Caspase cascade activation apoptosis

The orderly execution of apoptosis is orchestrated by an evolutionarily conserved pathway from worms to mammals Fig. A conserved apoptotic pathway in C. Functional homologs across species are indicated by the same color. Caspase-9 in mammals and Dronc in Drosophila are initiator caspases, whereas caspases-3 and -7 in mammals and DrICE in Drosophila belong to effector caspases.

CED-3 in C. The executioners of apoptosis are caspases, a family of conserved cysteine proteases that usually cleave after an aspartate residue in their substrates Thornberry and Lazebnik The critical involvement of a caspase in apoptosis was first documented in , when CED3 was found to play a central role in the programmed cell death in the nematode worm Caenorhabditis elegans Yuan et al.

Since then, the conserved mechanism of apoptosis has been identified in a number of species, particularly in Drosophila melanogaster and mammals Abrams ; Budihardjo et al. Caspases involved in apoptosis are classified into two groups, the initiator caspases, such as caspase-9 in mammals or its functional ortholog Dronc in Drosophila , and the effector caspases, such as caspases-3 and -7 in mammals and their homolog DrICE in Drosophila Fig.

All caspases are synthesized in cells as catalytically inactive zymogens, and must undergo an activation process. The activation of an effector caspase, such as caspase-3 or -7, is performed by an initiator caspase, such as caspase-9, through an internal cleavage to separate the large and small subunits.

An initiator caspase, however, is autoactivated under apoptotic conditions, a process usually requiring and facilitated by multicomponent complexes Adams and Cory ; Shi b. For example, the apoptosome is responsible for the activation of caspase-9 see later sections; Rodriguez and Lazebnik Genetic studies have identified four genes that act sequentially to control the onset of apoptosis in C.

In contrast to the mammalian pathway, CED-3 is the only known apoptotic caspase in C. In the absence of apoptotic signaling, the pro-apoptotic activity of CED-4 is constitutively suppressed by the antiapoptotic protein CED-9, through direct physical interactions. During C. In mammals, Bcl-2 and Bcl-xL are structurally and functionally homologous to the worm CED-9 proteins, whereas a large family of BH3-only proteins is distributed throughout the cell to sense apoptotic stress signals Cory and Adams Upon receiving apoptotic stimuli, the BH3-only proteins transduce the signal to mitochondria.

Through complex actions involving Bak and Bax, cytochrome c is released from the intermembrane space of mitochondria into the cytoplasm, where it binds to and activates Apaf-1, the mammalian ortholog of CED-4 Li et al.

Once activated, caspase-9 stays associated with the apoptosome as a holoenzyme to maintain its catalytic activity, as caspase-9 in isolation is marginally active Rodriguez and Lazebnik The primary target of the caspase-9 holoenzyme is caspase-3, one of the most deleterious effector caspases Fig.

The Inhibitor of Apoptosis IAP family of proteins suppresses apoptosis by interacting with and inhibiting the enzymatic activity of both initiator and effector caspases Deveraux and Reed ; Salvesen and Duckett ; Shi b ; Fig. A schematic diagram of representative IAPs. During apoptosis, IAP-mediated inhibition of caspases is effectively countered by a family of proteins that share an IAP-binding tetrapeptide motif Shi a.

The founding member of this family in mammals is the mitochondrial protein Smac Du et al. Smac, synthesized in the cytosol, is targeted to the intermembrane space of mitochondria. Upon stimulation of apoptosis, Smac is released back into the cytosol, together with cytochrome c.

Homologs of most components in the mammalian pathway have been identified in fruit flies Abrams ; Fig. The Drosophila Apaf-1, known as Dapaf-1 Kanuka et al. Dronc, in turn, cleaves and activates the effector caspase DrICE. During apoptosis, the antideath function of DIAP1 is countered by at least four pro-apoptotic proteins, Reaper, Hid, Grim, and sickle, through direct physical interactions.

These four proteins represent the functional homologs of the mammalian protein Smac, and they all share a conserved IAP-binding motif at their N termini Fig. A conserved family of IAP-binding motifs. Conserved residues are colored green and yellow. The invariant Ala, highlighted by an arrow, is necessitated by the mode of binding. The Drosophila proteins have an additional binding component conserved 6th—8th residues, colored blue.

It should be noted that the IAP-binding tetrapeptide motif has nothing to do with the well-characterized tetrapeptidic substrate specificity of caspases. The purpose of this review is not to cover all areas of advances in caspase regulation.

Rather, I would like to focus on the underlying molecular mechanisms of caspase regulation, most of which have been revealed in the last three to four years.

