Structural analyses of death domains and their interactions

The death domain (DD), which is a versatle protein interaction module, is the prime mediator of the interactions necessary for apoptosis, innate immunity and the necrosis signaling pathway. Because DD mediated signaling events are associated with critical human diseases, studies in these areas are of great biological importance. Accordingly, many biochemical and structural studies of DD have been conducted in the past decade to investigate apoptotic and innate immune signaling. Evaluation of the molecular structure of DD and their interactions with partners have shown the underlying molecular basis for the assembly of DD mediated complexes and for the regulation of apoptosis and innate immunity. This review summarizes the structure and function of various DDs and DD:DD complexes involved in those signaling pathways.     

The PYRIN domain: a member of the death domain-fold superfamily

PYRIN domains were identified recently as putative protein-protein interaction domains at the N-termini of several proteins thought to function in apoptotic and inflammatory signaling pathways. The approximately 95 residue PYRIN domains have no statistically significant sequence homology to proteins with known three-dimensional structure. Using secondary structure prediction and potential-based fold recognition methods, however, the PYRIN domain is predicted to be a member of the six-helix bundle death domain-fold superfamily that includes death domains (DDs), death effector domains (DEDs), and caspase recruitment domains (CARDs). Members of the death domain-fold superfamily are well established mediators of protein-protein interactions found in many proteins involved in apoptosis and inflammation, indicating further that the PYRIN domains serve a similar function. An homology model of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1, a member of the Apaf-1/Ced-4 family of proteins, was constructed using the three-dimensional structures of the FADD and p75 neurotrophin receptor DDs, and of the Apaf-1 and caspase-9 CARDs, as templates. Validation of the model using a variety of computational techniques indicates that the fold prediction is consistent with the sequence. Comparison of a circular dichroism spectrum of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1 with spectra of several proteins known to adopt the death domain-fold provides experimental support for the structure prediction.     

Death domain of p75 neurotrophin receptor: a structural perspective on an intracellular signalling hub

The death domain (DD) is a globular protein motif with a signature feature of an all‐helical Greek‐key motif. It is a primary mediator of a variety of biological activities, including apoptosis, cell survival and cytoskeletal changes, which are related to many neurodegenerative diseases, neurotrauma, and cancers. DDs exist in a wide range of signalling proteins including p75 neurotrophin receptor (p75^NTR), a member of the tumour necrosis factor receptor superfamily. The specific signalling mediated by p75^NTR in a given cell depends on the type of ligand engaging the extracellular domain and the recruitment of cytosolic interactors to the intracellular domain, especially the DD, of the receptor. In solution, the p75^NTR‐DDs mainly form a symmetric non‐covalent homodimer. In response to extracellular signals, conformational changes in the p75^NTR extracellular domain (ECD) propagate to the p75^NTR‐DD through the disulfide‐bonded transmembrane domain (TMD) and destabilize the p75^NTR‐DD homodimer, leading to protomer separation and exposure of binding sites on the DD surface. In this review, we focus on recent advances in the study of the structural mechanism of p75^NTR‐DD signalling through recruitment of diverse intracellular interactors for the regulation and control of diverse functional outputs.     

Generalized semi-refolding methods for purification of the functional death domain superfamily

The death domain (DD) superfamily comprising the death domain (DD) subfamily, the death effector domain (DED) subfamily, the caspase recruitment domain (CARD) subfamily and the pyrin domains (PYD) subfamily is one of the largest classes of protein interaction modules and plays a pivotal role in the apoptosis, inflammation, and immune cell signaling pathways. Despite the biological importance of the death domain superfamily, structural and in vitro biochemical studies have been limited because these domains are prone to aggregate under physiological conditions. Here, we describe a generalized method, termed semi-refolding, that is particularly applicable for purification of the functional death domain superfamily. The recombinant proteins Caspase-1 CARD, AIM2 PYD, NALP3 PYD, and RIP1 DD from inclusion bodies were successfully purified using this method.     

