Caspases are intracellular cysteinyl aspartate proteases with an important role in programmed cell death, proliferation, and immunity. The importance for caspases in the process of apoptosis was initially established by the discovery of the ced-3 gene, which encodes for a caspase important for programmed cell death in C. elegance. The discovery of the ced-3 caspase led to the identification of numerous apoptotic caspases in other species. Based on the initial discovery in apoptosis, caspases became synonymous with cell death for a long time. Interestingly, the mammalian homolog of ced-3 encodes Caspase-1, which mediates inflammation rather than cell death. More recently, it became clear that apoptotic caspases have additional and very important non-apoptotic functions in survival, proliferation, differentiation, and immunity.
Caspase structure and classification
Caspases can be classified into two types: initiator and effector caspases. Initiator caspases are activated first and trigger the activation of effector caspases by proteolytic cleavage of the inactive pro-form of effector caspases. Activated effector caspases cleave a broad range of structural and regulatory substrates that finally triggers cell death.
To prevent erroneous activation of the caspase pathway, caspases are synthesized as inactive zymogens. Caspases possess an N-terminal prodomain and a C-terminal caspase-domain that is composed of a small and a large subunit. Initiator caspases have longer prodomains compared to effector caspases, which contain protein interaction motifs that integrate the initiator caspases into macromolecular complexes. More specifically, the prodomain of initiator caspases contains specific protein interaction regions (e.g. caspase activation and recruitment domain (CARD) or death effector domain (DED)) that are important for their interaction with other adaptor proteins. The prodomain and the small and large subunits are connected by linker sequences that are often proteolytically cleaved at an aspartic acid (Asp) during the caspase activation process. C-terminal cleavage at the aspartic acid peptide bonds within the caspases and substrate proteins is a feature that is common to all caspases. The large subunit contains the catalytic site cysteine while the small subunit provides conserved residues to form the substrate binding pocket.
By now twelve caspases are identified in humans and most of the caspases to date are described in the context of programmed cell death. A lot of effort went towards the understanding of the regulatory functions of caspases during apoptosis.
Seven caspases have been described in Drosophila, with Nedd2-like caspase (Dronc) and Dredd defined as initiator caspases, and Drosophila interleukin-1 converting enzyme (Drice), Death caspase-1 (Dcp-1), Death executioner caspase related to Apopain/Yama (Decay), and Death Associated Molecule related to Mch2 (Damm) as effector caspases. The seventh caspase, Serine-threonine rich caspase (Strica), has a long prodomain typical for initiator caspases, but lacks any caspase recruitment domain or death effector domain. Since the cellular function of Strica is unclear, further research is required to clarify if Strica functions as a potential initiator caspase.
The full activation and stabilization of initiator caspases usually requires dimerization and auto-processing, while effector caspases require proteolytic cleavage by an initiator caspase. In contrast to initiator caspases, effector caspases form inactive homodimeric complexes in the absence of apoptotic signals. The linker between the small and large subunit of effector caspases sterically blocks the active site, and therefore activation requires the proteolytic cleavage of the inter-subunit linker by an initiator caspase. Proteolytic cleavage triggers rearrangement of the subunits in order to form the catalytic site.
The activation mechanism of initiator caspases is still not fully understood. Initiator caspases are inactive monomeric zymogens and they require signal mediated dimerization in order to become active. In general, this is initiated by the recruitment of the caspase to a signaling complex. Caspases bind the complex through prodomain-mediated homotypic interactions, and caspase recruitment to the complex triggers proximity-induced dimerization and activation of the caspase molecule. Dimerization triggers autoproteolytic cleavage in the inter-subunit linker that results in the separation of the small and large subunit. The additional cleavage of the linker between the prodomain and the large subunit releases the active caspase from the complex which consist of a tetrameric structure of the two small subunits surrounded by the two large subunits. The two heterodimers interact with each other predominantly through the interaction between the small subunits. The association of the subunits generates two active sites in the mature caspase molecule and it is proposed that each site function autonomously.
The requirement of dimerization or/and proteolytic cleavage for the activation of caspases is still under debate. For example, dimerization, but not cleavage is required for Caspase-8 function in T-cell proliferation whereas cleavage of Caspase-8 is required for induction of apoptosis. It is proposed that proteolytic cleavage stabilizes Caspase-8 to achieve adequate stability to function in the cytosol after its release from the complex. So far, it is not understood if the differences in the activation process trigger the diverse function of Caspase-8 in apoptotic and non-apoptotic processes. The specific requirements for Caspase-9 activation are still unclear since some studies suggested that cleavage and dimerization are not required for the full activation of Caspase-9. Similarly, the Caspase-9 ortholog Dronc appears to only require dimerization, and while cleavage occurs during apoptosis, it is not essential for Dronc activation. The molecular activation mechanism of the non-apoptotic initiator caspase Dredd, which is considered the Caspase-8 ortholog, is unknown.
While a lot of effort has been made to understand caspase activation in the process of apoptosis, further research is necessary to determine more details of caspase activation and how differences in their activation process might mediate the opposing physiological function in apoptosis, survival, and differentiation.