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Inhibition of CD95/Fas activation is currently under clinical investigation as a therapy for glioblastoma multiforme and preclinical studies suggest that disruption of the CD95-CD95L interaction could also be a strategy to treat inflammatory and neurodegenerative disorders. Besides neutralizing anti-CD95L/FasL antibodies, mainly CD95ed-Fc, a dimeric Fc fusion protein of the extracellular domain of CD95 (CD95ed), is used to prevent CD95 activation. In view of the fact that full CD95 activation requires CD95L-induced CD95 trimerization and clustering of the resulting liganded CD95 trimers, we investigated whether fusion proteins of the extracellular domain of CD95 with a higher valency than CD95ed-Fc have an improved CD95L-neutralization capacity. We evaluated an IgG1(N297A)-based tetravalent CD95ed fusion protein which was obtained by replacing the variable domains of IgG1(N297A) with CD95ed (CD95ed-IgG1(N297A)) and a hexavalent variant obtained by fusion of CD95ed with a TNC-Fc(DANA) scaffold (CD95ed-TNC-Fc(DANA)) promoting hexamerization. The established N297A and DANA mutations were used to minimize FcγR binding of the constructs under maintenance of neonatal Fc receptor (FcRn) binding. Size exclusion high-performance liquid chromatography indicated effective assembly of CD95ed-IgG1(N297A). More important, CD95ed-IgG1(N297A) was much more efficient than CD95ed-Fc in protecting cells from cell death induction by human and murine CD95L. Surprisingly, despite its hexavalent structure, CD95ed-TNC-Fc(DANA) displayed an at best minor improvement of the capacity to neutralize CD95L suggesting that besides valency, other factors, such as spatial organization and agility of the CD95ed domains, play also a role in neutralization of CD95L trimers by CD95ed fusion proteins. More studies are now required to evaluate the superior CD95L-neutralizing capacity of CD95ed-IgG1(N297A) in vivo., (© 2024 The Author(s). PROTEINS: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

Unraveling the concentration-dependent spatiotemporal organization of receptors in the plasma membrane is crucial to understand cell signal initiation. A paradigm of this process is the oligomerization of CD95 during apoptosis signaling, with different oligomerization models being discussed. Here, we establish the molecular-sensitive approach cell lifetime Förster resonance energy transfer image spectroscopy to determine CD95 configurations in live cells. These data are corroborated by stimulated emission depletion microscopy, confocal photobleaching step analysis, and fluorescence correlation spectroscopy. We probed CD95 interactions for concentrations of ~10 to 1000 molecules per square micrometer, over nanoseconds to hours, and molecular to cellular scales. Quantitative benchmarking was achieved establishing high-fidelity monomer and dimer controls. While CD95 alone is primarily monomeric (~96%) and dimeric (4%), the addition of ligand induces oligomerization to dimers/trimers (~15%) leading to cell death. This study highlights molecular concentration effects and oligomerization dynamics. It reveals a minimal model, where small CD95 oligomers suffice to efficiently initiate signaling.

Synthetic biology has emerged as a useful technology for studying cytokine signal transduction. Recently, we described fully synthetic cytokine receptors to phenocopy trimeric receptors such as the death receptor Fas/CD95. Using a nanobody as an extracellular-binding domain for mCherry fused to the natural receptor's transmembrane and intracellular domain, trimeric mCherry ligands were able to induce cell death. Among the 17,889 single nucleotide variants in the SNP database for Fas, 337 represent missense mutations that functionally remained largely uncharacterized. Here, we developed a workflow for the Fas synthetic cytokine receptor system to functionally characterize missense SNPs within the transmembrane and intracellular domain of Fas. To validate our system, we selected five functionally assigned loss-of-function (LOF) polymorphisms and included 15 additional unassigned SNPs. Moreover, based on structural data, 15 gain-of-function or LOF candidate mutations were additionally selected. All 35 nucleotide variants were functionally investigated through cellular proliferation, apoptosis and caspases 3 and 7 cleavage assays. Collectively, our results showed that 30 variants resulted in partial or complete LOF, while five lead to a gain-of-function. In conclusion, we demonstrated that synthetic cytokine receptors are a suitable tool for functional SNPs/mutations characterization in a structured workflow., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Fas and Fas ligand (FasL) pathway plays important roles in virus defense and cell apoptosis. In our previous work, nervous necrosis virus (NNV) was discovered in Pacific cod (Gadus macrocephalus), and the Fas ligand (PcFasL) was up-regulated when NNV outbreak, however, signal transmission of Fas/FasL in fish are still unclear. In the present study, Pacific cod Fas (PcFas), PcFasL and Fas-associating protein with a novel death domain (PcFADD) were characterized. The predicted protein of PcFas, PcFasL and PcFADD includes 333 aa, 90 aa and 93 aa, separately. 3-D models of PcFas, PcFasL and PcFADD were well constructed based on reported templates, respectively, even though the sequence homology with other fish is very low. The transcript levels of PcFas increased gradually from 15 day-post hatching (dph) to 75dph. PcFas was significantly up-regulated when cod larvae had NNV symptoms at 24dph, 37dph, 46dph, 69dph, and 77dph. Subcellular localization revealed that PcFasL was located in the cytoplasm, while PcFas was mainly located in the cell membrane. Exogenous expressed PcFasL of 900 μg/mL could kill the Epithelioma papulosum cyprinid (EPC) cells by MTT test, but low concentration has no effect on the cells. qPCR analysis showed that overexpression of PcFas could significantly up-regulate the expression of genes related to Fas/FasL signaling pathway, including bcl-2, bax, and RIP3, while overexpression of PcFasL significantly up-regulate the expression of caspase-3, caspase-9, and MLKL. Overexpression of PcFas or PcFasL could induce EPC apoptosis significantly by flow cytometry, which was consistent with the results of caspase-3 mRNA level increasing. The results indicated that NNV could induce apoptosis through Fas/FasL signal pathway., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Lipid rafts are heterogeneous membrane domains enriched in cholesterol, sphingolipids, and gangliosides that serve as sorting platforms to compartmentalize and modulate signaling pathways. Death receptors and downstream signaling molecules have been reported to be recruited into these raft domains during the triggering of apoptosis. Here, we provide two protocols that support the presence of Fas/CD95 in lipid rafts during apoptosis, involving lipid raft isolation and confocal microscopy techniques. A detailed protocol is provided for the isolation of lipid rafts, by taking advantage of their resistance to Triton X-100 solubilization at 4 °C, followed by subsequent sucrose gradient centrifugation and analysis of the protein composition of the different gradient fractions by Western blotting. In addition, we also provide a detailed protocol for the visualization of the coclustering of Fas/CD95 death receptor and lipid rafts, as assessed by using anti-Fas/CD95 antibodies and fluorescent dye-conjugated cholera toxin B subunit that binds to ganglioside GM1, a main component of lipid rafts, by immunofluorescence and confocal microscopy. These protocols can be extended to any protein of interest to be analyzed for its association to lipid rafts.

