Caspases, the principal effectors of apoptosis, are conserved in metazoans. These proteolytic enzymes are remarkable in their exquisite substrate specificity, cleaving the peptide bond after an Asp residue. Procaspase zymogens consist of an N-terminal prodomain followed by large and small subunits. Upon receipt of an apoptotic stimulus, proteolytic processing of the caspase generates the large and small subunits of the catalytic domain which are arranged as a dimer of heterodimers. Caspases also have roles in processes other than apoptosis, including cell proliferation, differentiation, and inflammation. For example, differentiation of human blood monocytes to macrophages is blocked by synthetic caspase inhibitors or baculovirus P35, indicating that caspase activity is required for differentiation. The finding that caspases involved in apoptosis also play a critical role in non-apoptotic functions suggests that these caspases can be activated through non-apoptotic pathways.
Apoptotic caspases are traditionally classified as either initiators or effectors, depending on their function in the apoptotic pathway, the length of their prodomain, and their mode of activation. Effector procaspase dimers are cleaved by upstream initiator caspases at their large/small subunit junctions, which leads to the formation of the active enzyme consisting of two active sites capable of binding and cleaving substrates. Once activated, effector caspases are responsible for the bulk of proteolysis that leads to the characteristic biochemical and morphological changes in the apoptotic cell. Upstream initiator caspases normally reside in the cell as monomers that require dimerization for full activation. Initiator caspase autocleavage between the large/small subunit junction leads to dimerization in vitro. In the cell, initiator caspase activation is mediated through the interaction of the long N-terminal prodomain of the initiator caspase with specific adaptor molecules. These interactions lead to the formation of large caspase activating complexes which activate the initiator caspases by "induced proximity". "Induced proximity" mediates dimerization of the caspase through conformational changes and/or by facilitating initiator caspase autocleavage. The mechanisms of initiator caspase activation are not completely understood, and are therefore still under investigation.
Caspase activation occurs via two principle cellular pathways, designated the "intrinsic" and "extrinsic", which involve the formation of caspase activating complexes. The extrinsic pathway in mammals is activated upon binding of the CD95 death ligand to Fas-Associated protein with Death Domain (FADD), which leads to the formation of the death-induced signaling complex (DISC). Initiator caspase human caspase-8 is recruited to the DISC through interactions of its N-terminal death effector domain, and once there is autoprocessed and activated. Once active, caspase-8 activates effector caspases-3 and -7 directly through proteolytic cleavage, or indirectly through activation of the mitochondrial pathway. Caspase-8 mediated cell death is critical to immune system regulation, and is a mechanism involved in activation-induced cell death of T cells.
The intrinsic apoptotic pathway is triggered upon receipt of any of a number of internal signals including developmental cues, DNA damage, or other cellular insults. The central step in the intrinsic pathway, mitochondrial outer membrane permeabilization (MOMP), is regulated by the B-cell lymphoma (Bcl)-2 family of proteins. Bcl-2 inhibits the pro-apoptotic activity of the Bcl-2 homology-3 (BH3) only proteins, including Bax and Bak. Bax and Bak may cause MOMP by forming pores in the mitochondria, though the exact mechanism of Bax and Bak-mediated MOMP is still under investigation. Release of pro-apoptotic factors from the mitochondria, such as Smac/DIABLO, relieves inhibitor of apoptosis protein (IAP) inhibition of initiator caspase-9. Cytochrome C is also released from the mitochondria upon permeabilization, and binding of cytochrome C to the caspase cofactor Apaf1 results in the formation of a large heptameric complex known as the apoptosome. With caspase-9 free from IAP inhibition, this caspase can then bind to the apoptosome, which induces caspase-9 dimerization by "induced proximity". Once activated, caspase-9 cleaves and activates caspases-3 and -7, leading to apoptosis.
Caspase substrate specificity
Caspases are specific for cleavage of the peptide bond after an Asp residue. All caspases have a positively charged Si substrate pocket, designed to bind the Asp sidechain of the P1 residue. In fact, caspases preferentially cleave a P1 Asp over Glu by up to 4 orders of magnitude. DRONC is the exception, as it tolerates a P1 Glu almost as well as an Asp. To a lesser extent, the S2 and S3 pockets also play a role in caspase substrate specificity. The S4 pocket generally is important for initiator vs. effector specificity, with effectors preferring an Asp residue, and initiators preferring Ile/Leu/Thr at this position. The specificity of caspases has been exploited to design synthetic substrates and inhibitors to measure and block caspase activity. For example, DEVD-AMC is used commonly as a substrate for effector caspase activity assays, and is used extensively in this thesis. While synthetic inhibitors, such as N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (z-VAD-fmk), are effective for broad inhibition of caspase activity, these molecules are generally not as effective as caspase-specific inhibitors.
Caspases have many natural substrates, of which caspases themselves are some of the most important. Initiator caspases proteolytically cleave effector caspases at the large/small subunit junction, and not surprisingly the sequence at this junction resembles the experimentally determined preferred peptide substrates of the initiator caspase. Caspase cleavage of cytoplasmic and nuclear scaffolding proteins results in the distinct morphology observed in apoptotic cells. Caspase substrates also include pro- and anti-apoptotic signaling proteins, as well as molecules involved in cell cycle regulation, DNA regulation and repair, and signal transduction. A complete understanding of caspases, their targets, and their target selectivity will aid in the development of therapies for the treatment of apoptosis-related diseases.