MOLECULAR PATHWAYS OF APOPTOSIS

Several stimuli that trigger apoptosis seem to do so by inducing activation of proteolytic enzymes— in particular, but not exclusively, caspases. In order to activate initiator caspases, several molecules of caspase zymogens have to be brought to close proximity to allow self-processing thanks to the moderate catalytic activity of the zymogens.

The Mitochondrial Pathway (The Intrinsic Pathway)

A large variety of different stimuli, including ultraviolet or gamma irradiation, oxidative stress, growth factor deprivation, and several cytotoxic compounds including kinase inhibitors such as staurosporine, have been reported to trigger caspase activation and apoptosis via a mitochondrial pathway (Figure 2.1).³¹ Although very different in nature, all these stimuli result in activation of proapoptotic members of the Bcl-2 family, which tightly regulates the mitochondrial function. The multidomain proapoptotic proteins, such as Bax and Bak, can be activated directly, or indirectly, via interaction with BH3-only proteins of the same family. For example, Bid is cleaved by caspase-8 to generate a C-terminal fragment (t-Bid) that interacts with Bax and Bak, triggering activation of these proteins and facilitating their insertion into the mitochondrial membrane, possibly through destabilization of the membrane. When activated, Bax and Bak permeabilize the outer mitochondrial membrane, causing the release of proapoptotic proteins, such as cytochrome c, Smac/DIABLO, HtrA2ZOmi, apoptosis-inducing factor (AIF), and endonuclease G, from the intermembrane space.

The Death Receptor Pathway (The Extrinsic Pathway)

The death receptors are a subset of the tumor necrosisZnerve growth factor (TNFZNGF) receptor superfamily. Members of the death receptor family include, among others, Fas (also called CD95 or APO-1), tumor necrosis factor-receptor 1 (TNF-R1, also called p55 or CD120a), and tumor necrosis factor-related apoptosis-inducing ligand-receptors 1 and 2 (TRAIL-R1 and TRAIL-R2, also called DR4 and DR5). When activated through interaction with their natural ligands, a group of complementary cytokines that belongs to the TNF family of proteins, these receptors are able to trigger an apoptotic signaling cascade.^42-44 The members of this family are all type-I transmem­brane proteins with a cysteine-rich, extracellular N-terminal domain, a membrane-spanning region, and a C-terminal intracellular tail containing a sequence of approximately 80 amino acids (the DD). The DD allows for the recruitment of adaptor molecules, which function as “docking proteins” to bind the proform of the initiator caspase-8, -10, and, perhaps, -2.

Signal transduction by death receptors is initiated by the oligomerization of the receptor that follows the engagement of the ligand to the receptor’s extracellular domain (Figure 2.2).

FIGURE 2.2 The death receptor/extrinsic pathway of apoptosis. Examples of signaling through the main death receptors (Fas/CD95/APO-1, TNF-R1, TRAIL-R1, and TRAIL-R2) are illustrated here. Engagement of death receptors by their cognate ligands results in oligomerization of the receptor and recruitment of adaptor proteins. The death domain (DD) on the adaptor protein interacts with the receptor’s DD, whereas the death effector domain (DED) binds the correspondent DED in the prodomain of the inactive initiator caspase-8 or -10. The resulting complex is referred to as the death-inducing signaling complex (DISC). The proximity of several procaspase molecules results in the activation of the caspase by self-processing, which, in turn, activates downstream caspases such as caspase-3. The inhibitors of apoptosis cFLIPs prevent the recruitment or inhibit the processing of procaspase-8 to the DISC.

consequence, different DD-containing adaptor proteins, such as FADD or tumor necrosis factor recep­tor-associated protein with DD (TRADD), are recruited to the receptor through homophilic interaction of the receptor’s and adaptor’s DDs. Some adaptor proteins also contain a DED that mediates the recruitment of caspases through the association with correspondent DED or CARD motifs in the prodomain of the inactive initiator caspases. The resulting complex is referred to as the DISC. The proximity of several procaspase molecules recruited to the receptor results in self-processing and activation of the caspase, which subsequently starts a cascade of caspase activation by directly pro­cessing and activating the effector caspases (i.e., caspase-3, -6, and -7). With regard to Fas, this type of signaling is referred to as “type I” and is generally associated with high levels of caspase-8 recruited and activated at the DISC (Figure 2.3).⁴⁵ Alternatively, the activation of effector caspases may occur indirectly, via a mitochondria-mediated process, with a signal referred to as “type II.” In type-II cells, the amount of caspase-8 activated at the DISC seems to be insufficient to directly cleave effector caspases, but it can efficiently cleave the BH3-only protein Bid to generate a fragment that translocates to the mitochondria and induces mitochondrial dysfunction.^45-47 After permeabilization of the outer mitochondrial membrane, several proapoptotic factors are released in the cytosol, where they contribute to caspase activation and apoptosis, as previously described for the mitochondrial pathway.

FIGURE 2.3 Fas∕CD95-mediated apoptotic pathways in type I and type II cells. The amount of active caspase- 8 generated at the DISC determines whether the cell activates a mitochondrial-independent (type I) or mitochondrial-dependent (type II) pathway of caspase activation and apoptosis.