

Interleukin-1β converting enzyme (ICE) family proteases (caspases) are known to be implicated as important effectors of apoptotic pathways. The purpose of this study was to elucidate the role of ICE family proteases in apoptosis of mouse cells derived from the terminal proximal tubule (S 3) treated with cisplatin, an anti-tumor drug, or staurosporine, a protein kinase C inhibitor. For this purpose, we measured the activities of ICE family proteases and examined the effects of tetrapeptide and viral ICE family protease inhibitors on the activities of ICE family proteases in and the degree of apoptosis of S 3 cells treated with cisplatin and staurosporine. RT-PCR analysis revealed that S 3 cells as well as mouse kidney express mRNA for ICE and CPP32, an ICE family protease. Results of enzymatic analysis, determination the degree of DNA fragmentation and cytotoxicity test suggest that CPP32 mediates cisplatin-induced apoptosis of S 3 cells, whereas ICE family proteases other than CPP32 mediate staurosporine-induced apoptosis of S 3 cells. In conclusion, distinct ICE family proteases mediate apoptosis of mouse proximal tubule cells depending on the stimuli to which the cells are exposed.

Involvement of ICE (Caspase) Family in γ‐Radiation‐Induced Apoptosis of Normal B Lymphocytes

Normal lymphocytes are highly sensitive to the damaging effects of ionizing radiation, and undergo cell death by apoptosis. We have investigated the possible involvement of the Interleukin-1 beta-converting enzyme (ICE) (Caspase) protease family, which appears to play an important role as intracellular mediator of apoptosis. Resting B lymphocytes isolated from human peripheral blood were irradiated (6 Gy) and cultured for 24 h, resulting in 25 +/- 5.1% apoptotic cells, as measured by the TUNEL assay (mean +/- SD, n = 6). Addition of the ICE family inhibitor Z-VAD.fmk (50 microM) completely inhibited apoptosis (2.0 +/- 1.5% at 24 h). By using fluorogenic substrates containing the peptide recognition sequences DEVD and YVAD, the type of ICE family protease involved was examined more closely. A marked transient increase in DEVD-, and absent YVAD-cleavage activity indicated the involvement of a CPP32-like protease, not an ICE-like protease. Western blot analysis demonstrated that untreated B lymphocytes expressed the proform of the ICE family members CPP32 and ICH1L, but no detectable ICE. The induction of cell death by radiation was accompanied by the activation of CPP32 as shown by the cleavage of the proform to the active subunit p17, and the cleavage of poly(ADP-ribose) polymerase (PARP), one of the known substrates of CPP32. In contrast, no activation of ICH1L could be detected. These results indicate the involvement of CPP32 and possibly other CPP32-like proteases in radiation-induced apoptosis of resting B lymphocytes.

Protease Activation Is Required for Glucocorticoid-Induced Apoptosis in Chronic Lymphocytic Leukemic Lymphocytes

AbstractRecent work has demonstrated that glucocorticoids, nucleoside analogues, and other cancer chemotherapeutics induce apoptosis in chronic lymphocytic leukemia (CLL) cells. In this study, we investigated the involvement of protease activation in these responses using selective peptide inhibitors of the interleukin-1β converting enzyme (ICE)/caspase family and a Ca2+-activated protease we recently implicated in thymocyte apoptosis. Apoptosis was associated with proteolytic cleavage of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) and increased caspase protease activity, and cell-permeant caspase antagonists [zVAD(OMe)fmk and Boc-D(OBzl)cmk] blocked apoptosis in response to the glucocorticoid methylprednisolone or the nucleoside analogue fludarabine, indicating that caspase activation was required for these responses. However, a peptide-based inhibitor of the Ca2+-dependent lamin protease (zAPFcmk) also completely suppressed DNA fragmentation and the cleavage of lamin B1 . Strikingly, treatment of cells with zAPFcmk alone led to characteristic PARP cleavage, depletion of the precursor forms of two ICE family proteases (CPP32 and ICH-1), and phosphatidylserine exposure, suggesting that blockade of the lamin protease led to activation of the ICE family. Our results implicate the lamin protease as a target for Ca2+ during chemotherapy-induced apoptosis in CLL lymphocytes, and they identify a novel functional interaction between the protease and members of the ICE family.

Inhibition of apoptosis by the actin-regulatory protein gelsolin.