Caspases are synthesized as single-chain zymogens. An effector caspase is known to exist constitutively as a homodimer, both before and after the intrachain activation cleavage. However, as a consequence of the intrachain cleavage, the catalytic activity of an effector caspase is increased by several orders of magnitude Salvesen and Dixit The mechanism of activation for a representative effector caspase, caspase-7, is revealed by the conforma tional changes of the active site following the activation cleavage Chai et al.

The active site comprises four surface loops, L1 through L4, all from the same monomer. L1 and L4 constitute two sides of the substrate-binding groove; L3 forms the base.

L2 lies across the groove and harbors the catalytic residue Cys, poised for catalysis Fig. Molecular mechanism of caspase-7 activation. A Structure of an activated and inhibitor-bound caspase-7 Wei et al.

Active site loops are labeled, with the catalytic cysteine highlighted in red. The covalently bound inhibitors are shown in orange. B Structure of a procaspase-7 zymogen Chai et al. Compared to that of the inhibitor-bound caspase-7, the conformation of the active site loops does not support substrate-binding or catalysis. C Comparison of the conformation of the active site loops. The broken connection in loops L3 and L4 indicates high mobility of these regions, as reflected by their high temperature factors from crystallographic refinement.

The active site conformation prior to the intrachain activation cleavage is revealed by the structure of the unprocessed procaspase-7 zymogen Chai et al. Compared to the inhibitor-bound caspase-7, the core structural elements of the procaspase-7 zymogen remain unchanged. However, the active site undergoes drastic conformational changes. Loop L2, which contains the catalytic cysteine, is rotated to a large extent, making this residue inaccessible to solvent Fig.

In addition, loops L3 and L4, which form the base and one side of the catalytic groove in the active caspase-7, respectively, become highly flexible Fig. These conformational rearrangements in the pro-caspase-7 zymogen do not allow formation of a substrate-binding groove, thus explaining why the procaspase-7 zymogen does not possess detectable catalytic activity.

In caspase-7, this ability is acquired through proteolytic cleavage after Asp Indeed, this prediction was confirmed for caspase-3 and -6 Srinivasula et al. The activation of an effector caspase zymogen is defined as the intrachain cleavage mediated by a specific initiator caspase. For the initiator caspases, however, the definition of activation carries an entirely different meaning. Although an initiator caspase undergoes an autocatalytic intrachain cleavage, this cleavage appears to have only modest effect on its catalytic activity Stennicke et al.

For example, the fully processed caspase-9 in isolation is only marginally active, much the same way as the unprocessed caspase-9 zymogen. In sharp contrast, association with the apoptosome leads to an enhancement of three orders of magnitude in the catalytic activity for the processed as well as the unprocessed caspase-9 Rodriguez and Lazebnik ; Srinivasula et al.

Thus, for caspase-9, activation has little to do with the intrachain cleavage; rather, it refers to the apopto-some-mediated enhancement of the catalytic activity of caspase At present, we do not understand the molecular mechanism for the activation of any initiator caspase.

Nonetheless, two models have been proposed. Based on results using heterologous fusion proteins, an Induced Proximity model was proposed to provide a general explanation for the activation of initiator caspases Salvesen and Dixit It states that the initiator caspases autoprocess themselves when they are brought into close proximity of each other.

However, this model merely summarizes what have been observed experimentally in laboratories, and does not reveal the molecular mechanisms for the activation of initiator caspases. More recently, dimerization of the initiator caspases, such as caspases-8 and -9, was proposed to be the driving force for their activation Renatus et al. This hypothesis provides a mechanism-based explanation for initiator caspase activation, and thus represents a qualitative advance over the previous Induced Proximity model.

Based on this model, the function of the apoptosome is to promote the homodimerization of caspase-9 due to its increased local concentrations in the apoptosome. Hence, this model is also known as Proximity-Induced Dimerization, and was proposed to be the unified mechanism for the activation of initiator caspases Boatright and Salvesen The validity of this model remains to be examined experimentally, as the supporting evidence is inclusive.

References: 1. Autoactivation of procaspase-9 by Apafmediated oligomerization. Mitomycin C induces apoptosis and caspase-8 and -9 processing through a caspase-3 and Fas-independent pathway.

Cell Death Differ. Nuclear translocation of granzyme B in target cell apoptosis. Verify Security Code. Quick order. All rights reserved. X Item added to cart successfully. No cart item available. Evidence is accumulating to suggest that proximal caspase activation events are typically initiated by molecules that promote caspase aggregation.

As expected, distal caspase activation events are likely to be controlled by caspases activated earlier in the cascade. However, recent data has cast doubt upon the functional demarcation of caspases into signalling upstream and effector downstream roles based upon their prodomain lengths.

In particular, caspase-3 may perform an important role in propagating the caspase cascade, in addition to its role as an effector caspase within the death programme.