Death-inducing signaling complex (DISC) and death effector domain (DED) filaments control extrinsic apoptosis.DED filaments comprise FADD, procaspase-8/10, and c-FLIP.Post-translational modifications (PTMs) of DED filament proteins create the additional checkpoint of apoptosis.A central role in understanding apoptosis control is in deciphering the 3D architecture of PTMs in the DED filament. 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. 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. In multicellular organisms, tissue homeostasis is maintained through a fine-tuned balance between cell proliferation and cell death [1.Eldeeb M.A. et al.Regulating apoptosis by degradation: the N-end rule-mediated regulation of apoptotic proteolytic fragments in mammalian cells.Int. J. Mol. Sci. 2018; 19E3414Crossref PubMed Scopus (24) Google Scholar, 2.Elmore S. Apoptosis: a review of programmed cell death.Toxicol. Pathol. 2007; 35: 495-516Crossref PubMed Scopus (8547) Google Scholar, 3.Wilson N.S. et al.Death receptor signal transducers: nodes of coordination in immune signaling networks.Nat. Immunol. 2009; 10: 348-355Crossref PubMed Scopus (425) Google Scholar, 4.Strasser A. et al.Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases.EMBO J. 2011; 30: 3667-3683Crossref PubMed Scopus (405) Google Scholar, 5.Taylor R.C. et al.Apoptosis: controlled demolition at the cellular level.Nat. Rev. Mol. Cell Biol. 2008; 9: 231-241Crossref PubMed Scopus (1905) Google Scholar]. Several physiological and pathological stimuli have been reported to trigger programmed cell death [6.Meier P. et al.Ubiquitin-mediated regulation of cell death, inflammation, and defense of homeostasis.Curr. Top. Dev. Biol. 2015; 114: 209-239Crossref PubMed Scopus (11) Google Scholar]. Apoptosis can be induced via two pathways: the extrinsic and the intrinsic or mitochondrial pathway [2.Elmore S. Apoptosis: a review of programmed cell death.Toxicol. Pathol. 2007; 35: 495-516Crossref PubMed Scopus (8547) Google Scholar] (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.Lavrik I.N. et al.Understanding apoptosis by systems biology approaches.Mol. BioSyst. 2009; 5: 1105-1111Crossref PubMed Scopus (41) Google Scholar]. 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.Kischkel F.C. et al.Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor.EMBO J. 1995; 14: 5579-5588Crossref PubMed Scopus (1782) Google Scholar,9.Krammer P.H. et al.Life and death in peripheral T cells.Nat. Rev. Immunol. 2007; 7: 532-542Crossref PubMed Scopus (485) Google Scholar], 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.Dickens L.S. et al.A death effector domain chain DISC model reveals a crucial role for caspase-8 chain assembly in mediating apoptotic cell death.Mol. Cell. 2012; 47: 291-305Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 11.Schleich K. et al.Stoichiometry of the CD95 death-inducing signaling complex: experimental and modeling evidence for a death effector domain chain model.Mol. Cell. 2012; 47: 306-319Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar] (Figure 1). DED belongs to the DD superfamily [3.Wilson N.S. et al.Death receptor signal transducers: nodes of coordination in immune signaling networks.Nat. Immunol. 2009; 10: 348-355Crossref PubMed Scopus (425) Google Scholar,13.Lamkanfi M. Dixit V.M. Manipulation of host cell death pathways during microbial infections.Cell Host Microbe. 2010; 8: 44-54Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar]. 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.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. 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.Meier P. et al.Ubiquitin-mediated regulation of cell death, inflammation, and defense of homeostasis.Curr. Top. Dev. Biol. 2015; 114: 209-239Crossref PubMed Scopus (11) Google Scholar,14.Feltham R. et al.IAPS and ubiquitylation.IUBMB Life. 2012; 64: 411-418Crossref PubMed Scopus (14) Google Scholar, 15.Lafont E. et al.Paving TRAIL's path with ubiquitin.Trends Biochem. Sci. 2018; 43: 44-60Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 16.Tourneur L. Chiocchia G. FADD: a regulator of life and death.Trends Immunol. 2010; 31: 260-269Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 17.Zamaraev A.V. et al.Post-translational modification of caspases: the other side of apoptosis regulation.Trends Cell Biol. 2017; 27: 322-339Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar]. 