1. IAPs
  2. FLIP
  3. Dominant negative Caspases
  4. Peptide Inhibitors
  5. Reviews


    Many viruses interact with the apoptotic pathway of their target cells. Viruses with lytic cycles encode anti-apoptotic proteins. Among these are p35 from baculovirus and CrmA (Cytokine response modifier A) from the cowpox virus which strongly inhibit caspase enzymatic activity. The IAPs (inhibitors of apoptosis proteins) are a family of anti-apoptotic proteins that are conserved across several species. The baculovirus Cp-IAP and Op-IAP, were the first members of this family to be identified based on their ability functionally to complement the cell death inhibitor, p35, in mutant viruses. Five human IAP homologs were identified: the neuronal apoptosis inhibitory protein (NAIP), cIAP1, cIAP2, XIAP and survivin. Ectopic expression of these human IAP genes can inhibit apoptosis induced by a variety of stimuli. BIR is an approximately 70 amino acid motif which is a common structural feature of all IAP family members. The BIR motif is present in one to three copies, and it may be necessary and sufficient for the anti-apoptotic effect of IAPs (Takahashi, 1998, J.Biol. Chem., 273: 7778-7790).

    XIAP, cIAP1 and cIAP2 prevented the proteolytic processing of Pro-Caspases -3, -6 and -7 by blocking the cytochrome c induced activation of Pro-Caspase-9 by binding directly to (Pro-)Caspase-9. But they did not prevent Caspase-8 induced activation of Pro-Caspase-3; however they subsequently inhibit processing of Caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases (Deveraux, Q.L. et al., 1998, EMBO vol.17, no.8: 2215-23).

    In another study, survivin and XIAP were shown to inhibit Bax- and Fas-induced apoptosis in 293 cells, and both were demonstrated to inhibit procaspase-7 processing. It was shown in vitro that Survivin and XIAP are binding to active Caspase-3 and Caspase-7, but not to their unactivated proforms (?). Therefore, it was confirmed that survivin and XIAP act on the level of the executioner caspases-3 and -7, but do not act on the level of initiator caspases (since they did not interacted with Caspase-8) In a cell-free system survivin inhibited cytochrome c induced caspase-activity and in vivo it prevented etoposide mediated apoptosis and caspase activity. While survivin is rarely present in normal adult tissues, it is commonly expressed in human cancers: possibly the level of expression can be correlated with the malignancy of a tumor.


    In 1997, another family of viral inhibitors of apoptosis was described: the v-FLIPs (viral FLICE-inhibitory proteins) which are present in several gamma-herpesviruses and in molluscipox virus. v-FLIPs contain two death effector domains (DEDs) so that they can interfere with the FADD-Caspase-8 interaction, thereby inhibiting the recruitment and activation of Caspase-8 during Fas-mediated apoptosis. v-FLIPs also block the early signaling events of other death cell surface receptors as TNFR1, TRAMP/DR3 and TRAIL-R/DR4. The identification of those viral inhibitors motivated the search for cellular homologs of the vFLIPs what resulted in the isolation of the cellular protein FLIP (Irmler et al., 1997, Nature, vol.388: 190-195). Several groups isolated FLIP what resulted in a number of aliases: CASH, Casper, I-FLICE, CLARP, FLAME-1, MRIT and Usurpin (see Rasper et al., 1998, Cell Death and Diff., 5: 271-288). FLIP is expressed as alternatively-spliced variants of a single FLIP gene. All those variants contain a long prodomain that harbors tandem DEDs. Following the prodomain, FLIPs (except the short form FLIP) possess a caspase protease region which is enzymatic inactive because essential amino acid residues are substituted, e.g. the catalytic diad forming Cys and His residues are substituted by Tyr or Arg, respectively. FLIP interacts with FADD what is mediated by the DEDs. FLIP also forms heterodimers with Capase-8 and is cleaved by Caspase-8. This may result in an even tighter binding of the cleaved FLIP to Caspase-8, blocking its proteolytic activity. This inhibition mechanism is reminiscent of the cleavage of baculovirus p35 which is a substrate for several caspases and which forms tight complexes with those caspases.

    Dominant negative Caspase

    It also should be mentioned that several caspase genes are expressed as multiple isoforms by alternativly splicing of the primary transcript (this is known for Caspase-1, -2, -3, -6, -7 and -8). Among those isoforms are enzymatically inactive variants which are expressed as modified mRNAs or truncated proteins which may play a crucial role in the negative or positive regulation of caspase activity. Caspase-8, for example, is expressed in at least seven isoforms that differ by deletions or sequence variations in the N-terminal prodomain (containing the DEDs) or by loss of the C-terminal part that normally encodes the p10/p20 caspase subunits. One isoform (MACH alpha-3 isoform) was shown to have a dominant negative effect on the activity of the active caspase-8 enzyme and to provide effective protection against Fas-mediated apoptosis (Boldin et al., 1996, Cell, vol. 85, 803-815).

    Peptide Inhibitors

    Finally, the synthetic peptide inhibitors of caspases should be mentioned which are used to elucidate the role of caspases in apoptosis and to study the hierarchic order of events in the caspase cascade. Several peptide-based inhibitors have been designed, mainly tetrapeptide-inhibitors. The peptide sequences are based on the recognition sequence of substrates which are cleaved by particular caspases. For example, the tetrapeptide aldehyde Ac-YVAD-CHO is based on the pro-IL-1beta cleavage site, and therefore is a strong inhibitor of Caspase-1, while the aldehyde tetrapeptide containing the PARP cleavage-site, c-DEVD-CHO, inhibits preferentially (but not specifically) caspase-3. The peptide z-VAD-fmk is a broad-range caspase inhibitor.


    Holcik and Korneluk, 2001, Nature Reviews, July (2): 550-56