Glycan biosynthesis relies on nucleotide sugars (NSs), abundant metabolites that serve as monosaccharide donors for glycosyltransferases. In vivo , signal-dependent fluctuations in NS levels are required to maintain normal cell physiology and are dysregulated in disease. However, how mammalian cells regulate NS levels and pathway flux remains largely uncharacterized. To address this knowledge gap, here we examined UDP-galactose 4'-epimerase (GALE), which interconverts two pairs of essential NSs. Using immunoblotting, flow cytometry, and LC-MS-based glycolipid and glycan profiling, we found that CRISPR/Cas9-mediated GALE deletion in human cells triggers major imbalances in NSs and dramatic changes in glycolipids and glycoproteins, including a subset of integrins and the cell-surface death receptor FS-7-associated surface antigen. In particular, we observed substantial decreases in total sialic acid, galactose, and GalNAc levels in glycans. These changes also directly impacted cell signaling, as GALE -/- cells exhibited FS-7-associated surface antigen ligand-induced apoptosis. Our results reveal a role of GALE-mediated NS regulation in death receptor signaling and may have implications for the molecular etiology of illnesses characterized by NS imbalances, including galactosemia and metabolic syndrome., (© 2020 Broussard et al.)

Proceeding our effort to study protein-protein interaction between the death receptor CD95 and phospholipase PLCγ1, we present in the current work chameleon-like traits of peptidomimetic inhibitors. Minute analysis of the interaction suggests that most of the binding energy relies on van der Waals contacts rather than more specific features, such as hydrogen bonds or salt bridges. The two most important positions of the peptoid for its interaction with PLCγ1 (Arg184 and Arg187) were modified to test this hypothesis. While Arg184 proves to be exchangeable for Trp, with no alteration in affinity, the nature of the amino acid replacing Arg187 is more dependent on its positive charge. However, affinity can be partially recovered by increasing van der Waals interactions. Overall, this study shows that for both positions, a subtle balance exists between hydrophobicity, surface contacts and affinity for CD95/PLCγ1, and provides information for the generation of new therapeutic compounds toward this druggable target., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Sepsis remains a significant health care burden, with high morbidities and mortalities. Patients with sepsis often require general anesthesia for procedures and imaging studies. Knowing that anesthetic drugs can pose immunomodulatory effects, it would be critical to understand the impact of anesthetics on sepsis pathophysiology. The volatile anesthetic sevoflurane is a common general anesthetic derived from ether as a prototype. Using a murine sepsis model induced by cecal ligation and puncture surgery, we examined the impact of sevoflurane on sepsis outcome. Different from volatile anesthetic isoflurane, sevoflurane exposure significantly improved the outcome of septic mice. This was associated with less apoptosis in the spleen. Because splenic apoptosis was largely attributed to the apoptosis of neutrophils, we examined the effect of sevoflurane on FasL-induced neutrophil apoptosis. Sevoflurane exposure significantly attenuated apoptosis. Sevoflurane did not affect the binding of FasL to the extracellular domain of Fas receptor. Instead, in silico analysis suggested that sevoflurane would bind to the interphase between Fas death domain (DD) and Fas-associated DD (FADD). The effect of sevoflurane on Fas DD-FADD interaction was examined using fluorescence resonance energy transfer (FRET). Sevoflurane attenuated FRET efficiency, indicating that sevoflurane hindered the interaction between Fas DD and FADD. The predicted sevoflurane binding site is known to play a significant role in Fas DD-FADD interaction, supporting our in vitro and in vivo apoptosis results.-Koutsogiannaki, S., Hou, L., Babazada, H., Okuno, T., Blazon-Brown, N., Soriano, S. G., Yokomizo, T., Yuki, K. The volatile anesthetic sevoflurane reduces neutrophil apoptosis via Fas death domain-Fas-associated death domain interaction.

CD95 is a member of the death receptor family and is well-known to promote apoptosis. However, accumulating evidence indicates that in some context CD95 has not only the potential to induce apoptosis but also can trigger non-apoptotic signal leading to cell survival, proliferation, cancer growth and metastasis. Despite extensive investigations focused on alterations in the expression level of CD95 and associated signal molecules, very few studies, however, have investigated the effects of post-translational modifications such as glycosylation, phosphorylation, palmitoylation, nitrosylation and glutathionylation on CD95 function. Post-translational modifications of CD95 in mammalian systems are likely to play a more prominent role than anticipated in CD95 induced cell death. In this review we will focus on the alterations in CD95-mediated signaling caused by post-translational modifications of CD95.

CD95/Fas ligand binds to the death receptor CD95 to induce apoptosis in sensitive cells. We previously reported that CD95L mRNA is enriched in sequences that, when converted to si/shRNAs, kill all cancer cells by targeting critical survival genes (Putzbach et al., 2017). We now report expression of full-length CD95L mRNA itself is highly toxic to cells and induces a similar form of cell death. We demonstrate that small (s)RNAs derived from CD95L are loaded into the RNA induced silencing complex (RISC) which is required for the toxicity and processing of CD95L mRNA into sRNAs is independent of both Dicer and Drosha. We provide evidence that in addition to the CD95L transgene a number of endogenous protein coding genes involved in regulating protein translation, particularly under low miRNA conditions, can be processed to sRNAs and loaded into the RISC suggesting a new level of cell fate regulation involving RNAi., Competing Interests: WP, AH, QG, AS, EB, AS, AQ, MH, MP No competing interests declared

Recent research has identified a potential role of the hyaluronic acid receptor stabilin-2 (Stab2) in cancer metastasis. Stab2 belongs to a group of scavenger receptors and is responsible for the clearance of more than ten ligands, including hyaluronic acid (HA). In vivo experiments on mice have shown that the absence of Stab2, or its blocking by an antibody, effectively opposes cancer metastasis, which is accompanied by an increase in the level of circulating HA. Knowledge of ligand recognition and signal transduction by Stab2 is limited and no three-dimensional structures of any protein fragments of this receptor have been solved to date. Here, a high-resolution X-ray structure of the seventh FAS1 domain of Stab2 is reported. This structure provides the first insight into the Stab2 structure.

Background: Fas ligand plays a key role in the human immune system as a major cell death inducing protein. The extracellular domain of human Fas ligand (hFasLECD) triggers apoptosis of malignant cells, and therefore is expected to have substantial potentials in medical biotechnology. However, the current application of this protein to clinical medicine is hampered by a shortage of the benefits relative to the drawbacks including the side-effects in systemic administration. Effective procedures for the engineering of the protein by attaching useful additional functions are required to overcome the problem., Results: A procedure for the site-specific chemical conjugation of hFasLECD with a fluorochrome and functional proteins was devised using an inverse-electron-demand Diels-Alder reaction between trans-cyclooctene group and methyltetrazine group. The conjugations in the present study were attained by using much less molar excess amounts of the compounds to be attached as compared with the conventional chemical modification reactions using maleimide derivatives in the previous study. The isolated conjugates of hFasLECD with sulfo-Cy3, avidin and rabbit IgG Fab' domain presented the functional and the structural integrities of the attached molecules without impairing the specific binding activity toward human Fas receptor extracellular domain., Conclusions: The present study provided a new fundamental strategy for the production of the engineered hFasLECDs with additional beneficial functions, which will lead to the developments of the improved diagnostic systems and the effective treatment methods of serious diseases by using this protein as a component of novel molecular tools.