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.Bentele M. et al.Mathematical modeling reveals threshold mechanism in CD95-induced apoptosis.J. Cell Biol. 2004; 166: 839-851Crossref PubMed Scopus (260) Google Scholar,19.Buchbinder J.H. et al.Quantitative single cell analysis uncovers the life/death decision in CD95 network.PLoS Comput. Biol. 2018; 14e1006368Crossref PubMed Scopus (15) Google Scholar]. In this review, we consider the role of PTMs in controlling the mechanisms of DISC and DED filament assembly, with a focus on the 3D organization of the DED filament.Box 1DED Filament ArchitectureThe core components of the death-inducing signaling complex (DISC) that contain a death effector domain (DED), form so-called DED filaments. The structure of the DED filaments has been recently solved by cryogenic electron microscopy (cryo-EM) structural analysis of the procaspase-8 N terminal prodomain [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. Procaspase-8 comprises DED1 and DED2 in its N terminal prodomain, followed by catalytic p18 and p10 domains (Figure IA). The cryo-EM analysis revealed that procaspase-8 DEDs assemble into oligomeric structures that were named DED filaments [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. The DED filaments provide a platform for the dimerization and subsequent activation of procaspase-8.The structure of DED naturally provides the basis for formation of filaments. Each DED can be characterized by the presence of six interfaces, Ia/b, IIa/b, and IIIa/b, which have the ability to bind to each other (Figure IB). The assembly of the DED filament is mediated by three types of DED interactions: type I, II, and III (Figure IB). Type I interactions involve the binding of Ia and Ib interfaces of adjacent DEDs and are largely mediated by hydrophobic amino acids, including the highly conserved 'FL' motif residues (Phe/Leu) [10.Dickens L.S. et al.A death effector domain chain DISC model reveals a crucial role for caspase-8 chain assembly in mediating apoptotic cell death.Mol. Cell. 2012; 47: 291-305Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar]. This motif is located at the Ib interface of the DEDs of caspase-8, caspase-10, and c-FLIP [10.Dickens L.S. et al.A death effector domain chain DISC model reveals a crucial role for caspase-8 chain assembly in mediating apoptotic cell death.Mol. Cell. 2012; 47: 291-305Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar]. Type I interactions mediate the formation of the so-called linear DED chains, which, in turn, represent the substructure of the DED filament (Figure IC). Importantly, each DED filament is comprised of three linear chains (Figure IC–E). Moreover, the linear chains of procaspase-8 were first suggested to be the platform for the dimerization and subsequent activation of procaspase-8, but, as it turned out later, they represent only the substructure of the DED filament [10.Dickens L.S. et al.A death effector domain chain DISC model reveals a crucial role for caspase-8 chain assembly in mediating apoptotic cell death.Mol. Cell. 2012; 47: 291-305Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 11.Schleich K. et al.Stoichiometry of the CD95 death-inducing signaling complex: experimental and modeling evidence for a death effector domain chain model.Mol. Cell. 2012; 47: 306-319Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar].Type II and type III interactions are mediated via the binding of IIa/IIb and IIIa/IIIb interfaces, correspondingly [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. Type II and III interactions are largely mediated by hydrophilic amino acid residues and provide a basis for the interactions between adjacent linear chains in the filament (Figure IB). In this way, the architecture of DED filament is based on the combination of all types of DED interactions: type I, II, and III. Remarkably, this includes so-called homotypic interactions between similar domains such as DED1/DED1 or DED2/DED2, as well as so-called heterotypic interactions between DED1 and DED2 (Figure IB). The intricate DED interaction network results in a DED filament structure with a helical symmetry and a right-handed rotation (Figure IC,D).As mentioned earlier, the DED filament structure was obtained using a structure of the N terminal DED-only part of procaspase-8 (e.g., PDB ID 5l08 [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]). In addition, the structure of the C terminal part of caspase-8 was solved (e.g., PDB ID 3H11 [54.Yu J.W. et al.Mechanism of procaspase-8 activation by c-FLIPL.