Caspases and Cell Death

Abstract

Caspases (Cysteinyl ASPartate‐specific proteASE) are cysteine proteases involved in cell death. The caspase family is comprised of 12 proteins in humans, seven in Drosophila melanogaster and a single protein in Caenorhabditis elegans. All caspases consist of three structural domains: a prodomain, a large subunit and a small subunit. The catalytically active enzymes are formed either by proteolytic cleavage of the subunits, or through a proximity‐induced activation process involving the prodomain. The cleavage site of caspase substrates is indicated by the positions P4, P3, P2 and P1. P1 is always aspartate, while amino acids in the other positions are highly variable. By cleaving over 1000 substrates in the cell, caspase activation results in the complete dismantling and ultimate death of the cell. Caspase activity can be regulated at a pharmacological level and has become a therapeutic target for the treatment of many diseases.

Key Concepts

  • Caspases (Cysteinyl ASPartate‐specific proteASE) are the key enzymes regulating and executing apoptosis. They include several proteases acting either at the upstream regulatory events, or in the effector phase of cell death.

  • Three groups of caspases have been classified and are involved either in inflammation or cell death.

  • Caspases cleave over 1000 proteins within the cell causing the morphological events of cell death.

  • Both natural (cellular, bacterial and viral) and pharmacological inhibitors of caspases have been identified offering novel therapeutic approaches to inflammatory and apoptotic pathways.

Keywords: apoptosis; caspase; cysteine protease; cell death; inflammation; caspase inhibitor

Figure 1.

The human caspases family. (a) Classification of the human caspases. According to the phylogenic tree, caspases are subdivided into two major subfamilies, ICE and ced‐3. The former are involved in inflammation, and the latter in apoptosis. The caspases with a very short prodomains (<30 aa) are boxed (type 3, 6, 7); all the other enzymes have long prodromains (>10 kDa) subject to complex regulation. The enzymes can be divided according to their proteolytic specificity into group I, involved in cytokine maturation, group II, involved in the final effector phase of apoptosis (indicated by a star) and group III, involved in the upstream regulation of apoptosis. At least two gene clusters have been identified, consistent with some caspases arising from tandem gene duplication. Caspase 13 is an error of sequencing; caspase 12 is mutated into a nonfunctional enzyme in up to 90% of the human population. Caspase 14 has no apparent role in apoptosis or inflammation preventing its classification. The scheme has been modified from Nicholson . (b) Structural organization and activation of caspases. The caspases are produced as pro‐enzymes (32–53 kDa), including a prodomain (3–24 kDa), the large subunit (17–21 kDa) containing the active site, and the small subunit (10–13 kDa). To become enzymatically active, these three components must be proteolytically cleaved (D‐x site), a phenomenon that is regulated by the prodomain itself; this allows the assembly of the large and small subunit, and dimerization into a functional enzyme.

Figure 2.

Caspases and cell death. In mammalian cells, apoptosis can be triggered by extracellular (death receptors) or intracellular signals. The signal converges to the mitochondrion/apoptosome where the final effector phase occurs. Caspases, the proteolytic enzymes responsible for cell death, are involved both in the upstream regulatory phase (regulatory caspases), and in the final terminal phase (effector caspases). They are activated by an adaptor molecule (FADD in the DISC, or Apaf‐1 in the apoptosome), and they are regulated by the Bcl‐2 family. The regulation at the level of the death receptor, and of the apoptosome is shown in greater details in the expansion boxes. Expansion 1 (e1): Formation of the DISC, following the activation of a death receptor such as CD95 (also called Fas or APO‐1). Three molecues of ligand (CD95L) bind three molecules of receptor (CD95), allowing the recruitment of the adaptor molecule FADD via their death domain (DD). In turn FADD, via its death effector domain (DED), recruits and activates caspase 8, which cleaves the specific substrate Bid. Truncated Bid (tBid) is in fact the molecular signal that propagates the death signal to the mitochondria and the apoptosome. This mechanism can be inhibited by the molecule FLIP (FLICE‐inhibitory protein), via its DED. Expansion 2 (e2): When activated by apoptotic signals (e.g. by tBid), mitochondria release several molecules, including cytochorme c and DIABLO/Smac. The former goes to the apoptosome (formed by cytochrome c, Apaf‐1, pro‐caspase 9 and dATP), and allows the activation of pro‐caspase 9. This in turns activates several molecules of downstream effector capsases (type 3, 6, 7), and consequently the cleavage of many cellular substrates results in cell death. Still at its terminal stage, cell death can be inhibited by IAPs, whereas Smac/DIABLO can remove the protection by IAPs. The cascade of proteolytic amplification created by caspase 9 (apical regulatory caspase) and caspases 3, 6, and 7 (downstream effector caspases) is extremely powerful. DISC, death initiation signalling complex and IAP, inhibitors of apoptosis proteins.

