Cellular cholesterol homeostasis is controlled by sterol- regulated proteolysis of membrane-bound transcription factors called sterol-regulatory element binding proteins (SREBPs). Caspase-3, a cysteine protease, was shown to cleave SREBP-1 and SREBP-2 in vitro at an aspartic acid between the basic helix-loop-helix leucine zipper domain and the first transmembrane domain, liberating a transcriptionally active fragment. It is shown that pro-caspase-3 exists in an inactive 32 kDa form in Chinese hamster ovary (CHO) cells. When apoptosis is induced with the protein kinase inhibitor staurosporine, pro-caspase-3 is cleaved to subunits of 20 and 10 kDa to form the active protease. Under these conditions membrane-bound SREBP-1 and SREBP-2 are both cleaved, and the transcriptionally active N-terminal fragments are found in nuclear extracts. Similar results are obtained in human U937 cells induced to undergo apoptosis by anti-Fas and etoposide. The apoptosis-induced cleavage of SREBPs is not suppressed by sterols, indicating that apoptosis-induced cleavage and sterol- regulated cleavage are mediated by different proteases. CHO cells expressing a mutant SREBP-2 with an Asp® Ala mutation at the caspase-3 cleavage site showed sterol-regulated cleavage but no apoptosis-induced cleavage (Wang et al., 1996).

The retinoblastoma (Rb) protein, an important cell cycle regulator with a known anti apoptotic function Rb, is an important mediator of cell cycle progression and regulation. Phosphorylation of Rb by cyclin-dependent kinases inactivates its growth-suppressive functions and drives cells through the cell cycle into mitosis. Several groups have described the cleavage of Rb during apoptosis (Janicke et al., 1996; An et al., 1996).

The C-terminal 42 amino acid peptide of Rb is specifically cleaved off by an ICE-like protease in tumour necrosis factor (TNF)- and staurosporine- induced apoptosis. Cleavage of Rb induced by TNF was blocked in vivo and in vitro by two specific inhibitors of caspases, and in vitro by a point mutation (Asp886 to Ala) within the caspase cleavage site of Rb, ⁸⁸³DEAD⁸⁸⁶. An antibody raised against the C-terminal 15 amino acid peptide of Rb recognised the full-length but not the cleaved form of Rb. The extent of Rb cleavage correlated directly with TNF-induced apoptosis in all tumour cell lines examined. Cleaved Rb bound cyclin D3 and inhibited the transcriptional activity of E2F-1, but failed to bind to the regulatory protein MDM2, which has been implicated in apoptosis. As Rb suppresses cell death and its C- terminus has important regulatory functions, Rb cleavage is an important event in apoptosis (Janicke et al., 1996).

When an HL-60 cell line resistant to cytosine arabinoside (Ara-C) is exposed to this anticancer drug, neither Rb cleavage nor apoptosis is detected. Consistent with that, processing of caspase-1 and -3 is also prevented in these cells. Treatment of these HL-60-Ara-C-resistant cells with etoposide induces apoptosis and Rb cleavage. Furthermore, the etoposide- induced Rb cleavage is inhibited by a specific tetrapeptide ICE-like inhibitor, demonstrating that activation of the Rb cleavage enzyme, a caspase, is required for overcoming drug resistance (An et al., 1996). Interestingly, different-sized cleavage products of Rb were observed in these studies, suggesting the possible involvement of different caspases.

A positional cloning approach has led to the identification of two closely related genes, the presenilins (PS), for autosomal dominant presenile Alzheimer disease (AD): PS-1 at 14q24.3 and PS- 2 at 1q31-q42. The PS-1 gene was identified by direct cDNA selection of yeast artificial chromosomes containing the candidate chromosomal region. Subsequently, the PS-2 gene was identified due to its high sequence homology with PS-1 and its location within the candidate region defined by linkage studies. The PS transcripts encode novel proteins that resemble integral transmembrane proteins of roughly 450 amino acids and at least seven transmembrane domains. The genomic organisation of the PS genes is very similar showing that full length PS-1 and PS-2 are encoded by 10 exons. However, different alternative splicing patterns have been observed for PS-1 and PS-2 indicating that the corresponding proteins (PS1 and PS2) may have similar but not identical biological functions (Cruts et al., 1996; Murgolo et al., 1996; Haass, 1996)

PS1 and PS2 are endoproteolytically processed in vivo and in cell transfectants to yield 27-35-kDa N-terminal and 15-24-kDa C-terminal fragments. The cleavage of PS1 and PS2 was studied in transiently and stably transfected hamster kidney and mouse and human neuroblastoma cells by immunoblot and pulse-chase experiments. C- terminal fragments were isolated by affinity chromatography and SDS-polyacrylamide gel electrophoresis and sequenced. The processing sites identified in PS1 and PS2 (Asp345/Ser346 and Asp329/Ser330, respectively) are typical for caspase-type proteases. Specific caspase inhibitors and cleavage site mutations confirmed the involvement of caspase(s) in PS1 and PS2 processing in cell transfectants. Fluorescent peptide substrates carrying the PS-identified cleavage sites were hydrolysed by proteolytic activity from mouse brain. The PS2-derived peptide substrate was also cleaved by recombinant human caspase-3. Additional processing of PS2 by non-caspase-type proteases was also observed (Kim et al., 1997; De Fea et al., 1994)

Huntington's disease (HD) is an inherited neurodegenerative disorder associated with expansion of a CAG repeat in the IT15 gene. The IT15 gene is translated to a protein product termed huntingtin that contains a polyglutamine (polyGln) tract. Comparison of repeat length and age at onset of disease symptoms shows an inverse correlation between the age at onset and the number of CAG repeat units, illustrating that the cause of HD is expansion of the polyGln tract. (Lucotte et al., 1994; Rosen, 1996; Bao et al., 1996). It was shown that apoptotic extracts and caspase-3 itself specifically cleave huntingtin. Interestingly, the rate of cleavage increases with the length of the huntingtin polyglutamine tract, providing an explanation for the gain-of-function associated with CAG expansion (Goldberg et al., 1996).

Shigella, the etiological agent of dysentery, kills macrophages by inducing apoptosis. Deletion mutants in the invasion invasion plasmid antigen B (ipaB) of Shigella flexneri are not cytotoxic. IpaB is localised to the cytoplasm of macrophages infected with S. flexneri. Purified IpaB induced apoptosis when microinjected into macrophages, indicating that IpaB is sufficient to induce apoptosis. Using a GST-IpaB fusion protein as a ligand in affinity purification, four IpaB binding proteins from macrophages were identified as the precursor and the mature polypeptides of caspase-1 or a highly homologous protease. IpaB binds directly to caspase-1 and this enzyme is activated during S. flexneri infection. Furthermore, specific inhibitors of caspase-1, Ac-YVAD.CHO, prevented Shigella-induced apoptosis (Chen et al., 1996). The specific mechanisms by which IpaB functions as an activator of caspase-1 remain to be determined.