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Apoptosis is a form of programmed cell death, deregulation of which occurs in multiple disorders, including neurodegenerative and autoimmune diseases as well as cancer. The formation of a death-inducing signaling complex (DISC) and death effector domain (DED) filaments are critical for initiation of the extrinsic apoptotic pathway. Post-translational modifications (PTMs) of DED-containing DISC components such as FADD, procaspase-8, and c-FLIP comprise an additional level of apoptosis regulation, which is necessary to overcome the threshold for apoptosis induction. In this review we discuss the influence of PTMs of FADD, procaspase-8, and c-FLIP on DED filament assembly and cell death induction, with a focus on the 3D organization of the DED filament. Death Effector Domain (DED) Proteins in Extrinsic Apoptosis Signaling In multicellular organisms, tissue homeostasis is maintained through a fine-tuned balance between cell proliferation and cell death [1-5]. Several physiological and pathological stimuli have been reported to trigger programmed cell death [6]. Apoptosis can be induced via two pathways: the extrinsic and the intrinsic or mitochondrial pathway [2] (Figure 1). The extrinsic pathway is triggered upon binding of death ligands, including CD95 ligand (CD95L) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) to death receptors (DRs) such as Fas/CD95 and TRAIL receptor-1/2 (TRAILR1/2), respectively [7]. This association leads to the recruitment of proteins with death domains (DD) such as Fas-associated protein with death domain (FADD) to the DD of CD95 or TRAIL-R1/2, resulting in the formation of the death-inducing signaling complex (DISC) [8,9], which in turn directs cleavage and activation of caspases for inducing apoptosis. DISC is comprised of procaspase-8/10 and cellular FLICE-like inhibitory protein (c-FLIP). Upon induction of apoptosis, the DED of FADD interacts with DEDs of procaspase-8, procaspase-10, and c-FLIP, resulting in the formation of DED-filaments, which serve as a platform for procaspase-8 dimerization and subsequent activation [10-12] (Figure 1). DED belongs to the DD superfamily [3,13]. DED comprises six α-helixes arranged in a Greek key structural motif. FADD contains one DD and one DED, whereas both caspase-8 and c-FLIP contain two DEDs at their N terminus. DED filaments are formed through so-called type I, II, and III interactions between DEDs (Box 1) [12]. Proper architecture of DED filaments at DR complexes provide an extra layer of regulatory control of cell death. Recent findings are revealing a role for post-translational modifications (PTMs) such as phosphorylation, ubiquitylation, SUMOylation, and nitrosylation in regulating these interactions [6,14-17]. These modifications assist in maintaining proper conformations of DEDs in DED filaments, which is required for efficient caspase-8 activation, and in overcoming a threshold for apoptosis induction [18,19]. In this review, we consider the role of PTMs in controlling the mechanisms of DISC and DED filament...

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The development of efficient combinatorial treatments is one of the key tasks in modern anti-cancer therapies. An apoptotic signal can either be induced by activation of death receptors (DR) (extrinsic pathway) or via the mitochondria (intrinsic pathway). Cancer cells are characterized by deregulation of both pathways. Procaspase-8 activation in extrinsic apoptosis is controlled by c-FLIP proteins. We have recently reported the small molecules FLIPinB/FLIPinBγ targeting c-FLIPL in the caspase-8/c-FLIPL heterodimer. These small molecules enhanced caspase-8 activity in the death-inducing signaling complex (DISC), CD95L/TRAIL-induced caspase-3/7 activation and subsequent apoptosis. In this study to increase the pro-apoptotic effects of FLIPinB/FLIPinBγ and enhance its therapeutic potential we investigated costimulatory effects of FLIPinB/FLIPinBγ in combination with the pharmacological inhibitors of the anti-apoptotic Bcl-2 family members such as ABT-263 and S63845. The combination of these inhibitors together with FLIPinB/FLIPinBγ increased CD95L-induced cell viability loss, caspase activation and apoptosis. Taken together, our study suggests new approaches for the development of combinatorial anti-cancer therapies specifically targeting both intrinsic and extrinsic apoptosis pathways.