Cell Death and Differentiation (1999) 6, 1028 ± 1042 ã 1999 Stockton Press All rights reserved 13509047/99 $15.00 http://www.stockton-press.co.uk/cdd Review Caspase structure, proteolytic substrates, and function during apoptotic cell death DW Nicholson*,1 strongly implicated ICE (or related family members, as it turned out) in a similar mammalian cell death pathway. 1 Merck Frosst Centre for Therapeutic Research, Merck Frosst Canada & Co., PO Importantly, this collective information also demonstrated an Box 1005, Pointe Claire ± Dorval, Quebec, Canada, H9R 4P8 essential role for specific proteolysis in apoptotic cell death, * Corresponding author: DW Nicholson Merck Frosst Centre for Therapeutic which led to the identification of proteolytic `victims' of the Research, Merck Frosst Canada & Co., PO Box 1005, Pointe Claire ± Dorval, caspases and shed light on the biochemical events that Quebec, Canada, H9R 4P8. Fax: +1 514 428-4900; E-mail: [email protected] occurred as a consequence of their cleavage. The importance of C. elegans genetics in defining the mammalian cell death Received 10.9.99; accepted 21.9.99; pathway is underscored by the fact that the individual cell Edited by G Melino death components, their molecular ordering and cellular functions have been largely conserved throughout evolu- tion6 (Figure 1). In addition to caspases, other proteases also Abstract contribute to the apoptotic cell death pathway. The serine Caspases play an essential role during apoptotic cell death. protease granzyme B, for example, has been well established These enzymes define a new class of cysteine proteases and as a caspase activator during CTL-mediated killing, and owing to its P Asp bias can function as a caspase surrogate comprise a multi-gene family with more than a dozen distinct 1 when caspases themselves are inoperative.7±9 The calpains mammalian family members. The discrete and highly limited have also been implicated in apoptosis,10 although their subset of cellular polypeptides that are cleaved by these precise role remains to be determined. proteases is sufficient to account for the majority of cellular and morphological events that occur during cell death. In some cases, caspases also play a contributory role in The mammalian caspase gene family and escalating the propensity for apoptosis, and in doing so may functional sub-families exacerbate disease pathogenesis. The caspase gene family11 thus far contains at least 14 mammalian members, of which 11 human enzymes are Keywords: apoptosis; caspase; protease; neurodegeneration known (Figure 2). A phylogenetic analysis indicates that the gene family is composed of two major sub-families which are Abbreviations: ICE, interleukin-1b converting enzyme; caspase, related to either ICE (caspase-1) or to the mammalian cysteinyl aspartate-speci®c proteinase; CED, product of cell-death- counterparts of CED-3. Further subdivisions can be made abnormal gene depending on whether the proenzymes harbour short prodomains (caspases-3, -6, -7) or long prodomains (the remainder). Alternatively, these proteases can be subdivided Proteases in apoptosis (the ICE age) on the basis of their substrate specificities which has been defined using a positional scanning combinatorial substrate ICE (interleukin-1b converting enzyme; caspase-1) is the library.12,13 Using the latter technique, the proteases fall into prototypical caspase and was initially identified as the only three specificity subgroups (Figure 3). As expected from protease responsible for the proteolytic maturation of proIL- earlier studies, the major specificity determinant is the S4 1,2 1b to its pro-inflammatory, biologically active form. When subsite (most of the enzymes are promiscuous at P2 and P3, originally discovered, ICE defined a new class of cysteinyl although they prefer His or Ile in P2 and prefer Glu in P3). proteases that was distinguishable from other cysteine Group I caspases (1, 4, 5, 13) are tolerant of liberal protease families based on general structural organization substitutions in P4 but prefer bulky hydrophobic amino acids and the absolute requirement for aspartic acid in the P1 such as Tyr or Trp. This preference is consistent with their role position of the scissile bond. A key role for ICE in inflammation in cytokine processing but does not support a substantial role had been largely established, which was quickly secured by in apoptosis since none of the polypeptides that are cleaved inhibitor studies and by the phenotype of ICE-deficient mice, during apoptosis contain hydrophobic residues in P4. The but other potential biological functions were not evident. At group II caspases (2, 3, 7) are substantially more stringent in approximately the same time, a genetic pathway for cell death S4, requiring a P4 Asp. This specificity and stringency is nearly was being defined in the nematode C. elegans.3 One of the indistinguishable from that of C. elegans CED-3. The genes in this genetic pathway, ced-3, encoded