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.

Metalloprotease-mediated cleavage of CD95 ligand.

CD95 is a member of the TNF receptor superfamily that is ubiquitously expressed in healthy and pathological tissues. Stimulation of CD95 by its physiological ligand CD95L induces its oligomerization leading in turn to the transduction of either apoptotic or nonapoptotic signals. CD95L can exist as both membrane-anchored and soluble forms (sCD95L), the latter resulting from the proteolytic cleavage of the former. Candidate proteases able to achieve CD95L cleavage were identified as matrix metalloproteases (MMP) due to their demonstrated ability to cleave other TNF superfamily ligands. The main goal of this study was to systematically identify the MMP family members capable of cleaving CD95L and subsequently determine the corresponding cleavage sites. By using different orthogonal biochemical approaches and combining them with molecular modelling, we confirmed data from the literature regarding CD95L cleavage by MMP-3 and MMP-7. Moreover, we found that MMP-2 and MMP-12 can cleave CD95L and characterized their resulting cleavage sites. This study provides a systematic approach to analyse the cleavage of CD95L, which until now had only been poorly described.

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.

Molecular mechanisms of FasL-mediated 'reverse-signaling'.

Effector lymphocytes, including NK and T cells, express FasL. Expression of Fas, the receptor for FasL in tumor cells, renders them susceptible to NK and T cell-mediated killing. The functional relevance of FasL in initiating death signals in tumor cells is well-characterized. However, the cytoplasmic interacting partners and the potential signaling pathways downstream of FasL are far from fully defined. FasL possesses an 81 amino acid long cytoplasmic tail with multiple unique recruitment motifs. We predict multiple interdependent signaling complexes form the core of the 'reverse signaling' downstream of FasL. A direct interaction between the proline-rich domain of FasL and the SH3 domain of PI(3)K-p85α initiates the first pathway. This cascade helps FasL to link to PLC-γ2 via PIP3 or the Akt-dependent activation of mTOR complexes. Independently, a GRB2/GADs-binding PXXP cytoplasmic motif of FasL can initiate a Ras-GTP-dependent PAK1→C-Raf→MEK1/2→ERK1/2 activation. FasL can recruit Fyn via the proline-rich domain leading to the recruitment of ADAP. Through its ability to directly interact with Carma1 and TAK1, ADAP initiates the formation of the Carma1/Bcl10/Malt1-based CBM signalosome that is primarily responsible for inflammatory cytokine production. Here, we explore the conserved cytoplasmic domains of FasL, the potential signaling molecules that interact, and the functional downstream consequences within the effector lymphocytes to define the FasL-mediated 'reverse signaling'.

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.

Confirmation of covalently-linked structure and cell-death inducing activity in site-specific chemical conjugates of human Fas ligand extracellular domain.

OBJECTIVE: In this study, we aimed to identify the structural components and to clarify the biological activity in the site-specific conjugates of human Fas ligand extracellular domain (hFasLECD) with either fluorescein moiety (FL) or chicken egg-white avidin (Avi). The conjugates were characterized by molecular-weight measurement using MALDI-TOF mass-spectrometric analysis and by cell-death inducing activity measurement against a human colorectal cancer cell line, HT-29, using MTT cell-viability assay. Pretreatment effect with human interferon-γ (IFN-γ) on the cell-death inducing activity was evaluated. RESULTS: The mass-spectrometric analysis of the hFasLECD-Avi conjugate showed that it was possible to detect the signal peak of molecular-weight to electric charge (m/z) derived from the component involved in the covalent linking as the sum of the molecular-weight of unconjugated hFasLECD- and Avi-derivative subunits, in addition to the signals from each corresponding subunit component irrelevant to the covalent linking. The cell-viability assay revealed that both conjugates possessed a remarkable death-inducing activity against HT-29 cells in synergy with the pretreatment using human IFN-γ. Following 24 h pretreatment with 100 IU/ml of human IFN-γ, almost no viable cells existed after 72 h treatment with either 100 or 1000 ng/ml of FL-hFasLECD and hFasLECD-Avi conjugates.

Impairment of Fas-ligand-caveolin-1 interaction inhibits Fas-ligand translocation to rafts and Fas-ligand-induced cell death.

