Structure /function

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The CD95 gene (APT1) consists of nine exons encoding a type I transmembrane protein harboring three CRDs, with exon 6 encoding the transmembrane domain [57] (Figure 1). Under denaturing conditions, CD95 migrates as a 40–50 kDa protein on SDS-PAGE. Similar to the TNF receptor [25], CD95 is pre-associated at the plasma membrane as a homotrimer, and this quaternary structure is mandatory for transmission of apoptotic signals in the presence of CD95L [19, 20]. Homotrimerization of CD95 occurs mainly through homotypic interactions involving the CD95-CRD1 domain [18-20]. Binding of CD95L or agonistic anti-CD95 mAbs to CD95 alters the receptor’s conformation and the extent to which the receptor is multimerized at the plasma membrane [58]. The intracellular region of CD95 encompasses an 80 amino-acid stretch designated as the DD (Figure 1), which consists of six anti-parallel α-helices [59]. Upon addition of CD95L, CD95 undergoes conformational modification of the DD, inducing a shift of helix 6 and fusion with helix 5, promoting both oligomerization of the receptor and recruitment of the adaptor protein FADD [60]. One consequence of the opening of the globular structure of CD95 is that the receptor becomes connected through this bridge, which increases the extent of its homo-aggregation. This long helix allows stabilization of the complex by recruitment of FADD. The CD95-DD:FADD-DD crystal structure provides several insights into the formation of the large CD95 clusters observed by imaging or biochemical methods in cells stimulated with CD95L. In addition, the structure also confirms that alteration in the conformation of CD95 plays an instrumental role in signal induction [60]. However, the idea of an elongated C-terminal α-helix favoring the cis-dimerization of CD95-DD was challenged by Driscoll et al., who did not observe the fusion of the last two helices at a more neutral pH (pH 6.2), in contrast to the acidic condition (pH 4) used in the initial study in which Scott et al. resolved the CD95-DD:FADD-DD structure [60]. At pH 6.2, association of CD95 predominantly interacted with FADD in a 5:5 complex, which arose via a polymerization mechanism involving three types of asymmetric interactions, but without major alteration of the DD globular structure [61, 62]. It is likely that the low-pH condition used by Scott et al. altered the conformation of CD95, resulting in the formation of non-physiological CD95:FADD oligomers [60]. Nonetheless, we cannot rule out the possibility that a local decrease in intracellular pH affects the initial steps of the CD95 signaling pathway in vivo, e.g., by promoting the opening of the CD95-DD and eventually contributing to formation of a complex that elicits a sequence of events distinct from that occurring at physiologic pH.

Once docked on CD95-DD, FADD self-associates [63], and binds procaspases-8 and -10, which are auto-processed and released in the cytosol as active caspases. Once activated, these caspases cleave many substrates, ultimately leading to the execution of the apoptotic program and cell death. The complex CD95/FADD/caspase-8/-10 is called DISC (Figure 2) [6]. Due to the importance of DISC formation to cell fate, it is not surprising that numerous cellular and viral proteins have evolved to hamper the formation of this structure: for example, both FLIP [64, 65] and PED/PEA-15 [66] interfere with the recruitment of caspase-8/-10 (Figure 2).