In apoptosis, activation of caspases triggers the proteolytic cleavage of vital cellular proteins and ultimately causes disassembly of the cell. Several mechanisms of caspase inhibition have been described to avoid unwanted caspase activation that could be devastating for the organism.
Viral inhibition: Viral inhibition extends host cell viability and blocks the activation of the host immune response, therefore increasing the time for viral replication in the host. Two well-studied viral caspase inhibitors are the cowpox virus inhibitor cytokine response modifier A (CrmA) and the baculovirus p35. Both inhibitors are so-called suicide inhibitors that irreversibly block the caspase. The viral inhibitor gets processed by caspases like a substrate, but generates a stable irreversible inhibitor-enzyme complex through a covalent thioester bond.
Chemical inhibition: Chemical caspase inhibitors have been developed as a research and clinical tool to prevent apoptosis. For example, the cell-permeable pan caspase inhibitor zVAD-FMK competes with the substrate for binding and irreversibly interacts with the catalytic site to prevent induction of apoptosis.
Cellular inhibition (IAPs): Inhibitor of apoptosis proteins (IAP) are a conserved family of proteins across species as diverse as viruses, yeast, flies, and humans. IAP proteins were originally identified in baculovirus infected insect cells. Two motifs are representative for the members of the IAP family and are highly conserved amongst IAP proteins. The ~70 amino acid long N-terminal baculovirus IAP repeat domain(s) (BIR) are Zink Finger like structures that mediate protein-protein interactions. The C-terminal Really Interesting Gene domain (RING) provides E3 ubiquitin ligase activity. Some IAPs also contain structures like the caspase activation recruitment domain or the conserved Ubiquitin-associated domain (UBA) that binds monomeric ubiquitin and ubiquitin chains.
The mammalian cellular Inhibitor of Apoptosis Protein 1 (c-IAP1), cellular Inhibitor of Apoptosis Protein 1 (c-IAP2), X-linked Inhibitor of Apoptosis Protein (XIAP), and Melanoma-Inhibitor of Apoptosis (ML-IAP) and the Drosophila dIAP1 and dIAP2 possess RING domains with E3 ligase activities. In general, E3 ligase activity enables auto-ubiquitination and cross-ubiquitination of the substrate. Ubiquitin is a small protein that has a central role in a wide array of degradative and non-degradative processes. Ubiquitin chains are synthesized of ubiquitin monomers joined by covalent bonds between the Cterminus of one ubiquitin and the internal K48 (48rd lysine of ubiquitin) or K63 (63rd lysine of ubiquitin) lysine residues in the next ubiquitin. The attachment of ubiquitin involves the sequential action of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). The type of lysine linkages of ubiquitin chains determines the cellular outcomes. While K48-linked chains target the substrate for proteasomal destruction, non-degradative K63-ubiquitin chains are involved in regulatory processes such as DNA repair, signal transduction, and receptor endocytosis.
IAPs are considered the guardians of the apoptotic machinery by binding caspases and inhibiting their active site. This sterically blocks the caspase, sequesters the caspase away from its substrate, or promotes proteasomal degradation of the caspase. Caspases are only bound by IAPs after the caspase is processed, and consequently IAPs are considered the very last line of defense to ensure the survival of a cell. The only known exception to date is the Drosophila caspase Dronc which is bound by dIAP1 in the monomeric zymogen state.
Currently eight human IAPs and four Drosophila IAPs have been described. In humans, XIAP inhibits Caspase-9 and -7. Caspase-8 is not inhibited by IAPs, but XIAP blocks the Caspase-8 substrate Casaspe-3. In Drosophila, dIAP1 inhibits the caspases Dronc, Drice, and Dcp-1.
By now, the name inhibitor of apoptosis is a bit of a misnomer since recent reports suggested that only some IAPs (like XIAP in mammals and dIAP1 in flies) block caspase function under physiological conditions. By now, IAPs have also been linked to processes like cell-cycle regulation, cell-signaling, protein degradation, and immunity. For example, cIAP1 and 2 ubiquitinate many components in the TNFR-complex I that mediates the recruitment of further downstream molecules important for NF-κB and MAPK activation. In Drosophila, dIAP2 is important to mediate appropriate immune responses. Initially, dIAP2 was described in the context of apoptosis. However, in contrast to dIAP1, dIAP2 mutants are viable and healthy, and don’t have developmental or stress-induced apoptosis defects. Importantly, follow up studies demonstrated that diap2 mutant flies are incapable to fight of gramnegative bacterial infection and die quickly after an infection. Combined, the data established dIAP2 as an essential component in immunity.