Info

This is not the only case where the nomenclature in apoptosis tends towards the morbid; these terms

however actually in use.

however actually in use.

and XIAP, thereby removing a further obstacle to apoptosis. Interestingly, several viruses - oncogenic or not - express their own IAPs to prevent a cell from apoptosis while they replicate.

The extrinsic pathway (Figure 7.4) is initiated when specific cell surface receptors are engaged by their specific ligands. As a rule, these 'death receptors' belong to the TNF receptor superfamily (Figure 7.5). Tumor necrosis factor a (TNFa) is one of several cytokine ligands of this receptor superfamily. This peptide is secreted by monocytes, macrophages and other cells of the immune system during inflammatory reactions and in response to cellular stress. It elicits various reactions, including apoptosis in some cells containing the TNFRI receptor.

Figure 7.4 The extrinsic pathway of apoptosis Caspase 8 and/or Caspase 10 may be activated depending on cell type and receptor. Activation of BID to coopt the intrinsic pathway (dotted box) is not obligatory in all cell types.

Figure 7.5 Some members of the TNFRSF family All members of the family share similar cysteine-rich domains in their extracellular domains, whose numbers vary. In the intracellular domain, they consistently contain a DED domain. Some members have additional signaling functions. The sFAS protein is otherwise identical to CD95/FAS, but lacks transmembrane and intracellular domains and acts as a decoy receptor. The ligands for TRAIL-R1 and TNFR1 are TRAIL and TNFa, respectively.

Figure 7.5 Some members of the TNFRSF family All members of the family share similar cysteine-rich domains in their extracellular domains, whose numbers vary. In the intracellular domain, they consistently contain a DED domain. Some members have additional signaling functions. The sFAS protein is otherwise identical to CD95/FAS, but lacks transmembrane and intracellular domains and acts as a decoy receptor. The ligands for TRAIL-R1 and TNFR1 are TRAIL and TNFa, respectively.

Other ligands of TNFRSFs are present mainly on the surface of immune cells, e.g. CD95L, and the ligand-receptor interaction is part of a cell-to-cell-interaction (^■9.5). CD95L is also called FAS ligand and activates TNFRSF6, alias CD95, FAS, or APO-1. The CD95/CD95L system is considered one of the most important components in killing of infected and tumor cells by cytotoxic T-cells. It is also employed in the selective elimination of auto-reactive immune cells. Defects in CD95 function occur in autoimmune diseases as well as in cancers (^7.3).

In fact, the borderline between membrane-bound and soluble ligands is blurred. Some cytokines, including TNFa, are also present as a active membrane-bound form on the cell surface and CD95L is also secreted. The regulation of receptor-ligand interactions in this system is in fact very complex. For instance, at the receiving end, the response is modulated by the presence of modulating and decoy receptors. The response to TNFa is modulated by the TNFRII receptor. When present at the cell surface, the TNFRII appears to bind the cytokine and pass it on to the TNFRI which mediates the actual response. When TNFRII is sheared off the cell, it acts as a 'decoy receptor', sequestering the ligand and preventing it from acting on TNFRI.

Similarly, in addition to the membrane-bound form of CD95 (also tmFAS), a soluble form is generated by alternative splicing (sFAS), which also acts as a decoy receptor to decrease the responsiveness to CD95L. Apoptotic responses to the TNFa-related cytokine TRAIL are also dependent on the relative expression of four different TNFRSF members, TRAIL-R1 through TRAIL-R4, two of which are true receptors and two are decoys.

Members of the TNFRSF family act through several pathways, notably the NFKB pathway (^6.9). Family members that can activate the extrinsic apoptosis pathway differ from their homologues by the presence of an additional intracellular domain, called the 'death domain' (Figure 7.4). This part of the protein is required for the activation of the extrinsic apoptotic pathway. Since the NFKB pathway as a rule counteracts apoptosis, the actual cellular response will often depend on the relative strengths of the two pathways activated in parallel. In specific cell types, cytokine receptors like TNFRI also stimulate cell proliferation.

Following ligand binding to an active TNFR, such as TNFRI or TNFRSF6, the ligand/receptor complexes trimerize and the receptor death domains bind FADD proteins by interaction with the homologous domains in this adaptor. In addition, FADD contains a death effector domain homologous to that in initiator caspases. By binding to the death receptor, this domain is exposed and binds an initiator pro-caspase, usually pro-caspase 8 or pro-caspase 10. The resulting complex appears sometimes in the literature as 'death inducing signaling complex' (DISC). Its function in apoptosis is to bring pro-caspase molecules into close proximity to dimerize and activate one another. The activated initiator caspases 8 or 10 then activate executioner caspases like caspase 3 setting the execution phase into motion. The FLIP protein acts as an inhibitor of the extrinsic pathway by interfering with initiator caspase dimerization.

In some cells, activation of the extrinsic pathway by certain death receptor ligands is sufficient to elicit apoptosis. In such cases, the expression levels of BCL2 and BCLXl are quite irrelevant. In others, induction of apoptosis requires the participation of the intrinsic pathway. In response to external signals, this is typically stimulated via the BID protein cleaved by caspase 8. Conversely, the intrinsic pathway also influences the extrinsic pathway. For instance, TP53 induces activators of the extrinsic pathway, but also increases the expression of CD95, thereby sensitizing cells to pro-apoptotic external signals.

The multiple biochemical and morphological changes that take place during the execution phase of apoptosis are caused by proteolytic cleavage of >300 cellular proteins by caspase 3 and other executioner caspases like caspase 6 (Table 7.2). The substrates comprise regulators of the cell cycle such as RB1, DNA repair proteins such as DNA-PK and poly-ADP-ribosyl polymerase (PARP), and cytosketelal proteins such as actin, lamins, and keratin 18. The characteristic 'nucleosomal ladder' DNA fragmentation is caused by several DNases, prominently CAD (caspase activated DNase) that are liberated by cleavage of inhibitory proteins to which they are normally bound. Cleavage of FAK (focal adhesion kinase), PAK2 (p21-associated kinase), and Gelsolin contributes to the loss of adhesion and the characteristic membrane changes such as blebbing and redistribution of membrane

156 CHAPTER 7

Table 7.2. Protein substrates of caspases during execution of apoptosis

Category

Examples

Cytoskeleton and structural Cell cycle and DNA replication DNA repair and metabolism

Transcription and splicing Signal transduction

Proteases, protease inhibitors and apoptotic regulators

Fodrin, ß-Catenin, Plakoglobin, actins, Gelsolin, cytokeratins, lamins MCM3, MDM2/HDM2, RB1, p21CIP1, p27KIP1, WEE1, CDC27, Cyclin A Topoisomerase I, Poly-ADP-ribosyl-polymerase (PARP), DNA-dependent protein kinase (DNA-PK), Inhibitor of caspase-activated DNases (ICAD) sterol regulatory element binding proteins (SREBPs), transcription factors STAT1, NFkB (p65, p50), and SP1, IkB, various SNRNPs protein kinases PKC5, PKC9, MEKK1, FAK, and others, PP2A, RAS-GAP, PLA2

po-caspases, Calpastatin, Huntingtin, presenilins, ataxins, BCL2, BCL-Xl proteins and phospholipids. These redistributions create signals for the subsequent burial phase. Importantly, phosphatidylserine which is normally restricted strictly to the inner layer of the membrane phospholipid bilayer, is flipped to the outer layer and recognized by receptors on macrophages that are attracted by further chemotactic signals diffusing out from the dying cell.

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