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Caspases at the Heart of the Apoptotic Cell Death Pathway Sophie Roy* Department of Biochemistry & Molecular Biology, Merck Frosst Centre for Therapeutic Research, Merck Frosst & Company, 16711 Trans Canada Highway, Kirkland, Que´ bec, Canada H9H 3L1 Received May 17, 2000
The caspases (cysteinyl aspartate specific proteases)1 are a family of cysteine proteases that are at the heart of the apoptotic pathway (see ref 1 for a review). Through cleavage of a discrete number of proteins involved in cellular homeostasis and structural integrity, they mediate the systematic disassembly of the dying cell into characteristic apoptotic bodies that are rapidly engulfed by macrophages and other phagocytes. The importance of these enzymes in mediating apoptosis is underscored by the fact that selective caspase inhibitors prevent cell death. Twelve caspase family members have been identified in human tissues to date. Central to our understanding of the apoptotic pathway is the elucidation of the mechanism by which these enzymes become catalytically active. In healthy cells, the caspases exist as dormant pro-enzymes. Upon receipt of a death-inducing signal, they undergo two (or more) cleavage events to liberate a large subunit and a small subunit that heterodimerize into the active enzyme. Caspase activity needs to be tightly regulated to prevent premature death of the cell. The cleavage sites within the caspases all contain an aspartic acid in the P1 position, suggesting that the caspases either participate in a proteolytic cascade with one caspase activating the other or undergo autolytic activation. The substrate specificities of 10 of the 12 family members have been identified using a positionally defined combinatorial tetrapeptide approach and are consistent with the caspases forming a proteolytic cascade (2). The caspases can be divided into three groups with distinct substrate specificities. The group I caspases (caspase-1, -4, and -5) recognize preferentially the tetrapeptide WEHD (other large hydrophobic amino acids are also tolerated in P4). The substrate specificity of group I caspases is not consistent with these caspases participating directly in the apoptotic pathway; rather, they have been shown to play an important role in cytokine maturation. The group II caspases (caspase-2, -3, and -7) recognize the tetrapeptide DEXD (where X is any amino acid) and are termed the “executioner” caspases because they are directly responsible for the cleavage and disabling of the homeostatic and structural proteins during apoptosis. The group III caspases (caspase-6, -8-, -9, and -10) recognize tetrapeptides with the sequence I/L/VEXD. They are termed the “activator” caspases because their * To whom correspondence should be addressed: Department of Biochemistry & Molecular Biology, Merck Frosst Centre for Therapeutic Research, Merck Frosst & Co., 16711 Trans Canada Hwy., Kirkland, Que´bec, Canada H9H 3L1. Telephone: (514) 428-3430. Fax: (514) 428-4939. E-mail:
[email protected]. 1 Abbreviations: caspases, cystienyl aspartate specific proteases; CTLs, cytotoxic T lymphocytes; IAPs, inhibitors of apoptosis proteins.
Figure 1. Mechanisms by which tumor cells interfere with the apoptotic pathway. The extrinsic and intrinsic pathways represent the two principal pathways by which apoptosis is initiated. Death ligands (e.g., TNFR and CD95L), via their cognate receptors, stimulate the extrinsic apoptotic pathway by recruiting and activating caspase-8. The intrinsic pathway is initiated by a number of stimuli, including DNA damage, causing the leakage of cytochrome c from mitochondria. Cytochrome c, in turn, binds to the adapter molecule Apaf-1 and stimulates the recruitment and activation of caspase-9. The activator caspases, caspase-8 and -9, cleave and activate the executioner caspases, caspase-3 and -7, that are responsible for the proteolytic events that mediate the apoptotic phenotype. Tumor cells have evolved a number of mechanisms to cripple the apoptotic pathway. Overexpression of Bcl-2 or disabling p53 function by mutation results in inhibition of the intrinsic pathway. Overexpression of IAPs (inhibitors of apoptosis proteins) acts downstream in the apoptotic pathway to inhibit directly the executioner caspases, caspase-3 and -7, and thus interferes with both intrinsic and extrinsic pathways. This figure had been reprinted, with minor modifications, from ref 6, with permission from Elsevier Science.