Structural characterization of transmembrane proteins in isotropic bicelles has become an increasingly popular application of solution NMR spectroscopy, as the fast-tumbling bicelles are membrane-like, yet can often yield spectral quality comparable to those of detergent micelles. While larger bicelles are closer to the true lipid bilayer, it remains unclear how large the bicelles need to be to allow accurate assessment of the protein transmembrane partition in the lipid bilayer. Here, we address the above question from the perspective of the protein residing in the bicelles, through systematic measurement of the protein chemical shift and transmembrane partition at different lipid/detergent ratios (q), ranging from 0.3 to 0.7, using the transmembrane domain of the human Fas receptor as model system. We found that the lipid environment of the bicelles, as reflected by the protein chemical shift, begins to be perturbed when q is reduced to below 0.6. We also implemented a solvent paramagnetic relaxation enhancement (PRE) approach for bicelles to show that the protein transmembrane partition in bicelles with q=0.5 and 0.7 are very similar, but at q=0.3 the solvent PRE profile is significantly different. Our data indicate that q values between 0.5 and 0.6 are a good compromise between high resolution NMR and closeness to the membrane environment, and allow accurate characterization of the protein position in the lipid bilayer., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

S-acylation is the covalent addition of a fatty acid, most generally palmitate onto cysteine residues of proteins through a labile thioester linkage. The death receptor CD95 is S-palmitoylated and this post-translational modification plays a crucial role on CD95 organization in cellular membranes and thus on CD95-mediated signaling. Here, we describe the nonradioactive detection of CD95 S-acylation by acyl-biotin exchange chemistry in which a biotin is substituted for the CD95-linked fatty acid. This sensitive technique, which depends on the ability of hydroxylamine to specifically cleave the thioester linkage between fatty acids and proteins, relies on three chemical steps: (1) blockage of free thiols of non-modified cysteine residues, (2) hydroxylamine-mediated cleavage of thioester-linked fatty acids to restore free thiols and (3) biotinylation of free thiols with a thiol reactive biotinylation agent. Resulting biotinylated proteins can be easily purified by an avidin capture and analyzed by SDS-PAGE and immunoblotting.

Phosphorylation of two tyrosines in the death domain of CD95 is a critical mechanism in determining the receptor's choices between cell death and survival signals. Recently, site-specific monoclonal antibodies against phosphorylated tyrosines of CD95 have been generated and used to successfully detect each phosphorylated death domain tyrosine of CD95 directly and separately by immunoblotting. Here we provide detailed protocols and useful tips for a successful site-specific detection of phosphorylated death domain tyrosine of CD95 following a protein separation by sizes (conventional SDS-PAGE) and by degrees of phosphorylation (phospho-protein affinity, mobility shift SDS-PAGE).

This work aimed at building a 3D model of trimeric apo CD95. By combining different molecular modeling approaches and experimental information, we have been able to obtain a consensual organization of the complex. Our strategy permitted the construction of a plausible trimer, and to sketch the interface between protomers. The final model will guide further experimental investigations and understanding of CD95 structure and functions.

A number of mathematical models of apoptosis generated recently allowed us to understand intrinsic mechanisms of life/death decisions in a cell. Nevertheless, the parameters for the mathematical models are often experimentally difficult to obtain and there is an emerging need for the development of efficient approaches for parameter estimation. In this study we suggest a new method for parameter estimation, which is based on stochastic simulations and can be used when the number of molecules in the system is small. Our approach comprised the following steps: we start from the selection of parameters that lead to a good ordinary differential equation (ODE) fit. We continued by carrying out stochastic simulations for each of these parameters. Comparing the correlation structure of these simulations with the data, we finally could identify the best parameter set. The method was applied for a model of CD95-induced apoptosis, the new best identified parameters fit well to the experimental data. The best parameter set allowed us to get new insights into CD95 apoptosis regulation and can be applied for the comprehensive analysis of other signaling networks. The modeling approach allowed us to get new insights into network regulation, in particular, to identify robustness in CD95 apoptotic response. Taken together, this new method provides valuable predictions and can be applied for the analysis of other signaling networks.

Fas (CD95, Apo-1, or TNFRSF6) is a prototypical apoptosis-inducing death receptor in the tumor necrosis factor receptor (TNFR) superfamily. While the extracellular domains of TNFRs form trimeric complexes with their ligands and the intracellular domains engage in higher-order oligomerization, the role of the transmembrane (TM) domains is unknown. We determined the NMR structures of mouse and human Fas TM domains in bicelles that mimic lipid bilayers. Surprisingly, these domains use proline motifs to create optimal packing in homotrimer assembly distinct from classical trimeric coiled-coils in solution. Cancer-associated and structure-based mutations in Fas TM disrupt trimerization in vitro and reduce apoptosis induction in vivo, indicating the essential role of intramembrane trimerization in receptor activity. Our data suggest that the structures represent the signaling-active conformation of Fas TM, which appears to be different from the pre-ligand conformation. Analysis of other TNFR sequences suggests proline-containing sequences as common motifs for receptor TM trimerization., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The Fas receptor is a representative death receptor, and the Fas-associated protein with death domain (FADD) is a crucial adapter protein needed to support the Fas receptor's activity. The Fas-FADD interactions constitute an important signaling pathway that ultimately induces apoptosis or programmed cell death in biological systems. The interactions responsible for this cell-death process are governed by the binding process of the Fas ligand to the Fas, followed by the caspase cascade activation. Using a computational approach, the present communication explores certain essential structural aspects of the Fas-FADD death domains and their interfacial interactions.

The extrinsic apoptotic pathway is initiated by binding of a Fas ligand to the ectodomain of the surface death receptor Fas protein. Subsequently, the intracellular death domain of Fas (FasDD) and that of the Fas-associated protein (FADD) interact to form the core of the death-inducing signaling complex (DISC), a crucial step for activation of caspases that induce cell death. Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells and specifically interacts with FasDD to regulate the apoptotic/survival signaling pathway. Inhibition of CaM activity in DISC stimulates apoptosis significantly. We have recently shown that CaM forms a ternary complex with FasDD (2:1 CaM:FasDD). However, the molecular mechanism by which CaM binds to two distinct FasDD motifs is not fully understood. Here, we employed mass spectrometry, nuclear magnetic resonance (NMR), biophysical, and biochemical methods to identify the binding regions of FasDD and provide a molecular basis for the role of CaM in Fas-mediated apoptosis. Proteolytic digestion and mass spectrometry data revealed that peptides spanning residues 209-239 (Fas-Pep1) and 251-288 (Fas-Pep2) constitute the two CaM-binding regions of FasDD. To determine the molecular mechanism of interaction, we have characterized the binding of recombinant/synthetic Fas-Pep1 and Fas-Pep2 peptides with CaM. Our data show that both peptides engage the N- and C-terminal lobes of CaM simultaneously. Binding of Fas-Pep1 to CaM is entropically driven while that of Fas-Pep2 to CaM is enthalpically driven, indicating that a combination of electrostatic and hydrophobic forces contribute to the stabilization of the FasDD-CaM complex. Our data suggest that because Fas-Pep1 and Fas-Pep2 are involved in extensive intermolecular contacts with the death domain of FADD, binding of CaM to these regions may hinder its ability to bind to FADD, thus greatly inhibiting the initiation of apoptotic signaling pathway.

