FLICE-Inhibitory Proteins: Regulators of Death Receptor-Mediated Apoptosis

Cell death is executed along several pathways. Apart from necrotic cell death occurring upon tissue injury, several distinct types of apoptosis have been observed. Apoptosis, or programmed cell death, is critical for tissue homeostasis in multicellular organisms. It plays an important role in many physiological processes, especially in development and in the immune system (39, 80). Many diseases are associated with either too much or too little apoptosis, such as AIDS, cancer, and autoimmunity (39).

On the molecular level, the cell death program can be divided into three parts: initiation, execution, and termination of apoptosis. Apoptosis is initiated by a variety of stimuli, including growth factor withdrawal (“death by neglect”), UV or γ-irradiation, chemotherapeutic drugs, and death receptor signals. In most cases the execution phase is characterized by membrane inversion and exposure of phosphatidylserine, blebbing (zeiosis), fragmentation of the nucleus, chromatin condensation, and DNA degradation. In the termination phase, “apoptotic bodies” are engulfed by phagocytes (39).

The growing subfamily of death receptors is part of the tumor necrosis factor (TNF)/nerve growth factor receptor superfamily. This superfamily is characterized by a sequence of two to five cysteine-rich extracellular repeats. The death receptors contain an intracellular death domain (DD), which is essential for transduction of the apoptotic signal. Six members of this subfamily are known so far, TNF-R1 (also called CD120a), CD95 (APO-1/Fas), DR3 (APO-3, LARD, TRAMP, and WSL1), TRAIL-R1 (APO-2 and DR4), TRAIL-R2 (DR5, KILLER, and TRICK2), and DR6 (67). Among these, CD95 is the best-characterized death receptor (65).

Death receptors are activated by their natural ligands, which have coevolved as a death ligand family, called the TNF family. Except for lymphotoxin α, the death ligands are type II transmembrane proteins which can be converted into a soluble form by the activity of metalloproteases. Several groups reported activity of the soluble CD95 ligand (CD95L) (38), whereas others ascribe the capacity to induce apoptosis to the membrane-bound form (66, 74). Recently, it has been proposed that the activity of soluble CD95L is enhanced by interaction with the extracellular matrix (4). The roles of soluble and membrane-bound CD95L remain to be shown in vivo.

The salient point of death receptor signaling is the formation of a multimolecular complex of proteins triggered by receptor cross-linking either with agonistic antibodies (77) or with death ligands. The structure formed is called the death-inducing signaling complex (DISC) (see Fig. 2A) (36, 37, 71, 82). Among the death receptors, the CD95 DISC has been characterized most extensively. It consists of oligomerized, most probably trimerized, CD95, the serine-phosphorylated adapter Fas-associated death domain protein (FADD)/Mort 1, two isoforms of caspase 8 (caspase 8/a [FLICE, Mach-α1, and Mch5β] and caspase 8/b [Mach-α2]) (50), and CAP3 (a molecule that contains the N-terminal death effector domains [DED] of caspase 8 and an as yet uncharacterized C terminus) (36). According to recent findings, FADD and caspase 8 are also recruited to the DISC of TNF-related apoptosis-inducing ligand R1 (TRAIL-R1) and TRAIL-R2 (7, 37, 71) and are essential for death induction via TRAIL-R2 (71).

Caspases are a family of aspartate-specific cysteine proteases that are necessary for execution of apoptosis. Caspases are synthesized as proenzymes (zymogens) that are activated by proteolytic cleavage. The active enzyme is a heterotetrameric complex of two large subunits containing the active site and two small subunits. Activation of caspases has been reported to occur after a variety of apoptotic stimuli, including death receptor signals (13).

The stoichiometry of the DISC components is not clear, but the necessity of their interactions has been elucidated and lies in the nature of their corresponding subdomains. Oligomerization of CD95 creates a conformation of the receptor DD which, by homophilic interaction, binds the adapter FADD/Mort1 via its DD. In addition to a DD, FADD possesses an N-terminal DED with which it binds procaspase 8 and CAP3. Procaspase 8 is then cleaved at the DISC in three consecutive steps, which lead to the formation of active caspase 8, consisting of a heterotetramer of two p10 and two p18 subunits (see Fig. 2B). The prodomain of caspase 8 remains at the DISC, while active caspase 8 dissociates from the DISC to start the cascade of caspase activation which constitutes the execution phase of apoptosis (47).

Recently, it was reported that death receptors are assembled prior to triggering via so-called pre-ligand binding assembly domains (8, 69). In this case signaling would be induced either by conformational changes of preformed death receptor trimers or, alternatively, by formation of multimeric complexes upon ligand binding.

Several knockout and transgenic mice underscore the central role of the DISC-associated molecules FADD and caspase 8 in signaling via death receptors (79, 88). FADD and caspase 8 knockout mice die at embryonic day 11. They show cardiac failure and abdominal hemorrhage. To study embryonic lethality, FADD^−/− chimeric mice were constructed. In thymocytes of these mice, CD95-mediated apoptosis was completely blocked. The same applied to FADD^−/−fibroblasts.

Death receptor-mediated apoptosis can be modulated at both the receptor level, e.g., by glycosylation (33, 56), and further downstream by interfering with the apoptotic signaling cascade. For example, inhibitor-of-apoptosis proteins directly inhibit caspases (for a review, see reference 11), and antiapoptotic members of the Bcl-2 family inhibit apoptosis of so-called CD95 type II cells, in which apoptosis is dependent on a mitochondrial pathway (62).

In this review, we discuss the role and mechanism of apoptosis modulation by FLICE-inhibitory proteins (FLIPs).

V-FLIPS

Database mining led to the identification of an entire family of DED-containing proteins (6, 25, 76). Some of these proteins are components of viruses of the gammaherpesvirus class, such as herpesvirus saimiri, human herpesvirus 8, a Kaposi's sarcoma-associated herpesvirus, and moluscum contagiosum virus. These proteins were called viral FLICE-inhibitory proteins (v-FLIPs) (76). v-FLIPs consist of two DEDs (Fig.1). They were shown to bind to the CD95 DISC and thus inhibit activation of caspase 8. v-FLIPs were capable of inhibiting apoptosis induced via several death receptors (CD95, TNF-R1, DR3, and DR4), suggesting that these receptors use similar signaling pathways (48). In human herpesvirus 8, the v-FLIP coding region is represented by a bicistronic mRNA following the coding region of v-cyclin (19). These coding regions are separated by an internal ribosome entry site (44). v-FLIP is expressed at low levels in latently infected cells (44, 60, 73), but its expression is increased in late Kaposi's sarcoma lesions or upon serum withdrawal from lymphoma cells (44, 73). Deletion of the v-FLIP gene from the genome of herpesvirus saimiri confirmed the antiapoptotic effect of v-FLIP but revealed that it is not essential for replication, transformation, or pathogenicity of herpesvirus saimiri (16).

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Fig. 1.

Structural similarities between caspase 8 and FLIP. For details, refer to the text.

Mice carrying a T-cell-specific v-FLIP-E8 transgene show strongly reduced thymocyte numbers, although thymocytes of these mice are resistant to CD95-mediated apoptosis (53). The reduction in thymocyte numbers seems to be independent of the CD95 system, since it was also observed in a CD95^−/− background. Interestingly, the thymic phenotype resembles that of T cells from FADD dominant-negative transgenic mice, suggesting that another death receptor system distinct from the CD95 system is critically involved in thymocyte selection (52, 84).