substrate specificity corresponds to the cleavage sites between the large and small subunits of most but not all group II and group III caspases. Sharing the same substrate specificity as the activator caspases is the serine protease granzyme B present in granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Both in vitro and in vivo data support a critical role for caspase-8 and -9 and granzyme B, in the initiation of the apoptotic proteolytic cascade leading to cell death. The activation of the group III activator caspases occurs via an oligomerization-induced autolytic mechanism (see ref 1 for a review). Two major cell death pathways have been described: the “extrinsic” pathway and the “intrinsic” pathway (Figure 1). Caspase-8 is the key activator caspase of the extrinsic pathway where it is activated in response to ligand binding to members of
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the death receptor family (e.g., TNF-R1, CD95). Caspase-8 is recruited to the plasma membrane upon stimulation of the death receptor via the adapter molecule FADD where it oligomerizes and undergoes autolytic maturation. Caspase-9 is the key activator caspase of the intrinsic pathway where it is activated in response to stimuli that cause the release of cytochrome c from mitochondria. Cytochrome c, in cooperation with dATP, binds to and induces a conformational change in the adapter molecule APAF-1, which then recruits caspase9, resulting in the oligomerization and activation of caspase-9. Cancer cells have evolved a number of mechanisms to cripple the apoptotic machinery, thus allowing them to acquire a growth advantage and resist the cytotoxic effects of chemotherapeutic agents. These include (1) mutation and inactivation of the tumor suppressor gene p53, (2) upregulation of Bcl-2, and (3) upregulation of IAPs (inhibitor of apoptosis proteins). (1) p53 is a transcription factor that preferentially induces cell-cycle arrest in normal cells but apoptosis in tumorigenic cells. Several mechanisms have been proposed for its pro-apoptotic effects. Recent evidence shows that caspase-9 and APAF-1 are required for the proapoptotic activity of p53, suggesting that the intrinsic cell death pathway is perturbed in tumor cells bearing p53 mutations (3). (2) Bcl-2 inhibits apoptosis by preventing the release of cytochrome c from mitochondria. The mechanism of action of Bcl-2 is still poorly understood. It has been shown to bind to and sequester pro-apoptotic proteins. It also has been shown to exhibit anti-oxidant properties and ion channel activity. Regardless of its mechanism of action, its importance in crippling the apoptotic pathway in tumor cells is underscored by multiple reports, suggesting that antisense oligonucleotides to Bcl-2 sensitize tumor cells to chemotherapeutic agents (reviewed in ref 4).
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(3) IAPs are part of a multigene family of macromolecular inhibitors of the executioner caspases, caspase2, -3, and -7. One of the IAPs, survivin, is expressed in transformed cell lines and human tumors but is undetectable in terminally differentiated adult tissues (5). These results suggest yet another mechanism by which tumor cells interfere with the apoptotic machinery. The elucidation of the anti-apoptotic strategies that tumor cells have developed to evade cell death sheds light on the apoptotic pathways that must be blunted to allow tumor cell survival and sets the stage for the development of chemotherapeutic strategies that sensitize tumor cells to apoptosis.
References (1) Nicholson, D. W. (1999) Caspase structure, proteolytic substrates and function during apoptotic cell death. Cell Death Differen. 6, 1028-1042. (2) Thornberry, N., Rano, T. A., Peterson, E. P., Rasper, D. M., Timkey, T., Garcia-Calvo, M., Houtzager, V. M., Nordstrom, P. A., Roy, S., Vaillancourt, J. P., Chapman, K. T., and Nicholson, D. W. (1997) A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 27, 17907-17911. (3) Soengas, M. S., Alarcon, R. M., Yoshida, H., Giaccia, A. J., hakem, R., Mak, T. W., and Lowe, S. W. (1999) Apaf-1 and caspase-9 in p53-dependent apoptosis and tumor inhibition. Science 284, 156159. (4) Coffer, F. E. (1999) Antisense therapy of hematologic malignancies. Semin. Hematol. 36 (4 Suppl. 6), 9-14. (5) Ambrosini, G., Adida, C., and Altieri, D. C. (1997) A novel antiapoptosis gene, survivin, expressed in cancer and lymphoma. Nat. Med. 3, 917-921. (6) Roy, S., and Nicholson, D. W. (2000) Programmed cell-death regulation: basic mechanisms and therapeutic opportunities. Mol. Med. Today 6, 264-266.
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