Fas-ligand/CD178 belongs to the TNF family proteins and can induce apoptosis through death receptor Fas/CD95. The important requirement for Fas-ligand-dependent cell death induction is its localization to rafts, cholesterol- and sphingolipid-enriched micro-domains of membrane, involved in regulation of different signaling complexes. Here, we demonstrate that Fas-ligand physically associates with caveolin-1, the main protein component of rafts. Experiments with cells overexpressing Fas-ligand revealed a FasL N-terminal pre-prolin-rich region, which is essential for the association with caveolin-1. We found that the N-terminal domain of Fas-ligand bears two caveolin-binding sites. The first caveolin-binding site binds the N-terminal domain of caveolin-1, whereas the second one appears to interact with the C-terminal domain of caveolin-1. The deletion of both caveolin-binding sites in Fas-ligand impairs its distribution between cellular membranes, and attenuates a Fas-ligand-induced cytotoxicity. These results demonstrate that the interaction of Fas-ligand and caveolin-1 represents a molecular basis for Fas-ligand translocation to rafts, and the subsequent induction of Fas-ligand-dependent cell death. A possibility of a similar association between other TNF family members and caveolin-1 is discussed.

Nonapoptotic functions of Fas/CD95 in the immune response.

CD95 (also known as Fas) is a member of the tumor necrosis factor receptor (TNFR) superfamily. Its cognate ligand, CD95L, is implicated in immune homeostasis and immune surveillance. Mutations in this receptor are associated with a loss of apoptotic signaling and have been detected in an autoimmune disorder called autoimmune lymphoproliferative syndrome (ALPS) type Ia, which shares some clinical features with systemic lupus erythematosus (SLE). In addition, deletions and mutations of CD95 have been described in many cancers, which led researchers to initially classify this receptor as a tumor suppressor. More recent data demonstrate that CD95 engagement evokes nonapoptotic signals that promote inflammation and carcinogenesis. Transmembrane CD95L (m-CD95L) can be cleaved by metalloproteases, releasing a soluble ligand (s-CD95L). Soluble and membrane-bound CD95L show different stoichiometry (homotrimer versus multimer of homotrimers, respectively), which differentially affects CD95-mediated signaling through molecular mechanisms that remain to be elucidated. This review discusses the biological roles of CD95 in light of recent experiments addressing how a death receptor can trigger both apoptotic and nonapoptotic signaling pathways.

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.

Production of the Non-apoptotic Metalloprotease-Cleaved CD95L and Its Cytotoxic Recombinant Counterpart Designed Ig-CD95L.

The ligand of CD95, CD95L (also known as FasL or CD178), is a type II transmembrane protein that belongs to the Tumor Necrosis factor (TNF) family (Fig. 1a). This membrane-bound cytokine is mainly expressed at the surface of activated T lymphocytes and natural killer cells, where it is used as an apoptotic factor to eliminate infected and transformed cells (Strasser et al., Immunity 30:180-192, 2009).

Balance between IL-3 and type Iinterferons and their interrelationship with FasL dictates lifespan and effector functions of human basophils.

BACKGROUND: In contrast to eosinophils and neutrophils, the regulation of the lifespan of human basophils is poorly defined, with the exception of the potent anti-apoptotic effect of IL-3 that also promotes pro-inflammatory effector functions and phenotypic changes. Type I IFNs (IFN-α, IFN-ß), which are well known for their anti-viral activities, have the capacity to inhibit allergic inflammation. OBJECTIVE: To elucidate whether type I IFNs have the potential to abrogate the lifespan and/or effector functions of human basophils. METHODS: We cultured human basophils, and for comparison, eosinophils and neutrophils, with IL-3, interferons, FasL and TRAIL, alone or in combination, and studied cell survival, effector functions and signalling pathways involved. RESULTS: Despite an identical pattern of early signalling in basophils, eosinophils and neutrophils in response to different types of interferons, only basophils displayed enhanced apoptosis after type I IFN treatment. IFN-γ prolonged survival of eosinophils but did not affect the lifespan of basophils. IFN-α-mediated apoptosis required STAT1-STAT2 heterodimers and the contribution of constitutive p38 MAPK activity. Whereas the death ligands FasL and TRAIL-induced apoptosis in basophils per se, IFN-α-mediated apoptosis did neither involve autocrine TRAIL signalling nor did it sensitize basophils to FasL-induced apoptosis. However, IFN-α and FasL displayed an additive effect in killing basophils. Interestingly, IL-3, which protected basophils from IFN-α-, TRAIL- or FasL-mediated apoptosis, did not completely block the additive effect of combined IFN-α and FasL treatment. Moreover, we demonstrate that IFN-α suppressed IL-3-induced release of IL-8 and IL-13. In contrast to IFN-α-mediated apoptosis, these inhibitory effects of IFN-α were not dependent on p38 MAPK signalling. CONCLUSIONS AND CLINICAL RELEVANCE: Our study defines the unique and granulocyte-type-specific inhibitory and pro-apoptotic function of type I IFNs and their cooperation with death ligands in human blood basophils, which may be relevant for the anti-allergic properties of type I IFNs.