a protein that preferred cleavage motif (DExD) for group II caspases was essential for all 131 programmed cell deaths that appears in many proteins that are cleaved during cell death, occurred during hermaphrodite development. When ced-3 consistent with group II caspases being the major effectors of was cloned and sequenced, it was found to be a C. elegans cell death. Group III caspases (6, 8, 9, 10), on the other hand, 4,5 homologue of mammalian ICE. This and other evidence prefer branched chain aliphatic amino acids in P4; residues Caspase structure and role during apoptotic cell death DW Nicholson 1029 that are found at the maturation site of most group II and group APAF-1/cytochrome c pathway16 (both of these examples III caspases. This specificity is consistent with the group III have been substantiated in caspase-8 or -9 knock-out mice as enzymes being upstream activators of the group II effector well).17 ± 20 One possible exception to this general ordering caspases (Figure 4). This molecular ordering of group III and based on substrate specificity, which remains unresolved, is group II caspases has been upheld in several cases and is whether caspase-6 (a group III caspase) plays an effector role best exemplified by caspase-8-mediated activation of (e.g. lamin proteolysis)21 instead of or in addition to a putative caspase-3 and -7 in the CD95 (Fas, APO-1) system,14,15 activation role. Another exception may be caspase-2 which and caspase-9-mediated activation of caspase-3 in the appears to be a self-activating effector caspase.12,22,23 Figure 1 C. elegans cell death genetic pathway and mammalian counterparts Figure 2 The human caspase gene family. Caspases segregate into two major phylogenic sub-families (ICE, CED-3). Based on their proteolytic specificities (see Figure 3), caspases further divide into three groups: group I enzymes (blue) mediate cytokine maturation whereas the apoptotic caspases are either group II (red) effectors of cell death or group III (green) upstream activators. Most caspases have long prodomains (410 kDa) except for caspases-3, -6 and -7 (box) which have short peptidic prodomains (530 aa). With the exception of caspase-13, the human chromosomal location for all of the caspases has been determined. At least two gene clusters have been identified, consistent with some caspases arising from tandem gene duplication. These include the caspases-1, -4, -5 gene cluster on 11q22.2-q22.3 and the caspases-8, -10, cFLIP/Usurpin gene cluster on 2q33-q34.71,72 (cFLIP/Usurpin (aka CASH, Casper, CLARP, FLAME-1, I-FLICE, MRIT) is homologous to caspases -5, -8 and -10, except that substrate binding and catalytic determinants are absent, making it a dominant-negative death repressor.) The human counterparts of murine caspases-11, -12 and -14 have not yet been identified (although murine caspase-12 may be equivalent to human caspase-5) Caspase structure and role during apoptotic cell death DW Nicholson 1030 General caspase structural features which together make up the active form of the enzyme, are liberated from the proenzyme by cleavage at Asp(P1)-X(P1') Caspases are synthesized as catalytically-dormant tripartite bonds. The presence of Asp at the maturation cleavage sites proenzymes (Figure 5). Both the large and the small subunits, is consistent with the ability of caspases to auto-activate or to Figure 3 Caspase proteolytic specificity. The caspases recognize a core tetrapeptide motif corresponding to the four residues N-terminal to the scissile bond (P4- P3-P2-P1). A positional-scanning combinatorial substrate library, comprised of all 8000 possible P1 Asp tetrapeptides, segregates the caspases into three 12,13 73 specificity groups with the indicated sub-site preferences. Caspase-2 also appears to have a P5 requirement. Figure 4 Molecular ordering of caspases. Exceptions to this general ordering may also exist. Caspase-2, for example, may be self activating. Caspase-6 may function as an effector protease. Amplification circuits also exist. The caspase complement varies considerably between different cell types, directly bearing on the pathways available and how they function within different cellular environments Caspase structure and role during apoptotic cell death DW Nicholson 1031 be activated by other caspases as part of an amplification recruitment-activation (e.g. caspases-2, -8, -9, -10). Two cascade. Components of the proteolytic device, including the caspase X-ray crystal structures have been published active site Cys and His residues, are harboured within the (caspases-1 and -3)13,24 ± 27 and in both cases, the enzyme large subunit whereas residues which form the S1 subsite that was found to be a tetramer containing two large and two small tethers the carboxylate side chain of the essential P1 Asp are subunits (a [p20:p10]2 (caspase-1) or [p17:p12]2 (caspase-3) derived from both the large and small subunits (Figure 6). homodimer of the large subunit: small subunit heterodimer).