Inhibition of both the extrinsic and intrinsic death pathways through nonhomotypic death-fold interactions

Death-fold domains constitute an evolutionarily conserved superfamily that mediates apoptotic signaling. These motifs, including CARD (caspase recruitment domain), DD (death domain), and DED (death effector domain), are believed to exert their effects solely through homotypic interactions. Herein we demonstrate that the CARD-containing protein ARC engages in nontraditional death-fold interactions to suppress both extrinsic and intrinsic death pathways. The extrinsic pathway is disrupted by heterotypic interactions between ARC's CARD and the DDs of Fas and FADD, which inhibit Fas-FADD binding and assembly of the death-inducing signaling complex (DISC). The intrinsic pathway is antagonized by ARC-Bax binding, involving ARC's CARD and the Bax C terminus. This inhibits Bax activation and translocation to the mitochondria. Knockdown of endogenous ARC facilitates DISC assembly and triggers spontaneous Bax activation and apoptosis. Conversely, physiological levels of ARC suppress these events. These studies establish a critical role for nonhomotypic death-fold interactions in the regulation of apoptosis.     

Differential signaling to apoptotic and necrotic cell death by Fas-associated death domain protein FADD

Two general pathways for cell death have been defined, apoptosis and necrosis. Previous studies in Jurkat cells have demonstrated that the Fas-associated death domain (FADD) is required for Fas-mediated signaling to apoptosis and necrosis. Here we developed L929rTA cell lines that allow Tet-on inducible expression and FK506-binding protein (FKBP)-mediated dimerization of FADD, FADD-death effector domain (FADD-DED), or FADD-death domain (FADD-DD). We show that expression and dimerization of FADD leads to necrosis. However, pretreatment of the cells with the Hsp90 inhibitor geldanamycin, which leads to proteasome-mediated degradation of receptor interacting protein 1 (RIP1), reverts FKBP-FADD-induced necrosis to apoptosis. Expression and dimerization of FADD-DD mediates necrotic cell death. We found that FADD-DD is able to bind RIP1, another protein necessary for Fas-mediated necrosis. Expression and dimerization of FADD-DED initiates apoptosis. Remarkably, in the presence of caspase inhibitors, FADD-DED mediates necrotic cell death. Coimmunoprecipitation studies revealed that FADD-DED in the absence procaspase-8 C/A is also capable of recruiting RIP1. However, when procaspase-8 C/A and RIP1 are expressed simultaneously, FADD-DED preferentially recruits procaspase-8 C/A.     

The role of TRADD in death receptor signaling

TRADD (TNFR1-associated death domain protein) was initially identified as an adaptor molecule that transduces the signal downstream of the TNFR1 (tumor necrosis factor receptor 1). TNFR1 belongs to the so-called death receptor (DR) family of receptors that depending on the context can induce either apoptosis or proliferation, as well as NF-κB and MAP kinase activation. The receptors of this group contain death domain (DD) that is necessary for the induction of apoptosis. This review summarizes the recent advances in the field of DR signaling and in particular the role of TRADD.     

Death domain assembly mechanism revealed by crystal structure of the oligomeric PIDDosome core complex

Proteins of the death domain (DD) superfamily mediate assembly of oligomeric signaling complexes for the activation of caspases and kinases via unknown mechanisms. Here we report the crystal structure of the PIDD DD and RAIDD DD complex, which forms the core of the caspase-2-activating complex PIDDosome. Although RAIDD DD and PIDD DD are monomers, they assemble into a complex that comprises seven RAIDD DDs and five PIDD DDs. Despite the use of an asymmetric assembly mechanism, all DDs in the complex are in quasi-equivalent environments. The structure provided eight unique asymmetric interfaces, which can be classified into three types. These three types of interactions together cover a majority of the DD surface. Mutagenesis on almost all interfaces leads to disruption of the assembly, resulting in defective caspase-2 activation. The three types of interactions may represent most, if not all, modes of interactions in the DD superfamily for assembling complexes of different stoichiometry.     

The role of TRADD in death receptor signaling

TRADD (TNFR1-associated death domain protein) was initially identified as an adaptor molecule that transduces the signal downstream of the TNFR1 (tumor necrosis factor receptor 1). TNFR1 belongs to the so-called death receptor (DR) family of receptors that depending on the context can induce either apoptosis or proliferation, as well as NF-κB and MAP kinase activation. The receptors of this group contain death domain (DD) that is necessary for the induction of apoptosis. This review summarizes the recent advances in the field of DR signaling and in particular the role of TRADD.     