Crystal Structure of the Complex of Human FasL and Its Decoy Receptor DcR3.

The apoptotic effect of FasL:Fas signaling is disrupted by DcR3, a unique secreted member of the tumor necrosis factor receptor superfamily, which also binds and neutralizes TL1A and LIGHT. DcR3 is highly elevated in patients with various tumors and contributes to mechanisms by which tumor cells to evade host immune surveillance. Here we report the crystal structure of FasL in complex with DcR3. Comparison of FasL:DcR3 structure with our earlier TL1A:DcR3 and LIGHT:DcR3 structures supports a paradigm involving the recognition of invariant main-chain and conserved side-chain functionalities, which is responsible for the recognition of multiple TNF ligands exhibited by DcR3. The FasL:DcR3 structure also provides insight into the FasL:Fas recognition surface. We demonstrate that the ability of recombinant FasL to induce Jurkat cell apoptosis is significantly enhanced by native glycosylation or by structure-inspired mutations, both of which result in reduced tendency to aggregate. All of these activities are efficiently inhibited by recombinant DcR3.

Distinct role of the Fas rs1800682 and FasL rs763110 polymorphisms in determining the risk of breast cancer among Han Chinese females.

BACKGROUND: In recent years, studies have demonstrated that polymorphisms in the promoters of Fas and FasL are significantly associated with breast cancer risk. However, the results of these studies were inconsistent. This case-control study was performed to explore the associations between Fas rs1800682 and FasL rs763110 polymorphisms and breast cancer. MATERIALS AND METHODS: A hospital-based case-control study of 560 Han Chinese females with breast cancer (583 controls) was conducted. The MassARRAY system was used to search for a possible association between the disease risk and the two single nucleotide polymorphisms, Fas rs1800682 and FasL rs763110. Statistical analyses were performed using SNPStats software to conduct Pearson's chi-square tests in five different genetic models. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated after adjustment to age and body mass index. PHASE v2.1 software was used to reconstruct all common haplotypes. RESULTS: A statistically significant association was found between Fas rs1800682 and increased breast cancer risk (AG vs AA: OR =1.37, 95% CI =1.06-1.78; AA+AG vs GG: OR =1.32, 95% CI =1.04-1.66), and also it was found that the FasL rs763110 polymorphism may decrease the risk. Stratified analyses demonstrated that the rs763110 polymorphism was associated with lower breast cancer risk among postmenopausal females (heterozygote model: OR =0.69, 95% CI =0.49-0.97; dominant model: OR =0.70, 95% CI =0.51-0.96). The T allele of rs763110 was also associated with a decreased risk of lymph node metastasis (allele model: OR =0.75, 95% CI =0.57-0.97) and an increased risk of the breast cancer being human epidermal growth factor receptor 2 positive (allele model: OR =1.37, 95% CI =1.03-1.18). Moreover, haplotype analysis showed that Ars1800682Trs763110 was associated to a statistically significant degree with lower risk of breast cancer (OR =0.70, 95% CI =0.53-0.91). CONCLUSION: These data suggest that the presence of Fas rs1800683 is an important risk factor for breast cancer, whereas FasL rs763110 may exert a protective effect against the onset of breast cancer.

The relationship between apoptosis, chromatin configuration, histone modification and competence of oocytes: A study using the mouse ovary-holding stress model.

