ABSTRACT
Capillary electrophoresis (CE) is an analytical technique widely utilized to resolve complex mixtures of nucleic acids. CE uses a variety of polymers in solution that act as a molecular sieve to separate nucleic acid fragments according to size. It has been shown previously that purified dsDNA can be resolved efficiently by solutions of hydroxyethylcellulose (HEC) polymer, providing a rapid and high resolution method of separation. We have applied this separation technique to viral double-stranded (ds) RNA segments derived from rotavirus process samples. HEC polymers of various molecular masses and concentrations were identified and compared for their ability to separate dsRNA based on the extent of expected polymer network formation. The HEC polymer exhibiting the most desirable separation characteristics was then used for subsequent optimization of various method parameters, such as, injection time, electric field strength, dye concentration and capillary equilibration. The optimized method was then applied to the quantification of genome concentration based on a representative segment of the rotavirus genome. This study demonstrated that purified viral dsRNA material of known concentration could be used to generate an external standard curve relating concentration to peak area. This standard curve was used to determine the concentration of unknown samples by interpolation. This novel RNA quantification assay is likely to be applicable to other types of virus, including those containing dsDNA.
Subject(s)
Cellulose/analogs & derivatives , Cellulose/chemistry , Electrophoresis, Capillary/methods , RNA, Double-Stranded/isolation & purification , RNA, Viral/isolation & purification , RNA, Double-Stranded/analysis , RNA, Viral/analysis , Solutions , ViscosityABSTRACT
Adenovirus encodes multiple gene products that regulate proapoptotic cellular responses to viral infection mediated by both the innate and adaptive immune systems. The E3-10.4K and 14.5K gene products are known to modulate the death receptor Fas. In this study, we demonstrate that an additional viral E3 protein, 6.7K, functions in the specific modulation of the two death receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The 6.7K protein is expressed on the cell surface and forms a complex with the 10.4K and 14.5K proteins, and this complex is sufficient to induce down-modulation of TRAIL receptor-1 and -2 from the cell surface and reverse the sensitivity of infected cells to TRAIL-mediated apoptosis. Down-modulation of TRAIL-R2 by the E3 complex is dependent on the cytoplasmic tail of the receptor, but the death domain alone is not sufficient. These results identify a mechanism for viral modulation of TRAIL receptor-mediated apoptosis and suggest the E3 protein complex has evolved to regulate the signaling of selected cytokine receptors.
Subject(s)
Adenovirus E3 Proteins/pharmacology , Apoptosis , Membrane Proteins , Receptors, Tumor Necrosis Factor/metabolism , Adenoviridae/metabolism , Adenovirus E3 Proteins/metabolism , Down-Regulation , Fas Ligand Protein , HT29 Cells/virology , Humans , Membrane Glycoproteins/metabolism , Receptors, Cytokine/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand , Signal Transduction , Subcellular Fractions , fas Receptor/metabolismABSTRACT
UNLABELLED: Background and Procedures NB-derived cell lines were tested for their sensitivity to apoptosis induced by the tumor-selective apoptotic ligand TRAIL. Noninvasive S-type cell lines are highly sensitive to TRAIL, whereas invasive N-type cell lines are resistant. RESULTS: Although both S- and N-type cell lines express TRAIL-R2, FADD, and caspases-3 and -10, only S-type cells express caspase-8. Reduced levels of caspase-8 protein were also observed in a stage IV NB tumor when compared to a ganglioneuroma. The caspase-8 gene is not deleted in either N-type NB cell lines or high-stage tumors, and expression can be induced by demethylation. CONCLUSIONS: Therefore, caspase-8 expression is silenced in malignant NB, which correlates to tumor severity and resistance to TRAIL-induced apoptosis.