Solution structure of the isolated Pelle death domain

The interaction between the death domains (DDs) of Tube and the protein kinase Pelle is an important component of the Toll pathway. Published crystallographic data suggests that the Pelle-Tube DD interface is plastic and implies that in addition to the two predominant Pelle-Tube interfaces, a third interaction is possible. We present the NMR solution structure of the isolated death domain of Pelle and a study of the interaction between the DDs of Pelle and Tube. Our data suggests the solution structure of the isolated Pelle DD is similar to that of Pelle DD in complex with Tube. Additionally, they suggest that the plasticity observed in the crystal structure may not be relevant in the functioning death domain complex.     

Apoptotic potential of Fas-associated death domain on regulation of cell death regulatory protein cFLIP and death receptor mediated apoptosis in HEK 293T cells

Fas-associated death domain (FADD) is a common adaptor molecule which plays an important role in transduction of death receptor mediated apoptosis. The FADD provides DED motif for binding to both procaspase-8 and cFLIP molecules which executes death receptor mediated apoptosis. Dysregulated expression of FADD and cFLIP may contribute to inhibition of apoptosis and promote cell survival in cancer. Moreover elevated intracellular level of cFLIP competitively excludes the binding of procaspase-8 to the death effector domain (DED) of FADD at the DISC to block the activation of death receptor signaling required for apoptosis. Increasing evidence shows that defects in FADD protein expression are associated with progression of malignancies and resistance to apoptosis. Therefore, improved expression and function of FADD may provide new paradigms for regulation of cell proliferation and survival in cancer. In the present study, we have examined the potential of FADD in induction of apoptosis by overexpression of FADD in HEK 293T cells and validated further its consequences on the expression of pro and anti-apoptotic proteins besides initiation of death receptor mediated signaling. We have found deficient expression of FADD and elevated expression of cFLIP(L) in HEK 293T cells. Our results demonstrate that over expression of FADD attenuates the expression of anti-apoptotic protein cFLIP and activates the cascade of extrinsic caspases to execution of apoptosis in HEK 293T cells.     

Caspase-independent Cell Killing by Fas-associated Protein with Death Domain

The binding of Fas ligand to Fas recruits caspase 8 to Fas via an adaptor, FADD/MORT1, and activates a caspase cascade leading to apoptosis. Here, we describe a human Jurkat-derived cell line (JB-6) that is deficient in caspase 8. This cell line was resistant to the apoptosis triggered by Fas engagement. However, the multimerization of Fas-associated protein with death domain, through the use of a dimerizing system, killed the JB-6 cells. This killing process was not accompanied by the activation of caspases or DNA fragmentation. The dying cells showed neither condensation nor fragmentation of cells and nuclei, but the cells and nuclei swelled in a manner similar to that seen in necrosis. These results suggested that Fas-associated protein with death domain can kill the cells via two pathways, one mediated by caspases and another that does not involve them.     

Revival of apoptotic cells that display early-stage dynamic membrane blebbing

The critical point at which apoptosis becomes irreversible and how cells attain an anti-apoptotic state remain unknown. Here, we report that apoptotic cells undergoing early-stage dynamic membrane blebbing revive. We examined this phenomenon in cell lines that stably express 2DED2DD, a modified FADD produced by fusing the tandem death effector domains (DEDs) and tandem death domains (DDs). Induction of apoptosis caused rapid blebbing. Eight hours later, most cells shrunk while some detached from the flask. Twenty-four hours later, when activated caspase 3 decreased, more than half the cells revived and appeared normal, probably due to the induction of unidentified anti-apoptotic proteins.     