The epigenetic factors causing competence differences between SN (surrounded nucleolus) and NSN (non-surrounded nucleolus) oocytes, the significance for the increased histone acetylation and methylation in SN oocytes, and whether chromatin configuration or histone modification determines oocyte competence, are unclear. This study has addressed these issues by using the ovary-holding (OH) stress models where oocyte SN configuration was uncoupled from histone modifications and developmental potential. Prepubertal mouse ovaries containing high percentages of NSN oocytes were preserved at 37 or 39 °C for 1 or 2 h before examination for oocyte chromatin configuration, developmental competence, histone modification and apoptosis. Whereas 1-h OH at 37 °C caused a moderate apoptosis with increased oocyte competence, improved histone modification and a normal NSN-to-SN transition, harsher OH conditions induced a severe apoptosis with decreased oocyte competence, impaired histone modification and a pseudo (premature) NSN-to-SN transition. Observations on Fas/FasL expression and using the gld (generalized lymphoproliferative disorder) mice harboring FasL mutations indicated that OH triggered oocyte apoptosis with activation of the Fas signaling. It was concluded that OH stress caused oocyte apoptosis with activation of the Fas/FasL system and that oocyte competence was more closely correlated with histone modification than with chromatin configuration.

Cell cycle and apoptosis regulation by NFAT transcription factors: new roles for an old player.

The NFAT (nuclear factor of activated T cells) family of transcription factors consists of four Ca(2+)-regulated members (NFAT1-NFAT4), which were first described in T lymphocytes. In addition to their well-documented role in T lymphocytes, where they control gene expression during cell activation and differentiation, NFAT proteins are also expressed in a wide range of cells and tissue types and regulate genes involved in cell cycle, apoptosis, angiogenesis and metastasis. The NFAT proteins share a highly conserved DNA-binding domain (DBD), which allows all NFAT members to bind to the same DNA sequence in enhancers or promoter regions. The same DNA-binding specificity suggests redundant roles for the NFAT proteins, which is true during the regulation of some genes such as IL-2 and p21. However, it has become increasingly clear that different NFAT proteins and even isoforms can have unique functions. In this review, we address the possible reasons for these distinct roles, particularly regarding N- and C-terminal transactivation regions (TADs) and the partner proteins that interact with these TADs. We also discuss the genes regulated by NFAT during cell cycle regulation and apoptosis and the role of NFAT during tumorigenesis.

MiR-21 up-regulation mediates glioblastoma cancer stem cells apoptosis and proliferation by targeting FASLG.

To investigate whether miR-21 can affect the apoptosis and proliferation of glioblastoma cancer stem cells (GSCs) from down-regulating FASLG. The expression of miRNA-21 was detected by quantitative real-time PCR in normal brain tissue and glioblastoma samples, and the changes of miRNA-21 expression between GSCs and non-GSCs were also detected. The apoptosis and proliferation ability of miR-21 in GSCs were analyzed by MTT and flow cytometry assay after anti-miR-21 transfection. For the regulation mechanism analysis of miR-21, TargetScan, PicTar and microRNA were selected to predict some potential target genes of miR-21. The predicted gene was identified to be the direct and specific target gene of miR-21 by luciferase activities assay and western blot. RNA interference technology was used to confirm the apoptosis and proliferation effects of miR-21 were directly induced by FASLG. The expression of miR-21 increased significantly in glioblastoma contrast to normal brain tissue, and miR-21 up-regulated in GSCs remarkably. The proliferation of GSCs cell could be inhibited with high-expression of miR-21 and this effect could be restored by miR-21 knocked down. Mechanism analysis revealed that FASLG was a specific and direct target gene of miR-21. The advanced effects of anti-miR-21 on GSCs apoptosis and proliferation were mediated by expression of silenced FASLG. In summary, aberrantly expressed miR-21 regulates GSCs apoptosis and proliferation partly through directly down-regulating FASLG protein expression in GSCs and this might offer a new potential therapeutic stratagem for glioblastoma.

Differential protein-protein interactions of full length human FasL and FasL fragments generated by proteolysis.