This protocol describes activation, isolation, and analysis of the CD95 (APO-1/Fas) death-inducing signaling complex (DISC) using affinity purification. Activation is achieved using a biotin-labeled anti-CD95 antibody and the native DISC complex is captured using streptavidin beads. This approach minimizes both the number of steps involved and any potential nonspecific interactions or cross-reactivity of antibodies commonly seen in immunoprecipitations using unlabeled antibodies and protein A/G beads. Composition of the isolated complex is analyzed via western blot to identify known DISC components, and dimerization-induced autocatalytic processing of procaspase-8 at the DISC can be confirmed by detection of caspase-8 cleavage products. The potential for DISC-associated caspase-8 to activate the caspase cascade can be determined by measuring caspase-8-dependent cleavage of the fluorigenic substrate Ac-IETD.AFC, or by performing a bioassay using exogenous protein substrates., (© 2015 Cold Spring Harbor Laboratory Press.)

Doxorubicin (DOX) is one of the preferred drugs for treating breast and liver cancers. However, its clinical application is limited due to severe side effects and the accompanying drug resistance. In this context, we investigated the effect on therapeutic efficacy of DOX by cholesterol depleting agent methyl-β-cyclodextrin (MCD), and explored the involvement of p53. MCD sensitizes MCF-7 and Hepa1-6 cells to DOX, Combination of MCD and marginal dose of DOX reduces the cell viability, and promoted apoptosis through induction of pro-apoptotic protein, Bax, activation of caspase-8 and caspase-7, down regulation of anti-apoptotic protein Bcl-2 and finally promoting PARP cleavage. Mechanistically, sensitization to DOX by MCD was due to the induction of FasR/FasL pathway through p53 activation. Furthermore, inhibition of p53 by pharmacological inhibitor pifithrin-α (PFT-α) or its specific siRNA attenuated p53 function and down-regulated FasR/FasL, thereby preventing cell death. Animal experiments were performed using C57BL/6J mouse isografted with Hepa1-6 cells. Tumor growth was retarded and survival increased in mice administered MCD together with DOX to as compared to either agent alone. Collectively, these results suggest that MCD enhances the sensitivity to DOX for which wild type p53 is an important determinant.

CD95 (Apo1/Fas) has been originally identified as the target of cell death-inducing antibodies. The recognition of CD95 as an apoptosis-triggering receptor represents one of the early milestones in the apoptosis field. Moreover, the research on CD95-induced cell death fostered various other discoveries of broad and general relevance in cell biology, for example, the identification of caspase 8 as the initiator caspase of the extrinsic apoptosis pathway. Activation of CD95-associated intracellular signaling pathways is not a simple consequence of ligand binding but is the fine-tuned result of a complex interplay of various molecular mechanisms that eventually determine the strength and quality of the CD95 response. There is growing evidence that different forms of CD95 stimulation trigger the assembly of CD95 signaling complexes of distinct composition. Moreover, the formation of signaling competent CD95 complexes is a multistep process and the subject of regulation by various cellular cues. This review addresses the relevance of the molecular nature of the CD95-stimulating agonist for the quality of the CD95 response and discusses the importance of modification, clustering, internalization, and lipid raft and actin association of CD95 for CD95 activity.

Fas apoptosis inhibitory molecule (FAIM) is a highly conserved anti-apoptotic protein which plays important roles in cells. There are two isoforms of FAIM, of which the short isoform FAIM-S is broadly expressed in all tissues, whereas the long isoform FAIM-L is exclusively expressed in the nervous system. No structure of human FAIM has been reported to date and the detailed molecular mechanisms underlying the anti-apoptotic function of FAIM remain unknown. Here, the crystal structure of the human FAIM-S N-terminal domain (NTD) and the NMR solution structure of the human FAIM-S C-terminal domain (CTD) were determined. The structures revealed that the NTD and CTD adopt a similar protein fold containing eight antiparallel β-strands which form two sheets. Both structural and biochemical analyses implied that the NTD exists as a dimer and the CTD as a monomer and that they can interact with each other. Several critical residues were identified to be involved in this interaction. Moreover, mutations of these critical residues also interfered in the anti-apoptotic activity of FAIM-S. Thus, the structural and functional data presented here will provide insight into the anti-apoptotic mechanism of FAIM-S.

The Fas death receptor-activated death-inducing signaling complex (DISC) regulates apoptosis in many normal and cancer cells. Qualitative biochemical experiments demonstrate that calmodulin (CaM) binds to the death domain of Fas. The interaction between CaM and Fas regulates Fas-mediated DISC formation. A quantitative understanding of the interaction between CaM and Fas is important for the optimal design of antagonists for CaM or Fas to regulate the CaM-Fas interaction, thus modulating Fas-mediated DISC formation and apoptosis. The V254N mutation of the Fas death domain (Fas DD) is analogous to an identified mutant allele of Fas in lpr-cg mice that have a deficiency in Fas-mediated apoptosis. In this study, the interactions of CaM with the Fas DD wild type (Fas DD WT) and with the Fas DD V254N mutant were characterized using isothermal titration calorimetry (ITC), circular dichroism spectroscopy (CD), and molecular dynamics (MD) simulations. ITC results reveal an endothermic binding characteristic and an entropy-driven interaction of CaM with Fas DD WT or with Fas DD V254N. The Fas DD V254N mutation decreased the association constant (Ka) for CaM-Fas DD binding from (1.79 ± 0.20) × 10(6) to (0.88 ± 0.14) × 10(6) M(-1) and slightly increased a standard state Gibbs free energy (ΔG°) for CaM-Fas DD binding from -8.87 ± 0.07 to -8.43 ± 0.10 kcal/mol. CD secondary structure analysis and MD simulation results did not show significant secondary structural changes of the Fas DD caused by the V254N mutation. The conformational and dynamical motion analyses, the analyses of hydrogen bond formation within the CaM binding region, the contact numbers of each residue, and the electrostatic potential for the CaM binding region based on MD simulations demonstrated changes caused by the Fas DD V254N mutation. These changes caused by the Fas DD V254N mutation could affect the van der Waals interactions and electrostatic interactions between CaM and Fas DD, thereby affecting CaM-Fas DD interactions. Results from this study characterize CaM-Fas DD interactions in a quantitative way, providing structural and thermodynamic evidence of the role of the Fas DD V254N mutation in the CaM-Fas DD interaction. Furthermore, the results could help to identify novel strategies for regulating CaM-Fas DD interactions and Fas DD conformation and thus to modulate Fas-mediated DISC formation and thus Fas-mediated apoptosis.