Subject(s)
Apoptosis/genetics , Caspases/genetics , Gene Expression Regulation, Neoplastic , Membrane Glycoproteins/genetics , Neuroblastoma/genetics , Tumor Necrosis Factor-alpha/genetics , Apoptosis Regulatory Proteins , Caspase 8 , Caspase 9 , Humans , TNF-Related Apoptosis-Inducing Ligand , Tumor Cells, CulturedABSTRACT
WSL-1/TRAMP (DR3) is a member of the tumour necrosis factor (TNF) receptor superfamily which exhibits effects on NF-kappaB activation and apoptosis. TWEAK, a novel TNF-related molecule, has been proposed as the ligand for this receptor. Utilising both human and murine TWEAK ligand, it is shown that TWEAK and WSL-1/TRAMP do not interact in an in vitro binding assay and that TWEAK binds strongly to cells that do not express WSL-1/TRAMP on the cell surface. Biological activity of TWEAK is also observed in these cells. Finally, cells isolated from WSL-1/TRAMP knockout mice are shown to retain their ability to interact with TWEAK. These results suggest that WSL-1/TRAMP is not the major receptor for TWEAK
Subject(s)
Carrier Proteins/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Animals , Apoptosis , Apoptosis Regulatory Proteins , Cell Line , Cytokine TWEAK , Humans , Interleukin-8/pharmacology , Lymphocytes/metabolism , Mice , Mice, Knockout , NF-kappa B/metabolism , Receptors, Tumor Necrosis Factor/deficiency , Receptors, Tumor Necrosis Factor, Member 25 , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis FactorsABSTRACT
Human neuroblastoma (NB) is a highly heterogeneous childhood cancer that is aggressively malignant or can undergo spontaneous regression that may involve apoptosis. NB-derived cell lines were tested for their sensitivity to apoptosis induced by the tumor-selective ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Noninvasive S-type cell lines (NB cell lines of substrate adherent phenotype) are highly sensitive to TRAIL, whereas invasive N-type cell lines (NB cell lines of neuronal phenotype) are resistant. Whereas both S- and N-type cell lines express TRAIL-R2, FADD, and caspase-3 and -10, only S-type cells express caspase-8. Reduced levels of caspase-8 protein were also observed in a malignant stage IV NB tumor when compared with a benign ganglioneuroma. The caspase-8 gene is not deleted in either N-type NB cell lines or high-stage NB tumors. Caspase-8 expression can be induced by demethylation with 5-aza-2'deoxycytidine, which enhances sensitivity to TRAIL. Therefore, caspase-8 expression is silenced in malignant NB, which correlates to tumor severity and resistance to TRAIL-induced apoptosis.
Subject(s)
Apoptosis/physiology , Caspases/biosynthesis , Membrane Glycoproteins/pharmacology , Neuroblastoma/enzymology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Apoptosis/drug effects , Apoptosis Regulatory Proteins , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/metabolism , Enzyme Activation , Fas Ligand Protein , Ganglioneuroma/enzymology , Ganglioneuroma/pathology , Humans , Immunohistochemistry , Neoplasm Invasiveness , Neoplasm Staging , Neural Cell Adhesion Molecules/biosynthesis , Neural Cell Adhesion Molecules/physiology , Neuroblastoma/pathology , Phenotype , Rabbits , Receptors, Tumor Necrosis Factor/biosynthesis , Receptors, Tumor Necrosis Factor/physiology , Recombinant Proteins/pharmacology , TNF-Related Apoptosis-Inducing Ligand , Tumor Cells, CulturedABSTRACT
Overexpression of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induces apoptosis and activation of NF-kappaB in cultured cells. In this study, we have demonstrated differential signaling capacities by both receptors using either epitope-tagged soluble TRAIL (sTRAIL) or sTRAIL that was cross-linked with a monoclonal antibody. Interestingly, sTRAIL was sufficient for induction of apoptosis only in cell lines that were killed by agonistic TRAIL-R1- and TRAIL-R2-specific IgG preparations. Moreover, in these cell lines interleukin-6 secretion and NF-kappaB activation were induced by cross-linked or non-cross-linked anti-TRAIL, as well as by both receptor-specific IgGs. However, cross-linking of sTRAIL was required for induction of apoptosis in cell lines that only responded to the agonistic anti-TRAIL-R2-IgG. Interestingly, activation of c-Jun N-terminal kinase (JNK) was only observed in response to either cross-linked sTRAIL or anti-TRAIL-R2-IgG even in cell lines where both receptors were capable of signaling apoptosis and NF-kappaB activation. Taken together, our data suggest that TRAIL-R1 responds to either cross-linked or non-cross-linked sTRAIL which signals NF-kappaB activation and apoptosis, whereas TRAIL-R2 signals NF-kappaB activation, apoptosis, and JNK activation only in response to cross-linked TRAIL.
Subject(s)
Apoptosis , Mitogen-Activated Protein Kinases/metabolism , Receptor Aggregation , Receptors, Tumor Necrosis Factor/metabolism , Signal Transduction , Antibodies, Monoclonal/immunology , Apoptosis Regulatory Proteins , Enzyme Activation , Flow Cytometry , Humans , Immunoglobulin G/immunology , Interleukin-6/metabolism , JNK Mitogen-Activated Protein Kinases , Membrane Glycoproteins/agonists , Membrane Glycoproteins/immunology , Membrane Glycoproteins/metabolism , NF-kappa B/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor/agonists , Staphylococcal Protein A/immunology , TNF-Related Apoptosis-Inducing Ligand , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/agonists , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Unlike other tumor necrosis factor family members, the cytotoxic ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo-2L contains an unpaired cysteine residue (Cys(230)) in its receptor-binding domain. Here we show that the biological activity of both soluble recombinant TRAIL and cell-associated, full-length TRAIL is critically dependent on the presence of Cys(230). Mutation of Cys(230) to alanine or serine strongly affected its ability to kill target cells. Binding to its receptors was decreased by at least 200-fold, and the stability of its trimeric structure was reduced. In recombinant TRAIL, Cys(230) was found engaged either in interchain disulfide bridge formation, resulting in poorly active TRAIL, or in the chelation of one zinc atom per TRAIL trimer in the active, pro-apoptotic form of TRAIL.