Fas ligand (FasL) is a death factor of the tumor necrosis factor superfamily. Like other members of this family of type II transmembrane proteins, FasL is subject to ectodomain shedding by a disintegrin and metalloproteinases (ADAMs) liberating soluble FasL and leaving membrane-integral N-terminal fragments (NTFs). These NTFs are further processed by intramembrane proteolysis through signal peptide peptidase-like 2a (SPPL2a), releasing intracellular domains (ICDs) which might translocate to the nucleus to regulate transcription. Previous work established that the proline-rich domain within the cytosolic N-terminus of FasL is required for protein-protein interactions with different Src homology 3 (SH3) or WW domain proteins. Distinct binding partners regulate FasL storage and surface appearance or are involved in other aspects of FasL biology. Given the large number of FasL interactors, we asked whether proteolytically processed FasL fragments associate with the same or distinct sets of SH3 domain proteins. To address this, we performed co-precipitation experiments using a monoclonal antibody directed against the FasL N-terminus for subsequent protein detection of full length FasL and NTFs/ICDs in Western blots. We demonstrate that members of the sorting nexin (SNX) family bind full length FasL and its N-terminal fragments whereas members of the Pombe Cdc15 homology (PCH) protein family bind full length FasL, but fail to associate with processed FasL. Thus, we provide first evidence that full length FasL and FasL fragments display selectivity regarding their association with intracellular binding partners. The differential binding most likely governs the fate and function of the intracellular FasL fragments.

[Detection of FasL mRNA, sFasL and their regulatory effect on T lymphocyte subsets in patients with severe acute pancreatitis].

OBJECTIVE: To investigate the levels of FasL mRNA in peripheral blood mononualear cells (PBMCs), serum soluble Fas ligand (sFasL) and their regulatory effect on T lymphocyte subsets in patients with severe acute pancreatitis (SAP). METHODS: Forty-eight patients with pancreatitis were randomly divided into two groups: 20 cases with SAP and 28 cases with mild acute pancreatitis (MAP). Twenty-eight healthy volunteers were selected as control group. The expression of FasL mRNA in PBMCs was detected by real-time quantitative PCR(qRT-PCR), and serum sFasL was measured by ELISA. T lymphocyte subsets in peripheral blood were detected by flow cytometry. RESULTS: Compared with control group and MAP group, FasL mRNA of PBMCs and serum sFasL increased significantly in SAP group (P<0.05), a little increase in MAP group, and there was no significant difference between MAP group and control group (P>0.05). The CD4(+) T cell ratio, CD4(+)/CD8(+) ratio decreased significantly in SAP group (P<0.05) vs control group and MAP group), and they were found negatively related to FasL mRNA, serum sFasL level. CONCLUSION: The SAP patients showed the significantly increased FasL mRNA of PBMCs and serum sFasL and decreased CD4(+) T-cell ratio, CD4(+)/CD8(+) ratio. FasL may mediate the apoptosis of T lymphocytes.

Enhancing production and cytotoxic activity of polymeric soluble FasL-based chimeric proteins by concomitant expression of soluble FasL.

Membrane FasL is the natural trigger of Fas-mediated apoptosis. A soluble homotrimeric counterpart (sFasL) also exists which is very weakly active, and needs oligomerization beyond its trimeric state to induce apoptosis. We recently generated a soluble FasL chimera by fusing the immunoglobulin-like domain of the leukemia inhibitory factor receptor gp190 to the extracellular region of human FasL, which enabled spontaneous dodecameric homotypic polymerization of FasL. This polymeric soluble human FasL (pFasL) displayed anti-tumoral activity in vitro and in vivo without systemic cytotoxicity in mouse. In the present work, we focused on the improvement of pFasL, with two complementary objectives. First, we developed more complex pFasL-based chimeras that contained a cell-targeting module. Secondly, we attempted to improve the production and/or the specific activity of pFasL and of the cell-targeting chimeras. We designed two chimeras by fusing to pFasL the extracellular portions of the HLA-A2 molecule or of a human gamma-delta TCR, and analyzed the consequences of co-expressing these molecules or pFasL together with sFasL on their heterotopic cell production. This strategy significantly enhanced the production of pFasL and of the two chimeras, as well as the cytotoxic activity of the two chimeras but not of pFasL. These results provide the proof of concept for an optimization of FasL-based chimeric proteins for a therapeutic use.

Epitope mapping of antibodies by mass spectroscopy: a case study.

Epitope mapping of antibodies is the identification and characterization of binding sites of monoclonal antibodies (mAbs) on target antigens. This knowledge can be useful in generating novel antibodies to a particular target as well as elucidating an antibody mechanism of action. Several techniques are available to identify antibody epitopes among which are preliminary and simple ones like sequence homology analysis ELISA and Western blotting. However, the more widely used robust methods typically involve the use of mass spectrometry to fully analyze and interpret the data and accurately identify the binding site. Such methods include epitope extortion/excision, hydrogen deuterium exchange.