Murine L929 fibrosarcoma cells were transfected with the human Fas (APO-1/CD95) receptor, and the role of various caspases in Fas-mediated cell death was assessed. Proteolytic activation of procaspase-3 and -7 was shown by Western analysis. Acetyl-Tyr-Val-Ala-Asp-chloromethylketone and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone, tetrapeptide inhibitors of caspase-1– and caspase-3–like proteases, respectively, failed to block Fas-induced apoptosis. Unexpectedly, the broad-spectrum caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone and benzyloxycarbonyl-Asp(OMe)-fluoromethylketone rendered the cells even more sensitive to Fas-mediated cell death, as measured after 18 h incubation. However, when the process was followed microscopically, it became clear that anti-Fas–induced apoptosis of Fas-transfected L929 cells was blocked during the first 3 h, and subsequently the cells died by necrosis. As in tumor necrosis factor (TNF)-induced necrosis, Fas treatment led to accumulation of reactive oxygen radicals, and Fas-mediated necrosis was inhibited by the oxygen radical scavenger butylated hydroxyanisole. However, in contrast to TNF, anti-Fas did not activate the nuclear factor κB under these necrotic conditions. These results demonstrate the existence of two different pathways originating from the Fas receptor, one rapidly leading to apoptosis, and, if this apoptotic pathway is blocked by caspase inhibitors, a second directing the cells to necrosis and involving oxygen radical production.

BelgiumTHE Fas/ APO-l receptor is one of the major regulators of apoptosisl-7. We report here that Fas/ APO-l-mediated apoptosis requires the activation of a new class of cysteine proteases, including interleukin-lJl-converting enzyme (ICE)s--lo, which are homologous to the product of the Caenorhabditis elegans cell-death gene ced-3 (refs 11, 12). Triggering of Fas/ APO-l rapidly stimulated the proteolytic activity of ICE. Overexpression of ICE, achieved by electroporation and microinjection, strongly potentiated Fas/ APO-l-mediated cell death. In addition, inhibition of ICE activity by protease inhibitors, as well as by transient expression of the pox virus-derived serpin inhibitor, CrmA or an antisense ICE construct, substantially suppressed Fas/ APO-l-triggered cell death. We conclude that activation of ICE or an ICE-related protease is a critical event in Fas/ APO-l-mediated cell death.The signal transduction pathway elicited by Fas/ APO-l is almost completely unknown. Initiation of apoptosis may involve a new class of cysteine proteases, including the product of the C. elegans cell-death gene eed-3, mammalian interleukin-lfJ-converting enzyme (ICE) and the related proteases Nedd-2/Ich-l, prICE and CPP-32 (refs 11-17). Overexpression ofCED-3, ICE or Nedd-2/Ich-1 in Rat-l fibroblasts has been shown to result in apoptotic cell death 12.15. We therefore investigated whether Fas/ APO-I-mediated apoptosis involved an ICE-related proteolytic activity. In L929-APO-l cells 18 or B-lymphoblastoid SKW 6.4 cells, apoptosis triggered by the agonistic monoclonal antibody anti-APO-I was strongly inhibited by the ICE inhibitor YVAD-CHO, a tetrapeptide aldehyde (Ki = 0.76 nM)8 (Fig. la).

Recent data show that a strong relation exists in certain cells between mitochondria and caspase activation in apoptosis. We further investigated this relation and tested whether treatment with the permeability transition (PT)-inducing agent atractyloside of Percoll-purified mitochondria released a caspase-processing activity. Following detection of procaspase-11 processing, we further purified this caspase-processing protease and identified it as cathepsin B. The purified cathepsin B, however, was found to be derived from lysosomes which were present as minor contaminants in the mitochondrial preparation. Besides procaspase-11, caspase-1 is also readily processed by cathepsin B. Procaspase-2, -6, -7, -14 are weak substrates and procaspase-3 is a very poor substrate, while procaspase-12 is no substrate at all for cathepsin B. In addition, cathepsin B induces nuclear apoptosis in digitonin-permeabilized cells as well as in isolated nuclei. All newly described activities of cathepsin B, namely processing of caspase zymogens and induction of nuclear apoptosis, are inhibited by the synthetic peptide caspase inhibitors z-VAD.fmk, z-DEVD.fmk and to a lesser extent by Ac-YVAD.cmk.z 1998 Federation of European Biochemical Societies.

In general, apoptotic stimuli lead to activation of caspases. Once activated, a caspase can induce intracellular signaling pathways involving proteolytic activation of other caspase family members. We report the in vitro processing of eight murine procaspases by their enzymatically active counterparts. Caspase-8 processed all procaspases examined. Caspase-1 and -11 processed the effector caspases procaspase-3 and -7, and to a lesser extent procaspase-6. However, vice versa, none of the caspase-1-like procaspases was activated by the effector caspases. This suggests that the caspase-1 subfamily members either act upstream of the apoptosis effector caspases or else are part of a totally separate activation pathway. Procaspase-2 was maturated by caspase-8 and -3, and to a lesser extent by caspase-7, while the active caspase-2 did not process any of the procaspases examined, except its own precursor. Hence, caspase-2 might not be able to initiate a wide proteolytic signaling cascade. Additionally, cleavage data reveal not only proteolytic amplification between caspase-3 and -8, caspase-6 and -3, and caspase-6 and -7, but also positive feedback loops involving multiple activated caspases. Our results suggest the existence of a hierarchic proteolytic procaspase activation network, which would lead to a dramatic increase in multiple caspase activities once key caspases are activated. The proteolytic procaspase activation network might allow that different apoptotic stimuli result in specific cleavage of substrates responsible for typical processes at the cell membrane, the cytosol, the organelles, and the nucleus, which characterize a cell dying by apoptosis.

