The Major Players in Apoptosis

Caspases: Orchestrators of Apoptosis Caspases are an evolutionarily conserved family of cysteine-dependent, aspartate-specific proteases, synthesized as zymogens (i.e. inactive enzyme precursors) [14]. Caspases consist of a prodomain of variable length, followed by a p20 and a p10 unit which contain the residues essential for substrate recognition and catalysis [13]. The proteolytic cleavage of the prodomain

Box 17.1: Inflammatory Diseases, Oxidative Stress, Antioxidants and Redox Modulators

A range of human disorders are characterized by a dysregulated immune response directed against self components. These highly damaging events are closely related to oxidative stress and the formation of reactive species by immune cells, such as macrophages and neutrophils. As part of their role in host defense, these cells contain a set of enzymes able to generate reactive oxygen species (ROS) and related compounds, such hypochlorous acid (HOCl), which is formed from H2O2 and Cl~ by the enzyme myeloperoxidase. Once formed, these highly aggressive chemical species then attack target cells in the vicinity, such as bacteria. Interestingly, the immune cells generating these chemicals also appear to possess a considerable resistance against them.

While the generation of reactive species by immune cells is therefore beneficial to the human body under normal conditions, an accidental activation of this defense in the absence of an (external) threat is counterproductive, as human diseases, such as rheumatoid arthritis, dramatically illustrate. There have been various attempts to alleviate this kind of attack. First, nonsteroidal antiinflammatory drugs (NSAIDs) are available, which inhibit cyclooxygenase (COX) enzymes and hence disrupt the cellular processes leading to inflammation. As an alternative, antioxidants have been considered that chemically sequester the dangerous cocktail of reactive species formed by immune cells.

The uses of NSAIDs and antioxidants cover a whole range of events, from the first step of trying to prevent the occurrence of inflammation to a situation where antioxidants are used to fight off the chemical weapons after they have been formed.

In addition, we find a couple of rather innovative approaches, most of which are still in their early stages of development. For instance, some anti-inflammatory agents work by inducing apoptosis of neutrophils. Within this context, it may be possible to resensitize ROS-resistant immune cells, so they may themselves be killed by the ROS they produce. Even more radical, it may be feasible to employ redox catalysts which could increase the toxicity of reactive species generated by macrophages and neutrophils: Since concentrations of reactive species are highest at the point of production, that is, at the location of the immune cell itself, such a catalytic conversion may well be sufficient to kill the hyperactive immune cell by using its own reactive species as a (selective) weapon against it. In the end, any approach inducing selective apoptosis of "hyperactive" immune cells may provide an interesting avenue to counteract inflammatory diseases.

results in the formation of the mature caspase, which consists of a heterotetramer p202-p102 derived from two precursor molecules.

To date, 11 human, 10 murine, 4 avian, 4 fish, 8 amphibian, 7 insect and 4 nematode caspases have been identified [15]. Caspases have evolved as separate groups and can be divided into three clusters: inflammatory caspases (cluster I), apoptosis initiator caspases (cluster II) and apoptosis effector caspases (cluster III)

(Figure 17.1a) [13]. The central role in the regulation and the execution of apoptotic cell death belongs to caspases from clusters II and III [13,14]. In mammals, caspases-2, -8, -9 and -10 are known as apoptosis initiator caspases, whereas caspases-3, -6 and -7 serve as effector caspases.

Initiator caspases possess a long prodomain composed of protein-protein interaction motifs such as two DED domains for caspases-8 and -10, and one CARD domain for caspases-2 and -9 (Figure 17.1a) [14]. These interaction motifs mediate the recruitment of caspases to death signaling complexes and subsequent autocatalytic activation [16]. Effector caspases lack a long prodomain and the ability to self-activate. They rather require cleavage by activated initiator caspases [16]. Effector caspases then cleave key substrates, such as polyadenosine diphosphate ribose polymerase (PARP) and inhibitor of caspase-3-activated DNase (ICAD), in the cell to produce the cellular and biochemical events defined as apoptosis.

ICAD is the inhibitor of the best-characterized apoptotic DNase CAD (caspase-activated DNase), a neutral Mg2 + -dependent endonuclease. The activity of CAD is regulated by ICAD, which is cleaved during apoptosis by caspases, resulting in DNA fragmentation [17]. PARP is a nuclear enzyme catalyzing the transfer of poly-ADP polymers to diverse proteins. It is involved in various processes, including DNA repair, gene expression and cell death [18]. PARP cleavage by caspases prevents the recruitment of the enzyme to sites of DNA damage. Although PARP cleavage is considered to be a classical hallmark of apoptosis, its function in the regulation of cell death is poorly understood [19]. By contrast, in some cases, PARP is able to mediate cell death through the recruitment of a mitochondrial protein into the nucleus termed apoptosis-inducing factor (AIF). Apoptosis-inducing factor normally resides in the mitochondrial intermembrane space, functioning as a scavenger of H2O2. In this regard, its structure is similar to that of glutathione reductase, another scavenger of H2O2 [20]. However, once in the nucleus, AIF fulfills a proapoptotic role, binding DNA and provoking large-scale DNA fragmentation [21].

Proteins of the Bcl-2 Family: Regulators of Apoptosis More than 15 Bcl-2 family proteins have been identified in mammalian cells [10] (see chapter 10). These proteins control the mitochondrial integrity and share at least one of the four conserved motifs known as Bcl-2 homology domains (BH1 to BH4). Bcl-2 family proteins can be classified into three subfamilies: one antiapoptotic (the Bcl-2 subfamily), and two proapoptotic (the Bax and BH3-only subfamilies) (Figure 17.1b). The balance between the pro- and antiapoptotic proteins determines the relative sensitivity of cells to an apoptotic stimulus.

In mammals, the Bcl-2 subfamily comprises five antiapoptotic proteins: Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A1. They contain all or most of the BH domains. The Bax subfamily comprises three proapoptotic members: Bax, Bak and Bok. They possess three BH domains and are often termed the BH123 proteins. The BH3 subfamily is the largest and comprises seven proapoptotic members in mammals: Bik, Bad, Bid, Bim, Bmf, Hrk, Noxa and Puma. These proteins possess only the BH3 domain and function upstream of the Bax-like proteins [22]. Most members of the whole Bcl-2 family possess a hydrophobic C-terminal segment which facilitates their targeting

(a) Cas pase family

Group I: Inflammatory caspases nh,—[

-coon Csspassl

J—C04H Caspase S

cooh caspasi, if coon Cajpase 11

J—cooh Caspase 14

-coon Csspassl

J—C04H Caspase S

cooh caspasi, if coon Cajpase 11

J—cooh Caspase 14

Croup II: Apoptosis initiator cas pases □—[

COOH Campase 1

j—cooh caspaws cooh caspa» 10

COOH Campase 1

j—cooh caspaws cooh caspa» 10

Group III: Apoptosis effector cas pases

HHL,

COOH Caspas« 3 1—cooh caipass s ]—COOrt Cajpasa 7

COOH Caspas« 3 1—cooh caipass s ]—COOrt Cajpasa 7

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