Subject(s)
Apoptosis , Cysteine/chemistry , Membrane Glycoproteins/chemistry , Tumor Necrosis Factor-alpha/chemistry , Amino Acid Sequence , Apoptosis Regulatory Proteins , Binding Sites , Disulfides/chemistry , Humans , Hydrogen-Ion Concentration , Membrane Glycoproteins/genetics , Membrane Glycoproteins/pharmacology , Molecular Sequence Data , Mutation , Protein Conformation , Receptors, Tumor Necrosis Factor/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , TNF-Related Apoptosis-Inducing Ligand , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/pharmacology , Zinc/chemistrySubject(s)
Apoptosis/physiology , Arabidopsis Proteins , Caspases/metabolism , Fatty Acid Desaturases/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Signal Transduction/physiology , Caspase 3 , Caspase 8 , Caspase 9 , Humans , Jurkat Cells/chemistry , Jurkat Cells/cytology , Jurkat Cells/enzymology , Receptors, TNF-Related Apoptosis-Inducing LigandABSTRACT
TNF receptor family members fused to the constant domain of immunoglobulin G have been widely used as immunoadhesins in basic in vitro and in vivo research and in some clinical applications. In this study, we assemble soluble, high avidity chimeric receptors on a pentameric scaffold derived from the coiled-coil domain of cartilage oligomeric matrix protein (COMP). The affinity of Fas and CD40 (but not TNFR-1 and TRAIL-R2) to their ligands is increased by fusion to COMP, when compared to the respective Fc chimeras. In functional assays, Fas:COMP was at least 20-fold more active than Fas:Fc at inhibiting the action of sFasL, and CD40:COMP could block CD40L-mediated proliferation of B cells, whereas CD40:Fc could not. In conclusion, members of the TNF receptor family can display high specificity and excellent avidity for their ligands if they are adequately multimerized.
Subject(s)
Membrane Glycoproteins/antagonists & inhibitors , Animals , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , CD40 Antigens/metabolism , CD40 Ligand , Cartilage Oligomeric Matrix Protein , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Extracellular Matrix Proteins/pharmacology , Fas Ligand Protein , Glycoproteins/genetics , Glycoproteins/metabolism , Glycoproteins/pharmacology , Humans , Jurkat Cells , Ligands , Lymphocyte Activation/drug effects , Matrilin Proteins , Membrane Glycoproteins/metabolism , Mice , Mice, Knockout , Receptors, Fc/genetics , Receptors, Fc/metabolism , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Solubility , Tumor Cells, Cultured , fas Receptor/metabolismABSTRACT
Cell death is achieved by two fundamentally different mechanisms: apoptosis and necrosis. Apoptosis is dependent on caspase activation, whereas the caspase-independent necrotic signaling pathway remains largely uncharacterized. We show here that Fas kills activated primary T cells efficiently in the absence of active caspases, which results in necrotic morphological changes and late mitochondrial damage but no cytochrome c release. This Fas ligand-induced caspase-independent death is absent in T cells that are deficient in either Fas-associated death domain (FADD) or receptor-interacting protein (RIP). RIP is also required for necrotic death induced by tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL). In contrast to its role in nuclear factor kappa B activation, RIP requires its own kinase activity for death signaling. Thus, Fas, TRAIL and TNF receptors can initiate cell death by two alternative pathways, one relying on caspase-8 and the other dependent on the kinase RIP.
Subject(s)
Adaptor Proteins, Signal Transducing , Caspases/metabolism , Cell Death/physiology , Proteins/metabolism , fas Receptor/metabolism , Animals , Apoptosis/physiology , Apoptosis Regulatory Proteins , Carrier Proteins/metabolism , Caspase 8 , Caspase 9 , Fas Ligand Protein , Fas-Associated Death Domain Protein , Humans , In Vitro Techniques , Jurkat Cells , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred BALB C , Models, Biological , Necrosis , Receptor-Interacting Protein Serine-Threonine Kinases , Signal Transduction , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , TNF-Related Apoptosis-Inducing Ligand , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family, designated BAFF (for B cell activating factor belonging to the TNF family), which is expressed by T cells and dendritic cells. Human BAFF was mapped to chromosome 13q32-34. Membrane-bound BAFF was processed and secreted through the action of a protease whose specificity matches that of the furin family of proprotein convertases. The expression of BAFF receptor appeared to be restricted to B cells. Both membrane-bound and soluble BAFF induced proliferation of anti-immunoglobulin M-stimulated peripheral blood B lymphocytes. Moreover, increased amounts of immunoglobulins were found in supernatants of germinal center-like B cells costimulated with BAFF. These results suggest that BAFF plays an important role as costimulator of B cell proliferation and function.