Seven members of the murine caspase (mCASP) family were cloned and functionally characterized by transient overexpression: mCASP-1 (mICE), mCASP-2 (Ichl), mCASP-3 (CPP32), mCASP-6 (Mch2), mCASP-7 (Mch3), mCASP-11 (TX) and mCASP-12. mCASP-11 is presumably the murine homolog of human CASP-4. Although mCASP-12 is related to human CASP-5 (ICE re i-III), it is most probably a new CASP-1 family member. On the basis of sequence homology, the caspases can be divided into three subfamilies: first, mCASP-1, mCASP-11 and mCASP-12; second, mCASP-2; third, mCASP-3, mCASP-6 and mCASP-7. The tissue distribution of the CASP-1 subfamily transcripts is more restricted than that of the CASP-3 subfamily transcripts, suggesting that the transcriptional regulation of the CASP members within one subfamily is related, but is quite different between the CASP-1 and the CASP-3 subfamilies. Transient overexpression of each of the seven CASPs induced apoptosis in mammalian cells. Only two, mCASP-1 as well as mCASP-3, were able to process precursor interleukin (IL)-lß to biologically active IL-lß. In addition, mCASP-3 is the predominant PARP-cleaving enzyme in vivo.

A20 is a Cys2/Cys2 zinc finger protein which is induced by a variety of inflammatory stimuli and which has been characterized as an inhibitor of cell death by a yet unknown mechanism. In order to clarify its molecular mechanism of action, we used the yeast two-hybrid system to screen for proteins that interact with A20. A cDNA fragment was isolated which encoded a portion of a novel protein (TXBP151), which was recently found to be a human T-cell leukemia virus type-I (HTLV-I) Tax-binding protein. The full-length 2386 bp TXBP151 mRNA encodes a protein of 86 kDa. Like A20, overexpression of TXBP151 could inhibit apoptosis induced by tumour necrosis factor (TNF) in NIH3T3 cells. Moreover, transfection of antisense TXBP151 partially abolished the anti-apoptotic effect of A20. Furthermore, apoptosis induced by TNF or CD95 (Fas/APO-1) was associated with proteolysis of TXBP151. This degradation could be inhibited by the broad-spectrum caspase inhibitor zVAD-fmk or by expression of the cowpox virus-derived inhibitor CrmA, suggesting that TXBP151 is a novel substrate for caspase family members. TXBP151 was indeed found to be specifically cleaved in vitro by members of the caspase-3-like subfamily, viz. caspase-3, caspase-6 and caspase-7. Thus TXBP151 appears to be a novel A20-binding protein which might mediate the anti-apoptotic activity of A20, and which can be processed by specific caspases.

No abstract

Emerging evidence suggests that multiple aspartatespecific cysteine proteases (caspases (CASPs)) play a crucial role in programmed cell death. Many cellular proteins have been identified as their substrates and serve as markers to assay the activation of CASPs during the death process. However, no substrate has yet been unambiguously identified as an effector molecule in apoptosis. PITSLRE kinases are a superfamily of Cdc2-like kinases that have been implicated in apoptotic signaling and tumorigenesis. In this paper we report that tumor necrosis factor (TNF)-mediated apoptosis is associated with a CrmA-and Bcl-2-inhibitable cleavage of PITSLRE kinases, indicating a role for CASPs. Testing of seven murine CASPs for their ability to cleave p110 PITSLRE kinase ␣2-1 in vitro revealed that only CASP-1 (ICE (interleukin-1␤-converting enzyme)) and CASP-3 (CPP32) were able to produce the same 43-kDa cleavage product as observed in cells undergoing TNF-induced apoptosis. Mutational analysis revealed that cleavage of p110 PITSLRE kinase ␣2-1 occurred at Asp 393 within the sequence YVPDS, which is similar to that involved in the CASP-1-mediated cleavage of prointerleukin-1␤. TNF-induced proteolysis of PITSLRE kinases was still observed in fibroblasts from CASP-1 0/0 mice. These data implicate CASP-3 as a potentially important CASP family protease responsible for the cleavage of PITSLRE kinases during TNF-induced apoptosis.Apoptosis is a fundamental process for normal development of multicellular organisms and is involved in the regulation of the immune system, normal morphogenesis, and maintenance of homeostasis (1). Aspartate-specific cysteine proteases belonging to the interleukin-1␤-converting enzyme (ICE) 1 family, recently renamed the caspase (CASP) family (2), have been implicated as principal effectors of apoptosis, presumably by their proteolytic action on specific targets, including members of the ICE-related protease family themselves, poly(ADP-ribose) polymerase, DNA-dependent protein kinase, the 70-kDa small U1 ribonucleoprotein, lamins, protein kinase C␦, D4-GDP dissociation inhibitor, and various components of the cytoskeleton (3, 4). Many of these proteins are likely to be involved in the morphological and biochemical changes that accompany apoptosis, or in aspects of DNA damage sensing and repair, and are used as markers to assay the activation of CASPs during the death process. However, no substrate has yet been unambiguously identified as a downstream effector molecule in apoptosis.PITSLRE kinases are a superfamily of protein kinases related to the master mitotic protein kinase Cdc2 (4 -6). Ectopic expression of the smallest member of this superfamily has previously been shown to induce apoptosis (5). In addition, deletion of the PITSLRE gene complex and complete loss of expression of specific isoforms occur in many neuroblastoma cell lines and is frequently observed in human cancers (8 -10). Induction of apoptosis via the Fas receptor in human T cells has recently been shown to be correlated with proteol...