Gelsolin is an actin-regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility in vivo through modulation of the actin network. In addition to its actin-regulatory function, gelsolin has also been proposed to affect cell growth. Our present experiments have tested the possible involvement of gelsolin in the regulation of apoptosis, which is significantly affected by growth. When overexpressed in Jurkat cells, gelsolin strongly inhibited apoptosis induced by anti-Fas antibody, C2-ceramide or dexamethasone, without changing the F-actin morphology or the levels of Fas or Bcl-2 family proteins. Upon the induction of apoptosis, an increase in CPP32(-like) protease activity was observed in the control vector transfectants, while it was strongly suppressed in the gelsolin transfectants. Pro-CPP32 protein, an inactive form of CPP32 protease, remained uncleaved by anti-Fas treatment in the gelsolin transfectants, indicating that gelsolin blocks upstream of this protease. The tetrapeptide inhibitor of CPP32(-like) proteases strongly inhibited Fas-mediated apoptosis, but only partially suppressed both C2-ceramide- and dexamethasone-induced apoptosis. These data suggest that the critical target responsible for the execution of apoptosis may exist upstream of CPP32(-like) proteases in Jurkat cells and that gelsolin acts on this target to inhibit the apoptotic cell death program.

Cisplatin-induced apoptosis of immortalized mouse proximal tubule cells is mediated by interleukin-1 beta converting enzyme (ICE) family of proteases but inhibited by overexpression of Bcl-2.

Cisplatin is known to induce serious renal damage including acute renal failure, the major site of renal injury appears to be localized to the third segment of the proximal tubule (S3). Apoptosis occurs during a variety of acute injuries to tubule cell. The purpose of this study was to determine whether cisplatin induces apoptosis of immortalized mouse S3 cells, and to define the intracellular pathways leading to cell death. S3 cells exposed to cisplatin exhibited biochemical, morphological, and flow cytometric changes characteristic of apoptosis associated with slight necrosis. Cisplatin-induced apoptosis could be inhibited by overexpression of crmA, a cowpox virus gene, of which the product is known to suppress activities of the interleukin-1 beta converting enzyme (ICE) family proteases. On the other hand, overexpression of bcl-2, an antiapoptotic oncogene, rendered S3 cells partially resistant to cisplatin. These results indicate that cisplatin-induced proximal tubule damage is associated with apoptosis, which is positively modulated by the ICE family of proteases and negatively by the product of bcl-2.

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.

Genetic control of cellular suicide

Genetic analysis of programmed cell death in C. elegans has led to the identification of two genes, ced-9, a cell death suppressor, and ced-3, a cell death inducer, that play critical roles in regulating programmed cell death. The ced-9 and ced-3 genes were found to encode proteins that share structural and functional similarities with the mammalian proto-oncogene product Bcl-2 and interleukin-1β converting enzyme (ICE), respectively. Multiple members of the Bcl-2 family and the ICE family have been identified in vertebrates. These results suggest that the mechanism of apoptosis in vertebrates may be evolved from a much simpler version of a similar pathway in primitive organisms.

Phosphatidylserine Externalization Is a Downstream Event of Interleukin-1β–Converting Enzyme Family Protease Activation During Apoptosis

AbstractPhosphatidylserine (PS), a class of acidic phospholipids, normally localizes on the internal surface of cellular plasma membranes. The internal PS is externalized when cells undergo apoptosis; however, the mechanism for this is largely unknown. To study the mechanism of PS externalization during development of apoptosis, we examined the correlation between the activation of interleukin-1β–converting enzyme (ICE) family protease and PS externalization in human monocytic leukemia U937 cells and in their apoptosis-resistant variants, UK711 and UK110, after treatment with etoposide and anti-Fas antibody. We found that PS externalization accompanied the development of apoptosis and the activation of ICE family proteases in these cell lines. Furthermore, inhibitors of ICE family proteases, Z-Asp and Z-VAD, prevented apoptosis and PS externalization in etoposide-treated U937 cells. These results indicate that PS externalization is a downstream event of ICE family protease activation during apoptosis development. Because ICE family proteases play a crucial role in apoptosis, PS externalization could be a rational and useful marker for the development of apoptosis.

Inhibition of interleukin 1beta converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage.

