First printed in R&D Systems' 2000 Catalog.
Caspases are a family of proteases that relay a "doomsday" signal in a step-wise manner reminiscent of signaling by kinases. Caspases are present in all cells as latent enzymes. They are recruited to receptor-associated cytosolic complexes whose formation is initiated by receptor oligomerization (e.g., TNF receptors, FAS, and TRAIL receptors) and to other cytoplasmic adaptor proteins, such as APAF-1. Recruitment of caspases to oligomerized receptors leads to activation via dimerization or dimerization accompanied by autoproteolytic cleavage. Active caspases can proteolyze additional caspases generating a caspase cascade that cleaves proteins critical for cell survival. The final outcome of this signaling pathway is a form of controlled cell death termed apoptosis.
The subgroup of caspases involved in apoptosis has been referred to as either initiators or effectors. Caspases-8, -9, and -10 (possibly, -2 and -5) can initiate the caspase activation cascade and are therefore called initiators. Based on the prototypes, caspases-8 and -9, initiators can be activated either by dimerization alone (caspase-9) Although an initiator caspase may not be responsible for starting the caspase cascade, it can become activated and involved in later steps of the cascade. Thus, in the latter scenario, the caspase would be more appropriately termed an effector.
A second subgroup of caspases is involved in processing a select group of cytokines. Caspase-1, the first caspase to be purified, was identified by its ability to cleave the precursor of IL-1 beta to its mature form. Caspase-11 is required for activation of caspase-1 and subsequently, caspase-11 deficient mice fail to activate caspase-1. Caspases-4, -5, -12, -13, and -14 are generally expected to be involved in cytokine processing. These caspases have an amino acid (aa) sequence homology closer to caspase-1 than to the caspases involved in apoptosis.
Members of the caspase family have several common characteristics. Additional constraints are placed on the specificity. Although many cellular proteins have the correct aa sequence required for caspase cleavage, only a select group are hydrolyzed. Thus, constraints on proteolysis limit hydrolysis to specific key proteins. Limitations on proteolysis results in the disassembly of the cell into membrane-limited cell fragments called apoptotic bodies. The apoptotic bodies are then engulfed and digested by phagocytic cells. It appears that limitations on proteolysis prevent the cell from lysing and releasing its contents into the surrounding environment, thereby avoiding an inflammatory response.
Nascent caspase polypeptides contain an amino-terminal pro-region, an internal aa stretch that becomes a large subunit, and a carboxyl-terminal aa stretch that becomes a small subunit (see figure 1). Caspase maturation involves homodimerization and proteolytic processing. Proteolytic cleavage removes the pro-region and splits the large subunit from the small subunit. Many caspases have two potential cleavage sites at the junction of the large and small subunits. These sites are separated by a few aa and cleavage may occur at one or both sites. Dimerization and proteolysis generates an active caspase composed of two large (~20 kDa) and two small (~10 kDa) subunits.
Crystal structures of caspase-3a-helices). Caspase-3 has two active site pockets each residing on an opposite side. Although X-ray crystallographic results could not definitively discriminate between possible modes of dimerization, each active site appears to be composed of a large subunit derived from one procaspase and a small subunit derived from the other procaspase.
Based on the caspase-3 structure, caspase dimerization is through the combining of proenzyme monomers oriented at 180° to each other. This orientation is exemplified in the simplistic stick diagram of caspase-8 shown in figure 2. Pro-regions are not required for proper in vitro refolding of caspase subunits to obtain the 2-large/2-small subunit active caspase. Thus, the pro-region may be irrelevant to the monomer-monomer interactions involved in dimerization (exemplified in figure 2 where the pro-regions extend beyond the overlap between large and small subunits). Caspase-8 cleaves itself during or after dimerization. The first proteolytic cleavage appears to occur at the junction of the small and large subunits (a in figure 2) to generate polypeptides of 12 and 41 kDa. Subsequent cleavage between the pro-region and the large subunit (b and c in figure 2) generates polypeptides of 17 and 24 kDa. The alternative step-wise autoproteolytic pathway (d-f in figure 2) involves a primary cleavage at the junction of the pro-region and large subunit.
Control of caspase dimerization is one level at which caspase activity is regulated. Pro-regions of some caspases function to facilitate caspase dimerization. These pro-regions contain domains that interact with adaptor proteins containing caspase recruitment domains (CARDs). APAF-1 binds to caspase-9 monomers, cytochrome c, and dATP. When cytochrome c is available to bind APAF-1, bound caspase-9 monomers are brought together, dimerize, and are activated. Caspase-8 dimerization is facilitated by binding to a death effector domain (DED) present in adaptors that form a complex on the cytosolic portion of trimerized apoptosis-inducing receptors. The DED that recruits caspase-8 differs from CARD. Recruitment of caspase-8 enables caspase-8 to dimerize and autoactivate. Based on the large pro-domains present in caspases-1, -2, -4, -5, and -10 (figure 1) and/or the presence of CARD in caspases-1, -2, and -4 and the presence of DED in caspase-10, the pro-region of these caspases likely facilitate dimerization. By analogy, these caspases may also undergo autoproteolytic activation after or during dimerization. Activation of caspases-3, -6, and -7, all lacking large pro-regions, requires cleavage between the large and small subunits by other caspases.
Proteolytically processed caspases are further degraded during apoptosis. Subunits can be generated and then may disappear during apoptosis. This has been observed in many cell types induced to undergo apoptosis by a variety of methods. Therefore, when examining potential caspase cleavage in a cell line, both the disappearance of the precursor and detection of subunits should be used as criteria. If a decrease in the level of caspase precursor is detected without the detection of individual subunits, earlier time points following induction of apoptosis should be evaluated.