TGFBI, a transforming growth factor β-induced extracellular matrix protein, circulates at a level of ~300ng/ml in humans and modulates several integrin-mediated cellular functions. The protein contains an N-terminal EMI domain, four consecutive FAS1 domains, and the RGD motif. Each FAS1 domain and the RGD motif have been known to interact with avb3 integrin. Here, we found that the binding affinity (Kd) of TGFBI for αvβ3 integrin was approximately 3.8×10(-8)M, a value ~2300-fold higher than that of a single FAS1 domain, and demonstrated that this greater affinity was due to the cooperative action of the four FAS1 domains and the RGD motif. Moreover, TGFBI exhibited more potent anti-angiogenic and anti-tumorigenic activities, even at a 100-fold lower molar dose than the reported effective dose of the FAS1 domain. Finally, our data showed that TGFBI specifically targeted the tumor vasculature and accumulated at the tumor site. Collectively, our results support the theory that TGFBI acts as a potent endogenous anti-tumor and anti-angiogenic molecule by targeting αvβ3 integrin, and highlights the importance of physiological circulating TGFBI levels in inhibiting tumor growth., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Fas binding to Fas-associated death domain (FADD) activates FADD-caspase-8 binding to form death-inducing signaling complex (DISC) that triggers apoptosis. The Fas-Fas association exists primarily as dimer in the Fas-FADD complex, and the Fas-FADD tetramer complexes have the tendency to form higher order oligomer. The importance of the oligomerized Fas-FADD complex in DISC formation has been confirmed. This study sought to provide structural insight for the roles of Fas death domain (Fas DD) binding to FADD and the oligomerization of Fas DD-FADD complex in activating FADD-procaspase-8 binding. Results show Fas DD binding to FADD stabilized the FADD conformation, including the increased stability of the critical residues in FADD death effector domain (FADD DED) for FADD-procaspase-8 binding. Fas DD binding to FADD resulted in the decreased degree of both correlated and anticorrelated motion of the residues in FADD and caused the reversed correlated motion between FADD DED and FADD death domain (FADD DD). The exposure of procaspase-8 binding residues in FADD that allows FADD to interact with procaspase-8 was observed with Fas DD binding to FADD. We also observed different degrees of conformational and motion changes of FADD in the Fas DD-FADD complex with different degrees of oligomerization. The increased conformational stability and the decreased degree of correlated motion of the residues in FADD in Fas DD-FADD tetramer complex were observed compared to those in Fas DD-FADD dimer complex. This study provides structural evidence for the roles of Fas DD binding to FADD and the oligomerization degree of Fas DD-FADD complex in DISC formation to signal apoptosis., (Copyright © 2012 Wiley Periodicals, Inc.)

We recently demonstrated that S-glutathionylation of the death receptor Fas (Fas-SSG) amplifies apoptosis (V. Anathy et al., J. Cell Biol. 184:241-252, 2009). In the present study, we demonstrate that distinct pools of Fas exist in cells. Upon ligation of surface Fas, a separate pool of latent Fas in the endoplasmic reticulum (ER) underwent rapid oxidative processing characterized by the loss of free sulfhydryl content (Fas-SH) and resultant increases in S-glutathionylation of Cys294, leading to increases of surface Fas. Stimulation with FasL rapidly induced associations of Fas with ERp57 and glutathione S-transferase π (GSTP), a protein disulfide isomerase and catalyst of S-glutathionylation, respectively, in the ER. Knockdown or inhibition of ERp57 and GSTP1 substantially decreased FasL-induced oxidative processing and S-glutathionylation of Fas, resulting in decreased death-inducing signaling complex formation and caspase activity and enhanced survival. Bleomycin-induced pulmonary fibrosis was accompanied by increased interactions between Fas-ERp57-GSTP1 and S-glutathionylation of Fas. Importantly, fibrosis was largely prevented following short interfering RNA-mediated ablation of ERp57 and GSTP. Collectively, these findings illuminate a regulatory switch, a ligand-initiated oxidative processing of latent Fas, that controls the strength of apoptosis.

The extracellular domains of death ligands and those of death receptors are closely related to many serious human diseases through the initiation of apoptosis. Recombinant production of the extracellular domains has been investigated due to demand for a large amount of purified samples, which are a prerequisite for their biochemical characterization and constitute the fundamentals of medical applications. This review focuses on the recombinant production of extracellular domains of the major members of death ligand and death receptor families using non-mammalian expression systems with an emphasis on Fas ligand and Fas receptor. In contrast to the efficient production of the functional extracellular domains of TRAIL, TNFα and LTα by intracellular expression systems using Escherichia coli or Pichia pastoris, that of Fas ligand requires the secretory expression systems using P. pastoris or Dictyostelium discoideum, and the productivity in P. pastoris was largely dependent on tag sequence, potential N-glycosylation site and expressed protein region. On the other hand, the exploitation of insect cell systems is generally useful for the preparation of functional extracellular domains of death receptors containing many disulfide bridges in the absence of extended secondary structure, and a Bombyx mori larvae secretion system presented a superior productivity for human Fas receptor extracellular domain. Based on the results obtained so far, further efforts should be devoted to the artificial control of death ligand - death receptor interactions in order to make a contribution to medicine, represented by the development of novel biopharmaceuticals.

The CD95 (Fas/APO-1) death-inducing signaling complex (DISC) is essential for the initiation of CD95-mediated apoptotic and nonapoptotic responses. The CD95 DISC comprises CD95, FADD, procaspase-8, procaspase-10, and c-FLIP proteins. Procaspase-8 and procaspase-10 are activated at the DISC, leading to the formation of active caspases and apoptosis initiation. In this study we analyzed the stoichiometry of the CD95 DISC. Using quantitative western blots, mass spectrometry, and mathematical modeling, we reveal that the amount of DED proteins procaspase-8/procaspase-10 and c-FLIP at the DISC exceeds that of FADD by several-fold. Furthermore, our findings imply that procaspase-8, procaspase-10, and c-FLIP could form DED chains at the DISC, enabling the formation of dimers and efficient activation of caspase-8. Taken together, our findings provide an enhanced understanding of caspase-8 activation and initiation of apoptosis at the DISC., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The capability of soluble CD95L trimers to trigger CD95-associated signaling pathways is drastically increased by oligomerization. The latter can be achieved, for example, by antibodies recognizing a N-terminal epitope tag in recombinant CD95L variants or by genetic engineering-enforced formation of hexamers. Using highly sensitive and accurate binding studies with recombinant CD95L variants equipped with a Gaussia princeps luciferase reporter domain, we found that oligomerization of CD95L has no major effect on CD95 occupancy. This indicates that the higher activity of oligomerized CD95L trimers is not related to an avidity-related increase in apparent affinity and points instead to a crucial role of aggregation of initially formed trimeric CD95L-CD95 complexes in CD95 activation. Furthermore, binding of soluble CD95L trimers was found to be insufficient to increase the association of CD95 with the lipid raft-containing membrane fraction. However, when Gaussia princeps luciferase-CD95L trimers were used as tracers to "mark" inactive CD95 molecules, increased association of these inactive receptors was observed upon activation of the remaining CD95 molecules by help of highly active hexameric Fc-CD95L or membrane CD95L. Moreover, in cells expressing endogenous CD95 and chimeric CD40-CD95 receptors, triggering of CD95 signaling via endogenous CD95 resulted in co-translocation of CD40-CD95 to the lipid raft fraction, whereas vice versa activation of CD95-associated pathways with Fc-CD40L via CD40-CD95 resulted in co-translocation of endogenous CD95. In sum, this shows that signaling-active CD95 molecules not only enhance their own association with the lipid raft-containing membrane fraction but also those of inactive CD95 molecules.