The interleukin 1beta converting enzyme (ICE) family plays a pivotal role in programmed cell death and has been implicated in stroke and neurodegenerative diseases. During reperfusion after filamentous middle cerebral artery occlusion, ICE-like cleavage products and tissue immunoreactive interleukin 1beta (IL-1beta) levels increased in ischemic mouse brain. Ischemic injury decreased after intracerebroventricular injections of ICE-like protease inhibitors, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.FMK), acetyl-Tyr-Val-Ala-Asp-chloromethylketone, or a relatively selective inhibitor of CPP32-like caspases, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone, but not a cathepsin B inhibitor, N-benzyloxycarbonyl-Phe-Ala-fluoromethylketone. z-VAD.FMK decreased ICE-like cleavage products and tissue immunoreactive IL-1beta levels in ischemic mouse brain and reduced tissue damage when administered to rats as well. Only z-VAD.FMK and acetyl-Tyr-Val-Ala-Asp-chloromethylketone reduced brain swelling, and N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone did not attenuate the ischemia-induced increase in tissue IL-1beta levels. The three cysteine protease inhibitors significantly improved behavioral deficits, thereby showing that functional recovery of ischemic neuronal tissue can follow blockade of enzymes associated with apoptotic cell death. Finally, we examined the effect of z-VAD.FMK on excitotoxicity and found that it protected against alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-induced or to a lesser extent N-methyl-D-aspartate-induced excitotoxic brain damage. Thus, ICE-like and CPP32-like caspases contribute to mechanisms of cell death in ischemic and excitotoxic brain injury and provide therapeutic targets for stroke and neurodegenerative brain damage.

The apoptotic cysteine protease CPP32

CPP32 (also called Yama and apopain) is a member of a growing family of cysteine proteases which includes the interleukin-1β-converting enzyme (ICE) and the product of the Caenorhabditis elegans cell death gene ced-3. CPP32 has been consistently implicated as a key protease of the ICE/CED-3 family that is activated in response to a variety of death stimuli. Active CPP32 consists of p17 and p12 subunits derived from a 32 kDa pro-enzyme. This activation can be mediated by some ICE-like proteases and the cytotoxic T-cell (CTL) protease granzyme B. Once activated, CPP32 can process some of the other ICE family members and cleaves several cellular proteins in apoptotic cells. Inhibitors of CPP32 and other ICE-like proteases are potent inhibitors of apoptosis and promise to be important therapeutic molecules for the treatment of diseases, such as neurodegenerative and autoimmune disorders, that arise from excessive cell death.

Role of Fas Ligand and Receptor in the Mechanism of T-Cell Depletion in Acquired Immunodeficiency Syndrome: Effect on CD4+ Lymphocyte Depletion and Human Immunodeficiency Virus Replication

AbstractDirect killing of CD4+ lymphocytes by human immunodeficiency virus-1 (HIV-1) probably cannot account for the magnitude of the loss of these cells during the course of HIV-1 infection. Experimental evidence supports a pathophysiologic role of the apoptotic process in depletion of CD4 cells in acquired immunodeficiency syndrome (AIDS). The Fas-receptor/Fas-ligand (Fas-R/Fas-L) system mediates signals for apoptosis of susceptible lymphocytes and lympoblastoid cell lines. A number of investigators have recently reported increased expression of the Fas receptor in individuals with HIV infection, along with increased sensitivity of their lymphocytes to anti-Fas antibody mimicking Fas ligand. We attempted to determine the role of Fas-mediated apoptosis in disease progression and viral replication. Increased Fas-receptor (CD95) expression on CD4+ and CD8+ lymphocytes was found in a large group of HIV-1–infected patients compared with normal controls; individuals with a diagnosis of AIDS and a history of opportunistic infection had significantly more Fas receptor expression than did asymptomatic HIV-infected persons and normal blood donor controls (P < .01). Triggering of the Fas-R by agonistic anti-Fas monoclonal antibody, CH11, was preferentially associated with apoptosis in the CD4+ cells; this effect was more pronounced in lymphocytes derived from HIV+ individuals. Soluble and membrane-bound forms of Fas-L were produced in greater amounts in peripheral blood mononuclear cells (PBMC) cultures and in plasma obtained from HIV-1–infected persons than from normal controls. Furthermore, triggering of lymphocytes from HIV-infected persons by CH11 increased levels of interleukin-1β converting enzyme (ICE), a protein associated with apoptosis. When PBMC were cultured in the presence of CH11, p24 production per number of viable cells was decreased as compared with the same PBMC without CH11 (P < .01). These findings suggest that multiple mechanisms, including increased production of Fas-L by infected PBMC, increased Fas-R expression, and induction of a protease of ICE family, may play roles in the apoptotic depletion of CD4+ cells in HIV infection.

