Caspases Death by Proteolysis

The link between apoptosis and proteolysis became apparent when a homology was established between the protein coded by the ced3 gene and a previously known protease in mammals, the ICE protease (review Martin, 1994 Kumar, 1995). The ICE 

Based on these observations, proteases related to the ICE protease were rapidly identified in mammals and these are known today collectively as the family of caspases (review Salvesen and Dixit, 1997 Thomberry, 1998 Eamshaw et al., 1999). The name caspase is based on particular properties of these proteases caspases use a s residue as a nucleophile and cleave the substrate after an Asp residue. 

Like many other proteases, the caspases are formed as inactive proenzymes of 30—50 kDa and are activated by proteolytic processing. The proenzymes have a prodomain and two cleavage sites for processing, which are consensus sites for the caspases, enabling activating by autoproteolysis. 

Stmctmal studies have shown that caspases can form tetramers with two active sites. The catalyticaUy active subunit of a caspase is made up of a large (17—12 kDa) and a small (10—13 kDa) subimit which form a heterodimer with an active site comprised of residues from both large and small subunits. Two heterodimers then align to form a tetramer with two catalytic centers (Fig. 15.4). 

The cleavage mechanism of the caspases is shown schematically in Fig. 15.5. They use a typical protease mechanism with a catalytic diad for cleavage of the peptide bond. The nucleophilic thiol of an essential Cys residue forms a covalent thioacyl bond to the substrate during the catalysis. The imidazole ring of an essential histidine is also involved in catalysis and this facilitates hydrolysis of the amide bond in the sense of an acid/base catalysis. 

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The granules contain two types of proteins that result in death. First, compounds that produce holes (pores) in the membrane of the cells these are the proteins, perforin and granulysin. Both insert into the membrane to produce the pores. These were once considered to result in death by lysis (i.e. exchange of ions with extracellular space and entry of water into the cell). However, it is now considered that the role of the pores is to enable enzymes in the granules, known as granzymes, to enter the cell. These granzymes contain proteolytic enzymes. They result in death of the cell by proteolysis but, more importantly, activation of specific proteolytic enzymes, known as caspases. These enzymes initiate reactions that result in programmed cell death , i.e. apoptosis (Chapter 20). 

Signalling for apoptosis can involve a plasma Fas ligand which binds to the PM Fas receptor with resultant activation of an associated cytosol-side Fas death domain of Fas and activation of caspase 8. Caspase 8 is a thiol protease and once activated initiates a so-called caspase cascade leading to activation of further caspases (with consequent proteolysis) and activation of a DNase (leading to DNA destruction with formation of a characteristic DNA fragment ladder ). Caspase 8 acts on mitochondria with resultant release of cytochrome c, which promotes caspase 3 activation by caspase 8 and hence the caspase cascade . Another signalling pathway for apoptosis involves tumour necrosis factor (TNF) binding to the TNF receptor with consequent activation of a cytosolic-side TNF receptor-associated death domain (TRADD) and resultant activation of the caspase cascade and cell death. 

Fig. 15.3 The major pathways of apoptosis. The extrinsic pathway uses extracellular death ligands (Fas ligand, tumor necrosis factor (TNF)) to activate death receptors which pass the apoptotic signal to initiator caspases (e. g. capsase 8) and to the executioner caspases (e. g. caspase 3 caspase 7). In the execution phase of apoptosis, various cellular substrates are degraded leading to cellular collapse. The intrinsic pathway uses the mitochondria as a central component for activation of apoptosis. In this pathway, a multitude of intracellular signals including various stresses, DNA damage and inappropriate cell signaling lead to activation of the pro-apoptotic protein Bax which induces release of cytochrome c from mitochindria, formation of the apoptosome and activation of the initiator caspase 9. Finally, the executioner caspases are activated and cells are destructed by proteolysis. Apoptosis via this pathway can be controlled by various antiapoptotic proteins including the Bcl-2 protein and inhibitors of apoptosis.

The presence of tumor necrosis factor a directly leads to apoptosis via interaction with the tumor necrosis factor receptor, one of a class of receptors referred to as death receptors. NF-kB, which must enter the nucleus to initiate apoptosis, is a transcription factor sequestered in the cytoplasm by inhibitor of kB (IkB). The binding of TNFa to its receptor leads to the ubiquitin-dependent proteolysis of IkB, allowing NF-kB to enter the nucleus. The activation of apoptosis results directly from the stimulation of NF-kB, a transcription factor whose phosphorylation is controlled by vanadium compounds. In a global gene expression study, it was found that diabetes increased the formation of IkB, whereas vanadium compound treatment lowered the production of this inhibitor [101]. The activation of the TNFR also activates the caspase proteins, a class of proteases that cleave proteins after specific aspartate residues. 

Proteolytic activation of procaspases uses two mechanisms, depending on whether the caspase functions as an effector caspase (caspases-3, -6 and -7) or as an initiator caspase (caspases-8 and -9). Initiator caspases are the first to be activated in response to a proapoptotic stimulus and are responsible for activating the effector caspases by limited proteolysis. The effector caspases are thought to be responsible for most of the substrate proteolysis observed during apoptosis. By digesting central proteins, the effector caspases direct the cell to death. 

Omi/Htra2, which lead to the activation of caspases— cysteine proteases that cleave a variety of intracellular contents. Through autoproteolytic activation, caspases create a feed-forward loop of proteolysis that inevitably leads to cell death. Accordingly, the cell contains the Bcl-2 family of proteins, which regulate mitochondrial permeability and caspase activity. A detailed description of apoptosis and its regulation by Bcl-2 proteins is beyond the scope of this chapter. Readers interested in additional details about the regulatory role of Bcl-2 proteins in apoptosis should read the work by Youle and Strasser (2008). 

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