We report an unexpected finding of common structural principles in two unrelated signaling systems: the FAS death domain transformation that initializes the extrinsic apoptotic pathway and signaling by calmodulin bending. The location and design of the hinge is postulated to be a general principle for creating potential signaling event. We suggest that already existing tool can predict the existence of such a hinge and formulate the hypothesis that the internal instabilities designed into the hinge sequences are necessary devices in effective signaling events., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Apoptosis has emerged as a fundamental process important in tissue homeostasis, immune response, and during development. CD95 (also known as Fas), a member of the tumor necrosis factor receptor (TNF-R) superfamily, has been initially cloned as a death receptor. Its cognate ligand, CD95L, is mainly found at the plasma membrane of activated T-lymphocytes and natural killer cells where it contributes to the elimination of transformed and infected cells. According to its implication in the immune homeostasis and immune surveillance, and since several malignant cells of various histological origins exhibit loss-of-function mutations, which cause resistance towards the CD95-mediated apoptotic signal, CD95 has been classified as a tumor suppressor gene. Nevertheless, this assumption has been recently challenged, as in certain pathophysiological contexts, CD95 engagement transmits non-apoptotic signals that promote inflammation, carcinogenesis or liver/peripheral nerve regeneration. The focus of this review is to discuss these apparent contradictions of the known function(s) of CD95.

Purpose: Autoimmune lymphoproliferative syndrome is a disorder of lymphocyte apoptosis. Although FAS molecules bearing mutations in the signal-transducing intracellular death domain exhibit dominant-negative interference with FAS-mediated apoptosis, mechanisms for pathology of non-death domain FAS mutations causing autoimmune lymphoproliferative syndrome are poorly defined., Methods: RNA stability, protein expression, ligand binding, and ability to transmit apoptosis signals by anti-FAS antibody or FAS ligand were determined for a cohort of 39 patients with non-death domain autoimmune lymphoproliferative syndrome. Correlations between mutation type and disease penetrance were established in mutation-positive family members., Results: Frameshifts or transcriptional stop mutations before exon 7 resulted in messenger RNA haploinsufficiency, whereas an amino-terminal signal sequence mutation and certain intracellular truncations prevented cell surface localization of FAS. All resulted in decreased FAS localization, inability to bind FAS ligand, and reduced FAS ligand-induced apoptosis. Extracellular missense mutations and in-frame deletions expressed defective FAS protein, failed to bind FAS ligand, and exhibited dominant-negative interference with FAS-mediated apoptosis. Mutation-positive relatives with haploinsufficient or extracellular mutations had lower penetrance of autoimmune lymphoproliferative syndrome clinical phenotypes than did relatives with death domain mutations., Conclusion: We have defined molecular mechanisms by which non-death domain FAS mutations result in reduced lymphocyte apoptosis, established a hierarchy of genotype-phenotype correlation among mutation-positive relatives of patients with autoimmune lymphoproliferative syndrome, and demonstrated that FAS haploinsufficiency can lead to autoimmune lymphoproliferative syndrome.

The CD95/CD95L receptor-ligand system is mainly recognised in the induction of apoptosis. However, it has also been shown that CD95L is over-expressed in many cancer types where it modulates immune-evasion and together with its receptor CD95 promotes tumour growth. Here, we show that CD95 surface modification of relatively large microparticles >0.5 μm in diameter, including those made from biodegradable polylactic-co-glycolic acid (PLGA), enhances intracellular uptake by a range of CD95L expressing cells in a process akin to phagocytosis. Using this approach we describe the intracellular uptake of microparticles and agent delivery in neurons, medulloblastoma, breast and ovarian cancer cells in vitro. CD95 modified paclitaxel-loaded PLGA microparticles are shown to be significantly more effective compared to conventional paclitaxel therapy (Taxol) at the same dose in subcutaneous medulloblastoma (∗∗∗P < 0.0001) and orthotopic ovarian cancer xenograft models where a >65-fold reduction in tumour bioluminescence was measured after treatment (∗P = 0.012). This drug delivery platform represents a new way of manipulating the normally advantageous tumour CD95L over-expression towards a therapeutic strategy. CD95 functionalised drug carriers could contribute to the improved function of cytotoxics in cancer, potentially increasing drug targeting and efficacy whilst reducing toxicity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

To comprehend the molecular processes that lead to the Fas death receptor clustering in lipid rafts, a 21-mer peptide corresponding to its single transmembrane domain (TMD) was reconstituted into mammalian raft model membranes composed of an unsaturated glycerophospholipid, sphingomyelin, and cholesterol. The peptide membrane lateral organization and dynamics, and its influence on membrane properties, were studied by steady-state and time-resolved fluorescence techniques and by attenuated total reflection Fourier transformed infrared spectroscopy. Our results show that Fas TMD is preferentially localized in liquid-disordered membrane regions and undergoes a strong reorganization as the membrane composition is changed toward the liquid-ordered phase. This results from the strong hydrophobic mismatch between the length of the peptide hydrophobic stretch and the hydrophobic thickness of liquid-ordered membranes. The stability of nonclustered Fas TMD in liquid-disordered domains suggests that its sequence may have a protective function against nonligand-induced Fas clustering in lipid rafts. It has been reported that ceramide induces Fas oligomerization in lipid rafts. Here, it is shown that neither Fas TMD membrane organization nor its conformation is affected by ceramide. These results are discussed within the framework of Fas membrane signaling events., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The death receptors CD95, TRAILR1 and TRAILR2 induce cell death in many types of tumor cells. Activation of these receptors has received considerable interest due to its potential use in cancer therapy. In particular the observation that most primary cells are not or only barely TRAIL-sensitive resulted in the development of targeted therapy concepts that base on activation of the TRAIL death receptors by recombinant TRAIL or agonistic antibodies. Indeed, a variety of preclinical studies and several phase I and II clinical trials show that activation of TRAIL death receptors effectively induces apoptosis in cancer cells in vivo without therapy-limiting toxicity on normal cells. Primary tumor cells are often sparsely sensitive for TRAIL death receptor-mediated apoptosis or acquire resistance during therapy. Sensitization/resensitization of tumor cells by chemotherapeutic drugs or radiation can therefore be necessary for TRAIL-based therapies, but this involves the danger of triggering side effects related to the breakage of apoptosis resistance of non-transformed cells. Thus, there is a foreseeable need to develop optimized combination therapies or to locally restrict TRAIL receptor activation to fully exploit the antitumoral potential of TRAIL death receptors in the clinic. Although the high sensitivity of hepatocytes for CD95-mediated apoptosis prohibits therapies resulting in systemic activation of CD95, several studies have shown that this limitation can be overcome by ex vivo treatment regimes or by CD95 activating agonists with cell type-specific activity. This patent review is focused on the death receptor agonists currently under consideration in clinical trials, but also addresses the hurdles that have to be cleared to broaden and to improve the applicability of the currently used clinical concepts related to death receptor activation.