LRDD, a novel leucine rich repeat and death domain containing protein

Death domains (DD) and leucine rich repeats (LRR) are two different types of protein interaction motifs. Death domains are found predominantly in proteins involved in signaling and are involved in homo- and heteromultimerization. Leucine rich repeats are found in proteins with diverse cellular functions, like cell adhesion and cellular signaling, and mediate reversible protein-protein interactions. In this paper we report the cloning of a new human gene called LRDD (leucine repeat death domain containing protein). LRDD encodes a protein of 83 kDa with six LRRs at the N-terminus and a DD at the C-terminus. LRDD appears to be processed into two fragments of about 33 and 55 kDa, containing LRRs and DD respectively. Interestingly, LRDD is shown to interact with two other death domain containing proteins, FADD and MADD, presumably through death domain interactions. LRDD may represent a new type of adapter protein that could be involved in signaling or other cellular functions.     

An apoptosis signaling pathway induced by the death domain of FADD selectively kills normal but not cancerous prostate epithelial cells.

The adaptor protein FADD directly, or indirectly via another adaptor called TRADD, recruits caspase 8 to death receptors of the tumor necrosis factor receptor family. Consequentially, a dominant-negative mutant (FADD-DN, which consists only of the FADD death domain) that binds to receptors but cannot recruit caspase 8 has been widely used to inhibit apoptosis by various stimuli that work via death receptors. Here, we show that FADD-DN also has another cell type- and cancer-dependent activity because it induces apoptosis of normal human prostate epithelial cells but not normal prostate stromal cells or prostate cancer cells. This activity is independent of FADD-DN's ability to bind to three known interacting proteins, Fas, TRADD or RIP suggesting that it is distinct from FADD's functions at activated death receptors. FADD-DN induces caspase activation in normal epithelial cells as demonstrated using a Fluorescence Resonance Energy Transfer assay that measures caspase activity in individual living cells. However, caspase-independent pathways are also implicated in FADD-DN-induced apoptosis because caspase inhibitors were inefficient at preventing prostate cell death. Therefore, the death domain of FADD has a previously unrecognized role in cell survival that is epithelial-specific and defective in cancer cells. This FADD-dependent signaling pathway may be important in prostate carcinogenesis.     

Helical assembly in the death domain (DD) superfamily

Death domain (DD) superfamily members play a central role in apoptotic and inflammatory signaling through formation of oligomeric molecular scaffolds. These scaffolds promote the activation of proinflammatory and apoptotic initiator caspases, as well as Ser/Thr kinases. Interactions between DDs are facilitated by a conserved set of interaction surfaces, type I, type II, and type III. Recently structural information on a ternary complex containing the DDs of MyD88, IRAK4, and IRAK2 and a binary complex containing Fas and FADD DDs has become available. This review will focus on how the three DD interaction surfaces cooperate to facilitate the assembly of these oligomeric signaling complexes.     

Xenopus death-domain-containing proteins FADD and RIP1 synergistically activate JNK and NF-kappaB

BACKGROUND INFORMATION: Death receptors (DRs) induce intracellular signalling upon engagement of their cognate ligands, leading to apoptosis, cell survival or pro-inflammatory responses. In mammals, DR signalling is mediated by the recruitment of several DD (death domain)-containing molecules, such as FADD (Fas-associated DD) and RIP1 (receptor-interacting protein 1). RESULTS: To elucidate the molecular mechanisms of intracellular DR signalling in Xenopus, we have isolated cDNAs encoding xFADD (Xenopus FADD), and xRIP1 and its short isoform xRIP1beta, which is produced by alternative splicing of the xRIP1 gene. These DD-containing proteins interacted with Xenopus DR members xDR-M1 and xDR-M2 through their DDs in co-transfected HEK-293T cells. Overexpression of xFADD activated not only xCaspase 8, but also AP-1 (activator protein 1), which reflects activation of JNK (c-Jun N-terminal kinase) and NF-kappaB (nuclear factor kappaB). A comparative analysis of xRIP1, a kinase-dead mutant of xRIP1 and xRIP1beta indicated that the kinase activity of xRIP1 was required for the activation of AP-1 and NF-kappaB. Interestingly, xFADD and xRIP1 interacted with each other via their DDs, and the expression of a mutant xRIP1 containing only the DD (xRIP1-DD) repressed the xFADD-induced activation of NF-kappaB and AP-1. xFADD and xRIP1 synergistically induced the activation of AP-1 and NF-kappaB, both of which were partially mediated by TRAF2 (tumour-necrosis-factor-receptor-associated factor 2) and TAK1 (transforming-growth-factor-beta-activated kinase 1). We also found that the activation pathways of NF-kappaB induced by xDR-M2 were inhibited by xRIP1-DD. CONCLUSIONS: Xenopus FADD, RIP1 and its splice variant RIP1beta have been characterized. Interaction of xFADD and xRIP1 induced synergistic activation of JNK and NF-kappaB. In addition, the NF-kappaB activation induced by xDR-M2 was partially mediated by xRIP1.     

