Although proteases related to the interleukin 1,8-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cellfree model system for apoptosis (S/M extracts A key question in cell death research is whether the apoptotic cascade is driven by the action of a single interleukin 1l3-converting enzyme (ICE)-related protease (IRP) (1-7) or by multiple IRPs acting in concert (8). In the nematode Caenorhabditis elegans, a single IRP is required for all developmental cell deaths (1, 9). In contrast, cDNA cloning experiments show that at least seven IRP mRNAs are expressed in a single human cell type (4, 7, 10, 11), raising the possibility that multiple IRPs might be required for completion of apoptosis in vertebrates. The individual roles of these multiple IRPs during apoptosis are currently unclear.To begin to address this question, we have compared proteolytic cleavage of two apoptotic substrates by cloned IRPs expressed in Escherichia coli and by cell-free extracts (named S/M extracts, prepared from chicken DU249 hepatoma cells committed to apoptosis by an S-phase aphidocolin block and subsequently collected in M phase) (12, 13). Exogenous nuclei incubated in S/M extracts recapitulate nuclear apoptotic events, including endonucleolytic cleavage of DNA, chromatin condensation, and fragmentation of the nucleus (12). Incubation of nuclei or purified poly(ADP-ribose) polymerase (PARP) in S/M extracts results in rapid, quantitative cleavage of the PARP to a 85-kDa fragment indistinguishable from that observed in a wide variety of apoptotic cells (13)(14)(15). This cleavage occurs at a conserved DEVDUG sequence and is mediated by an enzyme with substrate recognition properties and inhibitor sensitivity similar to ICE. We termed this proteolytic activity prICE [protease(s) resembling ICE (13)]. Subsequent investigations have shown that the cloned human IRPs CPP32, Mch2a, and Mch3a as well as the C. elegans IRP CED-3 all cleave PARP (5,7,11,16,17). ICE itself can also cleave a PARP subfragment when added in considerable excess (18); however, at near physiological levels, it does not cleave full-length native PARP (5, 13).Although PARP was the first apoptosis-specific IRP substrate to be identified, the physiological significance of PARP cleavage in apoptosis is presently unknown (for review, see ref.15). In contrast, cleavage of the nuclear lamins is a proteolytic event that appears to be required for completion of nuclear reorganization during apoptosis. Lamin A is cleaved in S/M extracts (8) to fragments that are indistinguishable from those produced in cells undergoing apoptosis (8,(19)(20)(21). The inhibitor profile of the lamin protease suggests that lamin cleavage depends upon the activity of an IRP distinct from the PARPcle...

The caspases are cysteine proteases that have been implicated in the execution of programmed cell death in organisms ranging from nematodes to humans.

Although specific proteinases play a critical role in the active phase of apoptosis, their substrates are largely unknown. We. previously identified poly(ADP-ribose) polymerase (PARP) as an apoptosis-associated substrate for proteinase(s) related to interleukin 1p8-converting enzyme (ICE). Now we have used a cell-free system to characterize proteinase(s) that cleave the nuclear lamins during apoptosis. Lamin cleavage during apoptosis requires the action of a second ICE-like enzyme, which exhibits kinetics of cleavage and a profile of sensitivity to specific inhibitors that is distinct from the PARP proteinase. Thus, multiple ICE-like enzymes are required for apoptotic events in these cell-free extracts. Inhibition of the lamin proteinase with tosyllysine "chloromethyl ketone" blocks nuclear apoptosis prior to the packaging of condensed chromatin into apoptotic bodies. Under these conditions, the nuclear DNA is fully cleaved to a nucleosomal ladder. Our studies reveal that the lamin proteinase and the fragmentation nuclease function in independent parallel pathways during the final stages of apoptotic execution. Neither pathway alone is sufficient for completion of nuclear apoptosis. Instead, the various activities cooperate to drive the disassembly of the nucleus.Proteinases of the interleukin 1(3-converting enzyme (ICE)! ced-3 family are essential for the execution of apoptotic cell death (1). These proteinases can induce apoptotic death when overexpressed in cultured mammalian (2-5) or insect (6) cells. Inhibition of ICE family enzymes by CrmA blocks apoptosis induced by factor withdrawal (7) or by the Fas and tumor necrosis factor (TNF) pathways (8). Although gene knockout experiments have revealed that ICE itself is not essential for many types of apoptotic death (9, 10), it does appear to be required for Fas-mediated cell death (10). In addition, the demonstration that apoptosis can be induced by introduction of trypsin, chymotrypsin, or proteinase K into cells (11) raises questions about the interpretation of experiments in which ICE family members are overexpressed in transfected cells. Additional studies using complementary approaches are therefore needed to clarify the role of ICE-like proteinases in apoptosis.Studies using a cell-free system in which endogenous enzymatic activities drive apoptotic events have provided independent biochemical evidence for the involvement of an ICE-like proteinase in apoptotic cell death (12). This cell-free system uses concentrated extracts from chicken DU249 cells in the condemned (committed) phase of apoptosis (13) which is similar to a sequence in pro-interleukin-1l3 that is cleaved by ICE; and it is selectively inhibited by Tyr-Val-AlaAsp-CH2Cl [YVAD "chloromethyl ketone" (YVAD-cmk)], a highly specific inhibitor of ICE family proteinases. These similarities led us to designate the PARP proteinase prICE (proteinase resembling ICE). In further experiments, treatment with YVAD-cmk at concentrations that inhibit prICE abolished all hallmark events of apoptosis ...

Two novel synthetic tetrapeptides, VEID-CHO and DMQD-CHO, could selectively inhibit caspase-6 and caspase-3, respectively. We used these inhibitors to dissect the pathway of caspase activation in Fas-stimulated Jurkat cells and identify the roles of each active caspase in apoptotic processes. Affinity labeling techniques revealed a branched protease cascade in which caspase-8 activates caspase-3 and -7, and caspase-3, in turn, activates caspase-6. Both caspase-6 and -3 have major roles in nuclear apoptosis. Caspase-6 cleaves nuclear mitotic apparatus protein (NuMA) and mediates the shrinkage and fragmentation of nuclei. Caspase-3 cleaves NuMA at sites distinct from caspase-6, and mediates DNA fragmentation and chromatin condensation. It is also involved in extranuclear apoptotic events: cleavage of PAK2, formation of apoptotic bodies, and exposure of phosphatidylserine on the cell surface. In contrast, a caspase(s) distinct from caspase-3 or -6 mediates the disruption of mitochondrial membrane potential (permeability transition) and the shrinkage of cytoplasm. These findings demonstrate that caspases are organized in a protease cascade, and that each activated caspase plays a distinct role(s) in the execution of Fas-induced cell death.