Death receptors (DRs) induce apoptosis but also stimulate proinflammatory "non-apoptotic" signaling (e.g. NF-κB and mitogen-activated protein kinase (MAPK) activation) and inhibit distinct steps of DR-activated maturation of procaspase-8. To examine whether isoforms of cellular FLIP (cFLIP) or its cleavage products differentially regulate DR signaling, we established HaCaT cells expressing cFLIP(S), cFLIP(L), or mutants of cFLIP(L) (cFLIP(D376N) and cFLIP(p43)). cFLIP variants blocked TRAIL- and CD95L-induced apoptosis, but the cleavage pattern of caspase-8 in the death inducing signaling complex was different: cFLIP(L) induced processing of caspase-8 to the p43/41 fragments irrespective of cFLIP cleavage. cFLIP(S) or cFLIP(p43) blocked procaspase-8 cleavage. Analyzing non-apoptotic signaling pathways, we found that TRAIL and CD95L activate JNK and p38 within 15 min. cFLIP variants and different caspase inhibitors blocked late death ligand-induced JNK or p38 MAPK activation suggesting that these responses are secondary to cell death. cFLIP isoforms/mutants also blocked death ligand-mediated gene induction of CXCL-8 (IL-8). Knockdown of caspase-8 fully suppressed apoptotic and non-apoptotic signaling. Knockdown of cFLIP isoforms in primary human keratinocytes enhanced CD95L- and TRAIL-induced NF-κB activation, and JNK and p38 activation, underscoring the regulatory role of cFLIP for these DR-mediated signals. Whereas the presence of caspase-8 is critical for apoptotic and non-apoptotic signaling, cFLIP isoforms are potent inhibitors of TRAIL- and CD95L-induced apoptosis, NF-κB activation, and the late JNK and p38 MAPK activation. cFLIP-mediated inhibition of CD95 and TRAIL DR could be of crucial importance during keratinocyte skin carcinogenesis and for the activation of innate and/or adaptive immune responses triggered by DR activation in the skin.

Protein modifications of death receptor pathways play a central role in the regulation of apoptosis. It has been demonstrated that O-glycosylation of TRAIL-receptor (R) is essential for sensitivity and resistance towards TRAIL-mediated apoptosis. In this study we ask whether and how glycosylation of CD95 (Fas/APO-1), another death receptor, influences DISC formation and procaspase-8 activation at the CD95 DISC and thereby the onset of apoptosis. We concentrated on N-glycostructure since O-glycosylation of CD95 was not found. We applied different approaches to analyze the role of CD95 N-glycosylation on the signal transduction: in silico modeling of CD95 DISC, generation of CD95 glycosylation mutants (at N136 and N118), modulation of N-glycosylation by deoxymannojirimycin (DMM) and sialidase from Vibrio cholerae (VCN). We demonstrate that N-deglycosylation of CD95 does not block DISC formation and results only in the reduction of the procaspase-8 activation at the DISC. These findings are important for the better understanding of CD95 apoptosis regulation and reveal differences between apoptotic signaling pathways of the TRAIL and CD95 systems.

The Fas/CD95 receptor mediates apoptosis but is also capable of triggering nonapoptotic signals. However, the mechanisms that selectively regulate these opposing effects are not yet fully understood. Here we demonstrate that the activation of Fas or stimulation with lysophosphatidic acid (LPA) induces cytoskeletal reorganization, leading to the association of Fas with actin stress fibers and the adaptor protein TRIP6. TRIP6 binds to the cytoplasmic juxtamembrane domain of Fas and interferes with the recruitment of FADD to Fas. Furthermore, through physical interactions with NF-κB p65, TRIP6 regulates nuclear translocation and the activation of NF-κB upon Fas activation or LPA stimulation. As a result, TRIP6 antagonizes Fas-induced apoptosis and further enhances the antiapoptotic effect of LPA in cells that express high levels of TRIP6. On the other hand, TRIP6 promotes Fas-mediated cell migration in apoptosis-resistant glioma cells. This effect is regulated via the Src-dependent phosphorylation of TRIP6 at Tyr-55. As TRIP6 is overexpressed in glioblastomas, this may have a significant impact on enhanced NF-κB activity, resistance to apoptosis, and Fas-mediated cell invasion in glioblastomas.

The death-inducing signaling complex (DISC) formed by the death receptor Fas, the adaptor protein FADD and caspase-8 mediates the extrinsic apoptotic program. Mutations in Fas that disrupt the DISC cause autoimmune lymphoproliferative syndrome (ALPS). Here we show that the Fas-FADD death domain (DD) complex forms an asymmetric oligomeric structure composed of 5-7 Fas DD and 5 FADD DD, whose interfaces harbor ALPS-associated mutations. Structure-based mutations disrupt the Fas-FADD interaction in vitro and in living cells; the severity of a mutation correlates with the number of occurrences of a particular interaction in the structure. The highly oligomeric structure explains the requirement for hexameric or membrane-bound FasL in Fas signaling. It also predicts strong dominant negative effects from Fas mutations, which are confirmed by signaling assays. The structure optimally positions the FADD death effector domain (DED) to interact with the caspase-8 DED for caspase recruitment and higher-order aggregation.

We have addressed complex formation between the death domain (DD) of the death receptor CD95 (Fas/APO-1) with the DD of immediate adaptor protein FADD using nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and size-exclusion chromatography with in-line light scattering. We find complexation to be independent of the C-terminal 12 residues of CD95 and insensitive to mutation of residues that engage in the high-order clustering of CD95-DD molecules in a recently reported crystal structure obtained at pH 4. Differential NMR linewidths indicate that the C-terminal region of the CD95 chains remains in a disordered state and (13)C-methyl TROSY data are consistent with a lack of high degree of symmetry for the complex. The overall molecular mass of the complex is inconsistent with that in the crystal structure, and the complex dissociates at pH 4. We discuss these findings using sequence analysis of CD95 orthologs and the effect of FADD mutations on the interaction with CD95., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

In a previous investigation, we demonstrated that after CD95/Fas triggering, raft-associated GD3 ganglioside, normally localized at the plasma membrane of T cells, can be detected in mitochondria, where they contribute to apoptogenic events. Here, we show the association of the glycosphingolipid GD3 with microtubular cytoskeleton at very early time points following Fas ligation. This was assessed by different methodological approaches, including fluorescence resonance energy transfer, immunoelectron microscopy, and coimmunoprecipitation. Furthermore, docking analysis also showed that GD3 has a high affinity for the pore formed by 4 tubulin heterodimers (type I pore), thus suggesting a possible direct interaction between tubulin and GD3. Finally, time-course analyses indicated that the relocalization of GD3 to the mitochondria was time related with the alterations of the mitochondrial membrane potential. Hence, microtubules could act as tracks for ganglioside redistribution following apoptotic stimulation, possibly contributing to the mitochondrial alterations leading to cell death.

