ICE-LAP3, a Novel Mammalian Homologue of the Caenorhabditis elegans Cell Death Protein Ced-3 Is Activated during Fas- and Tumor Necrosis Factor-induced Apoptosis

Abstract

Members of the ICE/ced-3 gene family have been implicated as components of the cell death pathway. Based on similarities with the structural prototype interleukin-1␤-converting enzyme (ICE), family members are synthesized as proenzymes that are proteolytically processed to form active heterodimeric enzymes. In this report, we describe a novel member of this growing gene family, ICE-LAP3, which is closely related to the death effector Yama/CPP32/Apopain. Pro-ICE-LAP3 is a 35-kDa protein localized to the cytoplasm and expressed in a variety of tissues and cell lines. Overexpression of a truncated version of ICE-LAP3 (missing the pro-domain) induces apoptosis in MCF7 breast carcinoma cells. Importantly, upon receipt of a death stimulus, endogenous ICE-LAP3 is processed to its subunit forms, suggesting a physiological role in cell death. This is the first report to demonstrate processing of a native ICE/ ced-3 family member during execution of the death program and the first description of the subcellular localization of an ICE/ced-3 family member.

Caspases: the executioners of apoptosis

“…Caspase-7 was cloned independently in three different laboratories and named Mch3\ICE-LAP3\CMH-1 [84][85][86]. A member of the CED-3 subfamily, it is a 303-amino-acid protein with high similarity to caspase-3 ( Figure 2a and Table 2).…”

“…Using a rabbit antipeptide antibody, caspase-7 protein migrates at " 35 kDa and is detected to a variable extent in a number of cell lines, including Jurkat T cells, where it is localized diffusely to the cytoplasm and juxtamembrane structures [85], consistent with the suggestion that the death effector machinery resides in the cytoplasm rather than the nucleus [8]. Overexpression of full-length caspase-7 in the MCF7 breast carcinoma cell line does not induce apoptosis, whereas expression of a truncated derivative, lacking the 53 Nterminal amino acids corresponding to the putative prodomain, induces apoptotic cell death [85]. Bacterially expressed caspase-7, like caspase-3, preferentially cleaves PARP and the peptide substrate Ac-DEVD-AMC, but not Ac-YVAD-AMC or pro-IL-1β.…”

Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8

“…The N-terminal His-tagged caspases-3, -6/a, and -8 were puri®ed from E. coli extracts by a nity chromatography with Ni 2+ -NTA agarose (Qiagen) according to the manufacturer's instructions. Human caspase-4 (Faucheu et al, 1995;Kamens et al, 1995;Munday et al, 1995) and caspase-7/a (Fernandes- Alnemri et al, 1995b;Duan et al, 1996a;Lippke et al, 1996) were expressed in DH5a E. coli as full-length proteins fused to glutathione-Stransferase at their N-termini. E. coli lysates containing active proteases were prepared as described (FernandesAlnemri et al, 1995a;Takahashi et al, 1996a).…”

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 ICE family is now known to comprise at least five members including CPP32/Yama/apopain [10][11][12], Nedd-2/ ICH-1 [13,14], TX/ICH-2/ICErealI [15][16][17], ICErellII [17] and Mch-2 [18]; Fernandes-Alnemri et al [19] and Duan et al [20] have recently described novel additions to the family, Mch-3 and ICE-LAP3 respectively, which are closely related to CPP32/Yama. All family members to date are synthesised as proenzymes that are proteolytically processed to form active heterodimeric proteases that contain the pentameric peptide, QACRG, which surrounds the putative catalytic residue cysteine [10][11][12][13][14][15][16][17][18][19][20].…”

“…All family members to date are synthesised as proenzymes that are proteolytically processed to form active heterodimeric proteases that contain the pentameric peptide, QACRG, which surrounds the putative catalytic residue cysteine [10][11][12][13][14][15][16][17][18][19][20]. Ectopic expression of ICE and its relatives resuits in the apoptosis of a variety of host cells which can be arrested by the cowpox serpin Crm A or by tetrapeptide (YVAD) inhibitors of ICE-like proteases [14,21,22].…”