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 8169-8174Crossref PubMed Scopus (113) Google Scholar]). However, the structure of full-length procaspase-8, comprising both the prodomain and C terminal part, has not been reported so far. This has to be addressed in future studies. The core components of the death-inducing signaling complex (DISC) that contain a death effector domain (DED), form so-called DED filaments. The structure of the DED filaments has been recently solved by cryogenic electron microscopy (cryo-EM) structural analysis of the procaspase-8 N terminal prodomain [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. Procaspase-8 comprises DED1 and DED2 in its N terminal prodomain, followed by catalytic p18 and p10 domains (Figure IA). The cryo-EM analysis revealed that procaspase-8 DEDs assemble into oligomeric structures that were named DED filaments [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. The DED filaments provide a platform for the dimerization and subsequent activation of procaspase-8. The structure of DED naturally provides the basis for formation of filaments. Each DED can be characterized by the presence of six interfaces, Ia/b, IIa/b, and IIIa/b, which have the ability to bind to each other (Figure IB). The assembly of the DED filament is mediated by three types of DED interactions: type I, II, and III (Figure IB). Type I interactions involve the binding of Ia and Ib interfaces of adjacent DEDs and are largely mediated by hydrophobic amino acids, including the highly conserved 'FL' motif residues (Phe/Leu) [10.Dickens L.S. et al.A death effector domain chain DISC model reveals a crucial role for caspase-8 chain assembly in mediating apoptotic cell death.Mol. Cell. 2012; 47: 291-305Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar]. This motif is located at the Ib interface of the DEDs of caspase-8, caspase-10, and c-FLIP [10.Dickens L.S. et al.A death effector domain chain DISC model reveals a crucial role for caspase-8 chain assembly in mediating apoptotic cell death.Mol. Cell. 2012; 47: 291-305Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar]. Type I interactions mediate the formation of the so-called linear DED chains, which, in turn, represent the substructure of the DED filament (Figure IC). Importantly, each DED filament is comprised of three linear chains (Figure IC–E). Moreover, the linear chains of procaspase-8 were first suggested to be the platform for the dimerization and subsequent activation of procaspase-8, but, as it turned out later, they represent only the substructure of the DED filament [10.Dickens L.S. et al.A death effector domain chain DISC model reveals a crucial role for caspase-8 chain assembly in mediating apoptotic cell death.Mol. Cell. 2012; 47: 291-305Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 11.Schleich K. et al.Stoichiometry of the CD95 death-inducing signaling complex: experimental and modeling evidence for a death effector domain chain model.Mol. Cell. 2012; 47: 306-319Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. Type II and type III interactions are mediated via the binding of IIa/IIb and IIIa/IIIb interfaces, correspondingly [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. Type II and III interactions are largely mediated by hydrophilic amino acid residues and provide a basis for the interactions between adjacent linear chains in the filament (Figure IB). In this way, the architecture of DED filament is based on the combination of all types of DED interactions: type I, II, and III. Remarkably, this includes so-called homotypic interactions between similar domains such as DED1/DED1 or DED2/DED2, as well as so-called heterotypic interactions between DED1 and DED2 (Figure IB). The intricate DED interaction network results in a DED filament structure with a helical symmetry and a right-handed rotation (Figure IC,D). As mentioned earlier, the DED filament structure was obtained using a structure of the N terminal DED-only part of procaspase-8 (e.g., PDB ID 5l08 [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]). In addition, the structure of the C terminal part of caspase-8 was solved (e.g., PDB ID 3H11 [54.Yu J.W. et al.Mechanism of procaspase-8 activation by c-FLIPL.