Death domain complexes are key protein arrangements in the regulation of various cellular signaling events. One of the most prominent death domain complexes first described in the initiation of apoptosis is formed by the transmembrane receptor Fas, the cytosolic adaptor protein FADD, and caspase-8 and is referred to as the Fas/FADD/caspase-8 death inducing signaling complex (DISC). The recent structure of the Fas/FADD death domain complex reveals how formation of this signaling platform can be stringently regulated utilizing only Fas receptor clustering to form a death domain network. This work reveals that an opening mechanism of Fas is needed to expose binding sites for the FADD death domain and sets the stage for a conditional interaction, which is characterized by weak interactions adapted for a regulatory function. The overall crystal structure reveals a tetrameric arrangement of four primary Fas/FADD complexes. Intriguingly all contacts mediating the tetramer are solely provided through Fas/Fas interactions and are entirely dependent on the open form. These findings are instrumental in depicting a mechanism for DISC regulation where Fas receptor clustering leads to the stabilization of the open Fas death domains which are then capable of binding FADD in a weak interaction. At the same time this mechanism ensures that in the absence of a sufficient stimulus no interaction between Fas and FADD is possible. Therefore the conformation dependent, conditional Fas/FADD death domain interaction represents the regulatory element per se. This interaction contrasts the classic constitutive interactions of adaptor domains, which cannot provide regulatory function themselves. This model portrays how sole death domains are able to mediate signaling upon receptor clustering in the complete absence of enzyme activity.

The death-inducing signaling complex (DISC) is critical for initiation of death-receptor-mediated apoptosis; however, paradoxically, CD95 also signals for cell survival. Here, we reconstitute a functional DISC using only purified CD95, FADD, and procaspase-8 and unveil a two-step activation mechanism involving both dimerization and proteolytic cleavage of procaspase-8 that is obligatory for death-receptor-induced apoptosis. Initially, dimerization yields active procaspase-8 with a very restricted substrate repertoire, limited to itself or c-FLIP. Proteolytic cleavage is then required to fully activate caspase-8, thereby permitting DISC-mediated cleavage of the critical exogenous apoptotic substrates, caspase-3 and Bid. This switch in catalytic activity and substrate range is a key determinant of DISC signaling, as cellular expression of noncleavable procaspase-8 mutants, which undergo DISC-mediated oligomerization, but not cleavage, fails to initiate CD95-induced apoptosis. Thus, using the reconstituted DISC, we have delineated a crucial two-step activation mechanism whereby activated death receptor complexes can trigger death or survival.

The death inducing signalling complex (DISC) formed by Fas receptor, FADD (Fas-associated death domain protein) and caspase 8 is a pivotal trigger of apoptosis. The Fas-FADD DISC represents a receptor platform, which once assembled initiates the induction of programmed cell death. A highly oligomeric network of homotypic protein interactions comprised of the death domains of Fas and FADD is at the centre of DISC formation. Thus, characterizing the mechanistic basis for the Fas-FADD interaction is crucial for understanding DISC signalling but has remained unclear largely because of a lack of structural data. We have successfully formed and isolated the human Fas-FADD death domain complex and report the 2.7 A crystal structure. The complex shows a tetrameric arrangement of four FADD death domains bound to four Fas death domains. We show that an opening of the Fas death domain exposes the FADD binding site and simultaneously generates a Fas-Fas bridge. The result is a regulatory Fas-FADD complex bridge governed by weak protein-protein interactions revealing a model where the complex itself functions as a mechanistic switch. This switch prevents accidental DISC assembly, yet allows for highly processive DISC formation and clustering upon a sufficient stimulus. In addition to depicting a previously unknown mode of death domain interactions, these results further uncover a mechanism for receptor signalling solely by oligomerization and clustering events.

Previous studies have demonstrated a calcium-dependent interaction of calmodulin (CaM) and Fas that is regulated during Fas-induced apoptosis in several cell lines, including cholangiocarcinoma, Jurkat cells, and osteoclasts. The binding of CaM and Fas has been identified on residues 231-254 of Fas; the V254N point mutation decreases the CaM/Fas binding, and the C-terminal deletion mutation increases the CaM/Fas binding. Recent studies have shown that CaM is recruited into the Fas-mediated death-inducing signaling complex (DISC) in a calcium-dependent manner. However, the molecular mechanisms whereby Fas mutations and CaM/Fas binding might regulate Fas-mediated DISC formation are unknown. In this study we investigated the binding thermodynamics and conformation of the CaM/Fas complexes with combined explicit solvent molecular-dynamics simulations and implicit solvent binding free-energy calculations. The binding free-energy analysis demonstrated that the Fas V254N point mutation reduced its binding affinity with CaM. In contrast, the Fas mutant with the deletion of the 15 amino acid at the C-terminus increased its binding to CaM. These observations are consistent with previous findings from biochemical studies. Conformational analyses further showed that the Fas V254N mutation resulted in an unstable conformation, whereas the C-terminal deletion mutation stabilized the Fas conformation, and both mutations resulted in changes of the degree of correlation between the motions of the residues in Fas. Analysis of the CaM/Fas complex revealed that CaM/Fas binding stabilized the conformation of both CaM and Fas and changed the degree of correlated motion of the residues of CaM and Fas. The results presented here provide structural evidence for the roles of Fas mutations and CaM/Fas binding in Fas-induced DISC formation. Understanding the molecular mechanisms of CaM/Fas binding in Fas-mediated DISC formation should provide important insights into the function of Fas mutations and CaM in regulating Fas-mediated apoptosis.

Selective compartmentalization and internalization have been shown as a means for regulating specific signals of cell surface receptors to correspond to cellular requirements and conditions. Here, we present a conserved extracellular glycosphingolipid-binding motif of Fas as one of the regulatory elements in the selection of its internalization route and consequently the signals transmitted upon ligand binding. This motif is required for clathrin-mediated internalization of Fas, which allows the transduction of its cell death signal. The loss of function of the motif drives the activated receptor to an alternative internalization route that is independent of clathrin and cholesterol-dependent rafts but dependent on ezrin, and thereby extinguishing its cell death signal while promoting its non-death functions. Through biochemical, biophysical, and genetic approaches, we present a protein/lipid-based mechanism as a key to the versatility of the signal transduction by the multifunctional Fas receptor-ligand system.

U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor 65 kDa (U2AF65) is an essential splicing factor in the recognition of the pre-mRNA 3' splice sites during the assembly of the splicing commitment complex. We report here that U2AF65 is proteolyzed during apoptosis. This cleavage is group I or III caspase dependent in a noncanonical single site localized around the aspartic acid(128) residue and leads to the separation of the N- and C-terminal parts of U2AF65. The U2AF65 N-terminal fragment mainly accumulates in the nucleus within nuclear bodies (nucleoli-like pattern) and to a much lesser extent in the cytoplasm, whereas the C-terminal fragment is found in the cytoplasm, even in localization studies on apoptosis induction. From a functional viewpoint, the N-terminal fragment promotes Fas exon 6 skipping from a reporter minigene, by acting as a dominant-negative version of U2AF65, whereas the C-terminal fragment has no significant effect. The dominant-negative behavior of the U2AF65 N-terminal fragment can be reverted by U2AF35 overexpression. Interestingly, U2AF65 proteolysis in Jurkat cells on induction of early apoptosis correlates with the down-regulation of endogenous Fas exon 6 inclusion. Thus, these results support a functional link among apoptosis induction, U2AF65 cleavage, and the regulation of Fas alternative splicing.