Synthesis of N-Glycosylated Soluble Fas Ligand.

Controlled cell death is essential for the regulation of the immune system and plays a role in pathogen defense. It is often altered in pathogenic conditions such as cancer, viral infections and autoimmune diseases. The Fas receptor and its corresponding membrane-bound ligand (FasL) are part of the extrinsic apoptosis pathway activated in these cases. A soluble form of FasL (sFasL), produced by ectodomain shedding, displays a diverse but still elusive set of non-apoptotic functions and sometimes even serves as a pro-survival factor. To gather more knowledge about the characteristics of this protein and the impact N-glycosylations may have, access to homogeneous posttranslationally modified variants of sFasL is needed. Therefore, we developed a flexible strategy to obtain such homogeneously N-glycosylated variants of sFasL by applying chemical protein synthesis. This strategy can be flexibly combined with enzymatic methods to introduce more complex, site selective glycosylations.

Synthetic receptor platform to identify loss-of-function single nucleotide variants and designed mutants in the death receptor Fas/CD95.

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.

Neutrophil elastase selectively kills cancer cells and attenuates tumorigenesis.

Cancer cell genetic variability and similarity to host cells have stymied development of broad anti-cancer therapeutics. Our innate immune system evolved to clear genetically diverse pathogens and limit host toxicity; however, whether/how innate immunity can produce similar effects in cancer is unknown. Here, we show that human, but not murine, neutrophils release catalytically active neutrophil elastase (ELANE) to kill many cancer cell types while sparing non-cancer cells. ELANE proteolytically liberates the CD95 death domain, which interacts with histone H1 isoforms to selectively eradicate cancer cells. ELANE attenuates primary tumor growth and produces a CD8+T cell-mediated abscopal effect to attack distant metastases. Porcine pancreatic elastase (ELANE homolog) resists tumor-derived protease inhibitors and exhibits markedly improved therapeutic efficacy. Altogether, our studies suggest that ELANE kills genetically diverse cancer cells with minimal toxicity to non-cancer cells, raising the possibility of developing it as a broad anti-cancer therapy.

Fas/FasL of pacific cod mediated apoptosis.

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.

Lipid Raft Isolation by Sucrose Gradient Centrifugation and Visualization of Raft-Located Proteins by Fluorescence Microscopy: The Use of Combined Techniques to Assess Fas/CD95 Location in Rafts During Apoptosis Triggering.

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.

Human UDP-galactose 4'-epimerase (GALE) is required for cell-surface glycome structure and function.

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.

Grape Seed Procyanidin B2 Protects Porcine Ovarian Granulosa Cells against Oxidative Stress-Induced Apoptosis by Upregulating let-7a Expression.

Oxidative stress is a causal factor and key promoter of all kinds of reproductive disorders related to granulosa cell (GC) apoptosis that acts by dysregulating the expression of related genes. Various studies have suggested that grape seed procyanidin B2 (GSPB2) may protect GCs from oxidative injury, though the underlying mechanisms are not fully understood. Therefore, whether the beneficial effects of GSPB2 are associated with microRNAs, which have been suggested to play a critical role in GC apoptosis by regulating the expression of protein-coding genes, was investigated in this study. The results showed that GSPB2 treatment protected GCs from a H2O2-induced apoptosis, as detected by an MTT assay and TUNEL staining, and increased let-7a expression in GCs. Furthermore, let-7a overexpression markedly increased cell viability and inhibited H2O2-induced GC apoptosis. Furthermore, the overexpression of let-7a reduced the upregulation of Fas expression in H2O2-treated GCs at the mRNA and protein levels. Dual-luciferase reporter assay results indicated that let-7a directly targets the Fas 3'-UTR. Furthermore, the overexpression of let-7a enhanced the protective effects of GSPB2 against GC apoptosis induced by H2O2. These results indicate that GSPB2 inhibits H2O2-induced apoptosis of GCs, possibly through the upregulation of let-7a.

Probing the side chain tolerance for inhibitors of the CD95/PLCγ1 interaction.

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.

The volatile anesthetic sevoflurane reduces neutrophil apoptosis via Fas death domain-Fas-associated death domain interaction.

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.

Modulation of CD95-mediated signaling by post-translational modifications: towards understanding CD95 signaling networks.

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 mRNA is toxic to cells.

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.

Crystal structure of the FAS1 domain of the hyaluronic acid receptor stabilin-2.

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.

Toxicity and non-harmful effects of the soya isoflavones, genistein and daidzein, in embryos of the zebrafish, Danio rerio.

Based on the assumed oestrogenic and apoptotic properties of soya isoflavones (genistein, daidzein), and following the current OECD test-guidelines and principle of 3Rs, we have studied the potential toxicity of phytochemicals on the zebrafish embryos test (ZFET). For this purpose, zebrafish embryos at 2-3 h post-fertilisation (hpf) were exposed to both soya isoflavones (from 1.25 mg/L to 20 mg/L) and assayed until 96 hpf. Lethal and sub-lethal endpoints (mortality, hatching rates and malformations) were estimated in the ZFET, which was expanded to potential gene expression markers, determining the lowest observed effect (and transcriptional) concentrations (LOEC, LOTEC), and the no-observable effect (and transcriptional) concentrations (NOEC, NOTEC). The results revealed that genistein is more toxic (LC50-96 hpf: 4.41 mg/L) than daidzein (over 65.15 mg/L). Both isoflavones up-regulated the oestrogen (esrrb) and death receptors (fas) and cyp1a transcript levels. Most thyroid transcript signals were up-regulated by genistein (except for thyroid peroxidase/tpo), and the hatching enzyme (he1a1) was exclusively up-regulated by daidzein (from 1.25 mg/L onwards). The ZFET proved suitable for assessing toxicant effects of both isoflavones and potential disruptions (i.e. oestrogenic, apoptotic, thyroid, enzymatic) during the embryogenesis and the endotrophic larval period.

An Exhaustive Search Algorithm to Aid NMR-Based Structure Determination of Rotationally Symmetric Transmembrane Oligomers.

Nuclear magnetic resonance (NMR) has been an important source of structural restraints for solving structures of oligomeric transmembrane domains (TMDs) of cell surface receptors and viral membrane proteins. In NMR studies, oligomers are assembled using inter-protomer distance restraints. But, for oligomers that are higher than dimer, these distance restraints all have two-fold directional ambiguity, and resolving such ambiguity often requires time-consuming trial-and-error calculations using restrained molecular dynamics (MD) with simulated annealing (SA). We report an Exhaustive Search algorithm for Symmetric Oligomer (ExSSO), which can perform near-complete search of the symmetric conformational space in a very short time. In this approach, the predetermined protomer model is subject to full angular and spatial search within the symmetry space. This approach, which can be applied to any rotationally symmetric oligomers, was validated using the structures of the Fas death receptor, the HIV-1 gp41 fusion protein, the influenza proton channel, and the MCU pore. The algorithm is able to generate approximate oligomer solutions quickly as initial inputs for further refinement using the MD/SA method.

Site-specific chemical conjugation of human Fas ligand extracellular domain using trans-cyclooctene - methyltetrazine reactions.

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.

Sketching of CD95 Oligomers by In Silico Investigations.

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.

Site-Specific Detection of Tyrosine Phosphorylated CD95 Following Protein Separation by Conventional and Phospho-Protein Affinity SDS-PAGE.

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).

Detection of S-Acylated CD95 by Acyl-Biotin Exchange.

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.

Optimal Bicelle Size q for Solution NMR Studies of the Protein Transmembrane Partition.

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.

Parameter identification using stochastic simulations reveals a robustness in CD95 apoptotic response.

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.