Death domain mutagenesis of KILLER/DR5 reveals residues critical for apoptotic signaling

The Fas/tumor necrosis factor (TNF)/TRAIL receptors signal death through a cytoplasmic death domain (DD) containing six alpha-helices with positively charged helix 2 interacting with negatively charged helix 3 of another DD. DD mutation occurs in head/neck and lung cancer (TRAIL receptor KILLER/DR5) and in lpr mice (Fas). We examined the apoptotic potential of known KILLER/DR5 lung tumor-derived mutants (n = 6) and DD mutants (n = 18) generated based on conservation with DR4, Fas, Fas-associated death domain (FADD), and tumor necrosis factor receptor 1 (TNFR1). With the exception of Arg-330 required in Fas or FADD for aggregation or for TNFR1 cytotoxicity, surprisingly major loss-of-function KILLER/DR5 alleles (W325A, L334A (lpr-like), I339A, and W360A) contained hydrophobic residues. Loss-of-function of I339A (highly conserved) has not been reported in DDs. Charged residue mutagenesis revealed the following points. 1) E326A, conserved in DR4, is dispensable for death; the homologous residue is positively charged in Fas, TNFR1, and FADD and is critical for DD interactions. 2) K331A, D336A, E338A, K340A, K343A, and D351A have partial loss-of-function suggesting multiple charges stabilize receptor-adapter interactions. Analysis of the tumor-derived KILLER/DR5 mutants revealed the following. 1) L334F has partial loss-of-function versus L334A, whereas E338K has major loss-of-function versus E338A, examples where alanine and tumor-specific substitutions have divergent phenotypes. 2) Unexpectedly, S324F, E326K, K386N, and D407Y have no loss-of-function with tumor-specific or alanine substitutions. Loss-of-function KILLER/DR5 mutants were deficient in recruitment of FADD and caspase 8 to TRAIL death-inducing signaling complexes. The results reveal determinants within KILLER/DR5 for death signaling and drug design.     

The adaptor protein FADD and the initiator caspase-8 mediate activation of NF-��B by TRAIL

Besides inducing apoptosis, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) activates NF-κB. The apoptosis signaling pathway of TRAIL is well characterized involving TRAIL receptors, Fas-associated protein with death domain (FADD) and caspase-8. In contrast, the molecular mechanism of TRAIL signaling to NF-κB remains controversial. Here, we characterized the receptor–proximal mediators of NF-κB activation by TRAIL. Deletion of the DD of TRAIL receptors 1 and 2 revealed that it is essential in NF-κB signaling. Because FADD interacts with the TRAIL receptor DD, FADD was tested. RNAi-mediated knockdown of FADD or FADD deficiency in JURKAT T-cell leukemia cells decreased or disabled NF-κB signaling by TRAIL. In contrast, TRAIL-induced activation of NF-κB was maintained upon loss of receptor interacting protein 1 (RIP1) or knockdown of FLICE-like inhibitory protein (FLIP). Exogenous expression of FADD rescued TRAIL-induced NF-κB signaling. Loss-of-function mutations of FADD within the RHDLL motif of the death effector domain, which is required for TRAIL-induced apoptosis, abrogated FADD''s ability to recruit caspase-8 and mediate NF-κB activation. Accordingly, deficiency of caspase-8 inhibited TRAIL-induced activation of NF-κB, which was rescued by wild-type caspase-8, but not by a catalytically inactive caspase-8 mutant. These data establish the mechanism of TRAIL-induced NF-κB activation involving the TRAIL receptor DD, FADD and caspase-8, but not RIP1 or FLIP. Our results show that signaling of TRAIL-induced apoptosis and NF-κB bifurcates downstream of caspase-8.