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 8169-8174Crossref PubMed Scopus (113) Google Scholar]). However, the structure of full-length procaspase-8, comprising both the prodomain and C terminal part, has not been reported so far. This has to be addressed in future studies. FADD is a pivotal signaling component of DR-mediated apoptosis. The DED of FADD initiates caspase-8 filament growth and is therefore essential for apoptosis induction [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar,20.Mouasni S. Tourneur L. FADD at the crossroads between cancer and inflammation.Trends Immunol. 2018; 39: 1036-1053Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar] (Figure 2A ). The proper structural conformation of FADD is a requirement for the initiation of DED filament growth (Box 2). Hence, its modulation by PTM might have major consequences for apoptosis initiation [20.Mouasni S. Tourneur L. FADD at the crossroads between cancer and inflammation.Trends Immunol. 2018; 39: 1036-1053Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar]. Several putative PTM sites in FADD DED have been predicted in silico and even identified by mass spectrometry, including phosphorylation and ubiquitylation. However, their relevance for signal transduction has not been confirmed [21.Han G. et al.Phosphoproteome analysis of human liver tissue by long-gradient nanoflow LC coupled with multiple stage MS analysis.Electrophoresis. 2010; 31: 1080-1089PubMed Google Scholar].Box 2FADD Architecture in the DED FilamentFADD plays the major role in triggering DED filament growth [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. FADD comprises both DD and DED (Figure IA). The recruitment of FADD via its DD to the DD of CD95 results in the formation of a platform that is able to nucleate caspase-8 DED filament growth [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. However, the structure of CD95/FADD/caspase-8-DED filament is not reported yet. Moreover, though it is known that FADD DED can form filamentous structures, the structure of oligomerized FADD DEDs is also not solved. Currently, only the structure of the FADD DED monomer has been obtained [70.Eberstadt M. et al.NMR structure and mutagenesis of the FADD (Mort1) death-effector domain.Nature. 1998; 392: 941-945Crossref PubMed Scopus (204) Google Scholar,71.Carrington P.E. et al.The structure of FADD and its mode of interaction with procaspase-8.Mol. Cell. 2006; 22: 599-610Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar]. Nevertheless, the known DED structures and the high similarity of the tertiary structure of FADD DED and caspase-8 DEDs allow in silico prediction of the structure of FADD within the DED filament. In particular, it might be suggested that FADD forms type I, II, and III interactions with caspase-8 DEDs [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar] (Figure IB–D). Further evidence for FADD DED interactions within DED filament is coming from immuno-gold labeling [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. According to these data, the nucleation point formed by the CD95/FADD complex is located at one end of the DED filament [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. This allows the hypothesis that the procaspase-8 DED filament is elongated in one direction and that only specific interfaces of FADD DED take part in procaspase-8 binding. On the basis of mutagenesis studies of FADD DED and caspase-8 DEDs, as well as of the structure PDB ID 5l08, it might be suggested that Ib, IIa, and IIIa interfaces of FADD are involved in the binding of procaspase-8 within the DED filament (Figure IC,D) [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar,34.Hughes M.A. et al.Co-operative and hierarchical binding of c-FLIP and caspase-8: a unified model defines how c-FLIP isoforms differentially control cell fate.Mol. Cell. 2016; 61: 834-849Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar,71.Carrington P.E. et al.The structure of FADD and its mode of interaction with procaspase-8.Mol. Cell. 2006; 22: 599-610Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar,72.Majkut J. et al.Differential affinity of FLIP and procaspase 8 for FADD's DED binding surfaces regulates DISC assembly.Nat. Commun. 