Figure 3.

Sequential activation of caspases. In general, caspases with a long prodomain are involved in the upstream regulation and activation of the apoptotic pathway; they require a tight regulation and activation, and cleave very specific substrates. Caspases with a short prodomain are involved in the amplification of the effector cascade; thus they have a very simple, fast and direct activation, and they also have many substrates, whose cleavage and inactivation finally kills the cell. Caspase 6 seems to be able to reactivate the upstream regulatory caspases, creating a feedback forward potentiation loop that strongly enhances the amplification of the death signal. Colour code is in keeping with Figure .

Figure 4.

Caspase proteolytic specificity. (a) Substrate recognition. The caspases recognize a tetrapeptide motif corresponding to the four residues P4‐P3‐P2‐P1. Though the position at P3 and P1 seems to be obligatory, the position P4 allows the classification of three subfamilies; see also Figure . This property has facilitated the identification of group‐specific inhibitors. Colour code is in keeping with Figure . (b) Substrates. This list of substrates is incomplete; a more comprehensive list of approximately 700 substrates, with their role in cell death is reported in http://bioinf.gen.tcd.ie/casbah/. The caspase substrates can have a single cleavage site (e.g. DQTD in Gelsonin), nested multiple sites (e.g. DEVDGVD in PARP), redundant clustered sites (e.g. DSLD‐(X13)‐DEED‐(X16)‐DLND‐(X32)‐DGTD in Huntigtin) or distal multiple sites (e.g. DEPD and DAVD in ICAD).

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Further Reading

Fischer U, Laenicke RU and Schultze‐Ostoff K (2003) Many cuts to ruin: a comprehensive update of caspase substrates. Cell Death and Differentiation 10: 76–100.

Franchi L, Eigenbrod T, Muñoz‐Planillo R and Nuñez G (2009) The inflammasome: a caspase‐1‐activation platform that regulates immune responses and disease pathogenesis. Nature Immunology 10(3): 241–247.

Fuentes‐Prior P and Salvesen GS (2004) The protein structures that shape caspase activity, specificity, activation and inhibition. Biochemical Journal 384: 201–232.

Hotchkiss RS and Nicholson DW (2006) Apoptosis and caspases regulate death and inflammation in sepsis. Nature Review of Immunology 6(11): 813–822.

Lüthi AU and Martin SJ (2007) The CASBAH: a searchable database of caspase substrates. Cell Death and Differentiation 14: 641–650.

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Shi Y (2004) Caspase activation: revisiting the induced proximity model. Cell 117(7): 855–888.

Slee EA, Adrian C and Martin SJ (1999) Serial killers: ordering caspase activation events in apoptosis. Cell Death and Differentiation 6: 1067–1074.

Zheng TS, Hunot S, Kuida K and Flavell RA (1999) Caspase knockouts: matter of life and death. Cell Death and Differentiation 6: 1043–1053.

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Hernandez, Lorraine D, Houde, Caroline, Hoek, Maarten, Butts, Brent, Nicholson, Donald W, and Mehmet, Huseyin(Dec 2009) Caspases and Cell Death. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021562]