Upregulation of Fas-induced apoptosis by interferon-? accompanied by increased ICE expression in renal cell cancer cells

The integration of Fas/Apo-1 (CD95) by Fas ligand or anti-Fas antibody induces apoptosis, and this system plays a pivotal role for the lysis of target cells by cytotoxic T lymphocytes. Fas-mediated apoptosis is also increased by a prior incubation of Fas-bearing cells with interferon(IFN)-γ. Interleukin-1-β converting enzyme (ICE) and/or CPP32, or other members of ICE family act as direct cell death executors downstream of this mechanism, and a tetrapeptide inhibitor of these cysteine proteases blocks Fas-mediated apoptosis. In this study, we examined the effect of IFN-γ on Fas-mediated apoptosis in ACHN cells. IFN-γ augmented apoptosis in a dose dependent manner and reached a plateau at 400 U/ml when exposed for 48 h before the end of culture. The kinetics revealed a significant increase in apoptosis after 24 h. Exposing ACHN cells to IFN-γ increased pro-ICE expression accompanied with a decrease of pro-CPP32. These results suggest that direct enhancement of ICE expression and/or upregulation of conversion of pro-CPP32 to active form increases Fas-mediated apoptosis by IFN-γ in ACHN cells.

Purification and Characterization of an Interleukin-1β-converting Enzyme Family Protease That Activates Cysteine Protease P32 (CPP32)

CPP32, a member of the interleukin-1beta-converting enzyme (ICE) family of cysteine proteases, cleaves poly(ADP-ribose) polymerase and sterol regulatory element binding proteins during apoptosis. CPP32 normally exists in the cytosol as a 32-kDa inactive precursor and only becomes activated when cells are undergoing apoptosis. The activation is a proteolytic event that generates a p20/p11 heterodimer. We report here the identification, purification, and characterization of a hamster CPP32-activating protease (CAP) that cleaves and activates CPP32. The biochemical properties of CAP suggest that it is another member of the ICE family of proteases. Purified CAP consists of two prominent polypeptides of 19 and 13 kDa. Protein sequencing revealed that CAP is derived from the hamster homolog of Mch2alpha, a member of the ICE family recently identified based on the sequence conservation among the ICE family members. CAP activity is inhibited by CrmA, a cowpox virus protein that prevents host cell apoptosis. CAP itself is also activated through proteolytic cleavage. These data are consistent with the idea that the activation of the ICE family of proteases during apoptosis proceeds through a cascade of proteolytic events.

Ligation of CD40 rescues Ramos-Burkitt lymphoma B cells from calcium ionophore- and antigen receptor-triggered apoptosis by inhibiting activation of the cysteine protease CPP32/Yama and cleavage of its substrate PARP

The new and growing family of interleukin-1β-converting enzyme (ICE) cysteine proteases are now recognised to be major effectors of cellular death by apoptosis. Like other members of this family, the CPP32/Yama proform is activated by processing to its active heterodimeric enzyme or apopain when it likely contributes to the process of apoptosis by cleaving poly(ADP-ribose) polymerase (PARP) and thereby inhibiting much of its DNA repair activity. Apoptosis plays a fundamental role in the regulation of the immune system where it is involved in the selection of both T and B lymphocytes bearing antigen receptor (AgR) for non-self. Cells of the Ramos Epstein-Barr virus (EBV)-genome-negative Burkitt lymphoma (BL) B cell line (Ramos-BL) can be triggered into growth arrest and apoptosis by treating with the calcium ionophore ionomycin or by crosslinking their surface AgR with antibodies directed against immunoglobulin (Ig)M (anti-IgM). Ionomycin- and AgR-triggered growth arrest and apoptosis are arrested by signals transduced through the surface CD40 of Ramos-BL B cells. Both ionomycin and anti-IgM trigger activation of CPP32 and cleavage of PARP prior to the onset of apoptosis; this process is abrogated by treatment with anti-CD40 and is independent of Bcl-2 expression. A tripeptide inhibitor of ICE family cysteine proteases, Z-Val-Ala-Asp-fluoromethylketone (zVAD- fmk) inhibits ionomycin- and AgR-triggered CPP32 activation, PARP cleavage and apoptosis, but not growth arrest, in Ramos-BL B cells. Thus, in this report we demonstrate that in a physiological system, activation of endogenous members of the ICE family, including CPP32, and cleavage of the death substrate PARP act as major effectors of apoptotic death.