2014; 5: 3350Crossref PubMed Scopus (54) Google Scholar].To consider the role of FADD within the DED filament, in silico structural models must be created. In particular, as it was done in [55.Hillert L.K. et al.Long and short isoforms of c-FLIP act as control checkpoints of DED filament assembly.Oncogene. 2019; 39: 1756-1772Crossref PubMed Scopus (20) Google Scholar], the procaspase-8 DED1 and DED2 from cryo-EM structure (PDB ID 5L08) [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar] were replaced by FADD DED (PDB ID 1A1W) [70.Eberstadt M. et al.NMR structure and mutagenesis of the FADD (Mort1) death-effector domain.Nature. 1998; 392: 941-945Crossref PubMed Scopus (204) Google Scholar], with an approach based on the structural alignment of these structures. This type of in silico model can be used for the analysis of the FADD function in the DED filament (see Figure 2 in main text).Figure IFas-Associated Protein with Death Domain (FADD) Death Effector Domain (DED) Architecture in the DED Filament.Show full caption(A) Scheme of FADD, including death domain (DD) and DED. (B) Scheme of type I/II/III interfaces of FADD DED. (C) A 2D scheme of the DED filament model with FADD in 2D presentation. Three DED chains are shown (chains 1, 2, and 3). Procaspase-8 DED1 is shown in dark blue, while procaspase-8 DED2 is shown in light blue. FADD DED is shown in brown. The presentation is modified from [55.Hillert L.K. et al.Long and short isoforms of c-FLIP act as control checkpoints of DED filament assembly.Oncogene. 2019; 39: 1756-1772Crossref PubMed Scopus (20) Google Scholar]. (D) A 3D scheme of the model of procaspase-8 filament with FADD. The direction of the type I interaction is shown with a broken arrow. The same color code for procaspase-8 and FADD DEDs as in (B) is used. This model is constructed on the basis of [55.Hillert L.K. et al.Long and short isoforms of c-FLIP act as control checkpoints of DED filament assembly.Oncogene. 2019; 39: 1756-1772Crossref PubMed Scopus (20) Google Scholar].View Large Image Figure ViewerDownload Hi-res image Download (PPT) FADD plays the major role in triggering DED filament growth [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. FADD comprises both DD and DED (Figure IA). The recruitment of FADD via its DD to the DD of CD95 results in the formation of a platform that is able to nucleate caspase-8 DED filament growth [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. However, the structure of CD95/FADD/caspase-8-DED filament is not reported yet. Moreover, though it is known that FADD DED can form filamentous structures, the structure of oligomerized FADD DEDs is also not solved. Currently, only the structure of the FADD DED monomer has been obtained [70.Eberstadt M. et al.NMR structure and mutagenesis of the FADD (Mort1) death-effector domain.Nature. 1998; 392: 941-945Crossref PubMed Scopus (204) Google Scholar,71.Carrington P.E. et al.The structure of FADD and its mode of interaction with procaspase-8.Mol. Cell. 2006; 22: 599-610Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar]. Nevertheless, the known DED structures and the high similarity of the tertiary structure of FADD DED and caspase-8 DEDs allow in silico prediction of the structure of FADD within the DED filament. In particular, it might be suggested that FADD forms type I, II, and III interactions with caspase-8 DEDs [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar] (Figure IB–D). Further evidence for FADD DED interactions within DED filament is coming from immuno-gold labeling [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. According to these data, the nucleation point formed by the CD95/FADD complex is located at one end of the DED filament [12.Fu T.M. et al.Cryo-EM structure of caspase-8 tandem DED filament reveals assembly and regulation mechanisms of the death-inducing signaling complex.Mol. Cell. 2016; 64: 236-250Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar]. This allows the hypothesis that the procaspase-8 DED filament is elongated in one direction and that only specific interfaces of FADD DED take part in procaspase-8 binding. On the basis of mutagenesis studies of FADD DED and caspase-8 DEDs, as well as of the structure PDB ID 5l08, it might be suggest