Ordering of caspases in cells undergoing apoptosis by the intrinsic pathway

Abstract

Caspases are a family of aspartate-specific cysteine proteases responsible for the biochemical and morphological changes that occur during the execution phase of apoptosis. The hierarchical ordering of caspases has been clearly established using dATPactivated cell lysates to model the intrinsic pathway induced by initial mitochondrial perturbation. In this model, caspase-9, the apical caspase, directly processes and activates the effector caspases, caspase-3 and -7, and then active caspase-3 but not caspase-7, processes caspase-2 and -6, and subsequently the activated caspase-6 processes caspase-8 and -10. To address the possibility that this model in vitro system might not reflect the precise ordering of caspases in intact cells, we have examined this possibility in cells induced to undergo apoptosis by activation of the intrinsic pathway. We have used caspase deficient cells, small interference RNA for caspase-6 and -7, and a specific caspase-3 inhibitor. In contrast to the earlier in vitro studies, we now show that in intact cells caspase-7 can also directly process and activate caspase-2 and -6. The processing of caspase-2 and -6 occurs within the cytoplasm and active caspase-6 is then responsible for both the processing of caspase-8 and the cleavage of caspase-6 substrates, including lamin A/C. Apoptosis, a major form of cell death used to remove unwanted or excess cells, is characterized by a plethora of morphological and biochemical changes resulting primarily from the activation of a family of cysteine proteases, which cleave their substrates at specific aspartate residues. 1-4 Two major apoptotic pathways have been described, the intrinsic pathway involving initial mitochondrial perturbation resulting from cellular stress or cytotoxic insults and the extrinsic pathway, which is triggered by the activation of death receptors of the TNF family. 5-7 Caspase-8 and -9, the apical caspases in the extrinsic and intrinsic pathways, possess a protein interaction prodomain, which result in their recruitment and subsequent activation within cellular complexes, namely the death-inducing signaling complex (DISC) and the Apaf-1 apoptosome, respectively, and the subsequent activation of effector caspases, such as caspase-3, -6 and -7. 5,[8][9][10] Caspases are synthesized as single-chain zymogens, containing an N-terminal prodomain, as well as large (B20 kDa) and small (B10 kDa) subunits, with the basic catalytic domain forming from a large and small subunits. 1,3,4 Caspase-2 also has a sizeable prodomain, but it is unclear whether it should be considered an initiator or an effector caspase. 3 Although caspase-2 has been proposed to act upstream of mitochondria and to be activated within the PIDDosome, it may also be activated downstream of caspase-3. 11-14 Using recombinant proteins, caspase-3, -8 and to a lesser extent caspase-7 can process caspase-2. 15 The physiological role of caspase-2 is not known and no marked phenotype is observed in caspase-2 À/À mice. Caspase-6 is generally considered to be an effector casp...

Protease Activity of Procaspase-8 Is Essential for Cell Survival by Inhibiting Both Apoptotic and Nonapoptotic Cell Death Dependent on Receptor-interacting Protein Kinase 1 (RIP1) and RIP3

“…After oligomerization within a DISC, procaspase-8 undergoes auto-cleavage, and then translocates into the cytosol as an activated dimer harboring the active pocket with the active site Cys-283 (40,41). Procaspase-8 was also reported to be cleaved by caspase-6 resulting in the formation of the active dimer, which process might function to form the positive feedback loop of caspase activation in induction of apoptosis (42,43). Our DE mutant does not retain the Asp residue in linker sequences where procaspase-8 is cleaved by caspase-8 itself and other caspases.…”

Caspases and cancer

“…Caspase-6 is believed to be an effector caspase that is activated downstream of caspase-3 and -7 in apoptosome-mediated apoptosis. 74 Apart from lamin A/C, few other specific substrates for caspase-6 have been described, calling into question to which extent this particular caspase actually participates in drug-induced apoptosis. 75 However, it has been suggested that caspase-6 cleavage of tau protein leads to neurofibrillary tangle formation during Alzheimer's disease pathogenesis.…”

Cellular Mechanisms Controlling Caspase Activation and Function

“…It has been reported that caspase-6 is activated only following activation of caspase-3 or caspase-7 and thus is not activated directly by an initiator caspase (Inoue et al 2009). Once active, caspase-6 can cleave initiator caspases-2 and -8, although the functional consequences of this cleavage (away from the initiator caspase activation platform) are unclear (Slee et al 1999;Inoue et al 2009). In contrast, other studies have showed that caspase-6 can be activated in the absence of caspase-3 or -7 activity (LeBlanc et al 1999;Allsopp et al 2000;Doostzadeh-Cizeron et al 2000).…”

“…Studies directed toward ordering of the caspase cascade and characterization of caspase substrates indicates that caspase-3 has both distinct and overlapping roles with caspase-7 and caspase-6 (Slee et al 1999;Lakhani et al 2006;Luthi and Martin 2007;Inoue et al 2009). Although it might appear that by the time apoptotic signals reach the point of caspase-3 activation, there is no salvaging the cell, caspase-3 is still regulated through a variety of posttranslational modifications.…”