Pro-caspases

Two main apoptotic pathways have been identified in mammalian cells the extrinsic pathway that is activated by the binding of ligands to cell-surface death receptors, and the intrinsic pathway that involves the mitochondrial release of cytochrome cP The activation of extrinsic and intrinsic apoptotic pathways promotes the cleavage into the active form of the pro-caspase-8 and pro-caspase-9, respectively, that mainly determine the activation of effector caspase-3. ° The intrinsic pathway is the main apoptotic pathway activated by chemotherapeutic drugs, while the cytotoxic drug-induced activation of the extrinsic pathway is a more controversial issue. ... 

Stoka, V., Turk, B., Schendd, S.L., Kim, T.-H., Cirman, T, Srripas, SJ., EUerby, L.M., Bredesen, L., Freeze, H, Abrahamson, M., Bromme, D., Krajewski, S., Reed, J.C., Yin, X.-M., Turk, V., and Salvesen G.S., 2001, Lysosomal Protease Pathways to Apoptosis. Cleavage of Bid, not pro-caspases, is the most likely route. J. Biol. Chem. 276 3149-3157 Srm, X. and Ross, D., 1996, Quinone-induced apoptosis in human colon adenocarcinoma ceUs via DT-diaphorase mediated bioactivation. Chem. Biol. Interact. 100 267-76 Taatjes, D.J. and Koch, T.H., 2001, Nuclear targeting and retention of anthracycUne antitumor dmgs in sensitive and resistant tumor ceUs. Curr. Med Chem. 8 15-29 Tarr, M. and van Helden, PT)., 1990, Inhibition of transcription by adriamycin is a... 

Caspase-10 FLICE-2, Mch4 Apoptosis Caspase-7 PARP pro-Caspase-9 Caspase-3... 

Granzyme B will cleave proteins at aspartate residues and will therefore activate pro-caspase 10 and can cleave factors like ICAD (inhibitor of caspase-activated DNase). However, Granzyme B can also directly activate caspase 3. In this way, the upstream signaling pathways are bypassed and there is direct induction of the execution phase of apoptosis. Recent findings indicate that this method of granzyme B cytotoxicity is critical as a control mechanism for T-cell expansion of type 2 helper T (Th2) cells. Moreover, findings indicate that neither death receptors nor caspases... 

Receptors that contain an intracellular DD are known as death receptors, which are exemplified by Fas and TNF-Rl (Ashkenazi and Dixit, 1998 Nagata, 1997). Fas is an effective prototypical cell killing receptor. The intracellular DD of Fas directly recruits a DD-containing protein known as Fas-associated DD (FADD) via DD-DD interactions (Chinnaiyan et al, 1995). FADD also contains a death effector domain (DED), which further recruits the DED-containing pro-caspase-8 or pro-caspase-10 to elicit cas-pase activation and apoptosis (Boldin et al, 1996 Muzio et al, 1996 Wang et al, 2001b). 

Like other receptors, death receptors are activated by binding ligands. The activated death receptor-ligand complex is linked with its death domain (DD) to a homologous DED (death effector region), repeated in tandem, and recruits a zymogen form of a protease, pro-caspase 8 (Fig. 13.1). But the linker is, like other linkers, versatile. 

Four or more amino acids, NH2-terminal to the aspartate after which the polypeptide chain is cleaved, make up the recognition site in pro-caspases. The sequence of the recognition-site tetrapeptide differs among different pro-caspases. The only conserved residue is aspartate. 

Fig. 13.4 Common structural properties of caspases. Pro-caspases have a smaller (10 kDa) subunit, a larger (20 kDa) subunit and an amino-terminal pro-domain. Cleavage of consensus sites in the pro-enzymes separates the two subunits from the remaining NH2-termlnal Pro-domain, which controls processing. Processing is either by autocatalysis or by another caspase with compatible specificity. After processing is completed, the two subunits, the large and the small one, combine, and associate to a tetramer with two catalytic sites, each functioning independently.

Activation of caspases is irreversible, because it involves peptide-bond cleav e. This is unlike most other protein modifications which play a role in cellular regulation. Therefore, proteolysis is involved only in unidirectional, irreversible processes, such as the cell cycle and cell death. But, the possibilities to regulate irreversible reactions are rather limited. In a cascade of proteolytic reactions, the first enzyme in the chain is the most likely point of control. This is the initiator caspase. The signals controlling initiator caspases vary, there are both external and internal signals (Fig. 13.5). Several mechanisms control the irreversible activation of caspases, including phosphorylation, separation, and compartmentalization of pro-caspases and positive and n ative regulators. 

Caspase-9 Apaf-3, ICE-LAP6, Mch6 Caspase-3, pro-caspase-9, caspase-7, PARP Apoptosis... 

Activation of Akt kinase proceeds in a multi-step process with regulation by PtdIns(3,4,5)P3 as the critical step. PtdIns(3,4,5)P3 binds to the PH domain of Akt kinase and thereby recruits the enzyme to the cell membrane. This step is thought to be a prerequisite for a subsequent phosphorylation of a Thr residue in the activation loop (see Chapter 7 on protein kinases) and an N-terminal Ser residue of Akt. The protein kinase responsible for this step is named phosphoinositide-dependent protein kinase 1 (PDK1, review Vanhaesebroeck and Alessi, 2000), and this also contains a PH domain with high affinity for PtdIns(3,4,5)P3. Membrane targeting and the double phosphorylation activate Akt, allowing the phosphorylation of downstream substrates like the Bad protein (see Section 15.7.1), pro-caspase 9, the transription factor CREB (see Section 1.4.5.2), glycogen synthase kinase (GSK), and 6-phosphofructo-2-kinase. 

In a further step of apoptosis, the cytochrome c released from the mitochondria promotes the assembly of a multiprotein complex, termed apoptosome, which contains cytochrome c, the adaptor protein Apafl, and procaspase-9. The apoptosome requires ATP for its formation and is able to cleave and activate procaspase-3, an effector caspase. The adaptor protein Apafl appears to play a major structural role in this assembly. Apafl contains WD motifs for interaction with cytochrome c and a CARD motif, which directs binding to the CARD motifs of procaspase-9 and procaspase-3. Structural studies on the apoptosome by electron microscopy have revealed a wheel shaped heptameric complex, with the CARD domains of Apafl located at the central hub and the WD40 repeats at the extended spokes (Acehan et al., 2002). The location of pro-caspase 9 in this complex is still open as is the mechanism of caspase 9 activation. 

Acehan, D., X. Jiang, D. G. Morgan, J. E. Heuser, X. Wang, and C. W. Akey. 2002. Three-dimensional structure of the apoptosome Implications for assembly, pro-caspase-9 binding, and activation. Mol Cell 9 423-32. 

Name (Previous name) Function Substrate specificity Pro-caspase Others Comments... 

Pro-caspases are activated by cleavage at the same consensus site, either by themselves or by other caspases. Those that activate other caspases, including themselves, are initiator caspases (caspases-8, -9, and -10) and those that cleave other protein substrates are effector caspases (caspases-3, -6 and -7). The likely roles of effector caspases in apoptosis are ... 

---