Subject(s)
B-Lymphocytes/immunology , Membrane Proteins/physiology , Receptors, Tumor Necrosis Factor/physiology , Tumor Necrosis Factor-alpha/physiology , Amino Acid Sequence , Animals , B-Cell Activating Factor , B-Lymphocytes/cytology , Base Sequence , Cell Division , Cell Line , Chromosome Mapping , Chromosomes, Human, Pair 13/genetics , Cloning, Molecular , DNA Primers/genetics , Dendritic Cells/immunology , Humans , Ligands , Lymphocyte Activation , Membrane Proteins/genetics , Mice , Molecular Sequence Data , Receptors, Tumor Necrosis Factor/genetics , Sequence Homology, Amino Acid , T-Lymphocytes/immunology , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family designated APRIL (for a proliferation-inducing ligand). Although transcripts of APRIL are of low abundance in normal tissues, high levels of mRNA are detected in transformed cell lines, and in human cancers of colon, thyroid, and lymphoid tissues in vivo. The addition of recombinant APRIL to various tumor cells stimulates their proliferation. Moreover, APRIL-transfected NIH-3T3 cells show an increased rate of tumor growth in nude mice compared with the parental cell line. These findings suggest that APRIL may be implicated in the regulation of tumor cell growth.
Subject(s)
Growth Substances/physiology , Membrane Proteins/physiology , Tumor Cells, Cultured/pathology , Tumor Necrosis Factor-alpha/physiology , 3T3 Cells , Amino Acid Sequence , Animals , Cell Division/drug effects , Humans , Ligands , Lymphoma, B-Cell , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Mice , Mice, Inbred BALB C , Mice, Nude , Molecular Sequence Data , Neoplasm Transplantation , RNA, Messenger/biosynthesis , Transfection , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism , Tumor Necrosis Factor Ligand Superfamily Member 13ABSTRACT
Members of the tumor necrosis factor receptor (TNFR) superfamily have an important role in the induction of cellular signals resulting in cell growth, differentiation and death. TNFR-1 recruits and assembles a signaling complex containing a number of death domain (DD)-containing proteins, including the adaptor protein TRADD and the serine/threonine kinase RIP, which mediates TNF-induced NF-kappa B activation. RIP also recruits caspase-2 to the TNFR-1 signaling complex via the adaptor protein RAIDD, which contains a DD and a caspase-recruiting domain (CARD). Here, we have identified a RIP-like kinase, termed CARDIAK (for CARD-containing interleukin (IL)-1 beta converting enzyme (ICE) associated kinase), which contains a serine/threonine kinase domain and a carboxy-terminal CARD. Overexpression of CARDIAK induced the activation of both NF-kappa B and Jun N-terminal kinase (JNK). CARDIAK interacted with the TNFR-associated factors TRAF-1 and TRAF-2, and a dominant-negative form of TRAF-2 inhibited CARDIAK-induced NF-kappa B activation. Interestingly, CARDIAK specifically interacted with the CARD of caspase-1 (previously known as ICE), and this interaction correlated with the processing of pro-caspase-1 and the formation of the active p20 subunit of caspase-1. Together, these data suggest that CARDIAK may be involved in NF-kappa B/JNK signaling and in the generation of the proinflammatory cytokine IL-1 beta through activation of caspase-1.
Subject(s)
Caspase 1/metabolism , Mitogen-Activated Protein Kinases , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Base Sequence , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Line, Transformed , Humans , JNK Mitogen-Activated Protein Kinases , Molecular Sequence Data , NF-kappa B/metabolism , Protein Kinases , Protein Serine-Threonine Kinases/genetics , Proteins/chemistry , Proteins/metabolism , Receptor-Interacting Protein Serine-Threonine Kinase 2 , Receptor-Interacting Protein Serine-Threonine Kinases , Receptors, Tumor Necrosis Factor/metabolism , Sequence Homology, Amino Acid , TNF Receptor-Associated Factor 1 , TNF Receptor-Associated Factor 2ABSTRACT
MyD88 has a modular organization, an N-terminal death domain (DD) related to the cytoplasmic signaling domains found in many members of the tumor necrosis factor receptor (TNF-R) superfamily, and a C-terminal Toll domain similar to that found in the expanding family of Toll/interleukin-1-like receptors (IL-1R). This dual domain structure, together with the following observations, supports a role for MyD88 as an adapter in IL-1 signal transduction; MyD88 forms homodimers in vivo through DD-DD and Toll-Toll interactions. Overexpression of MyD88 induces activation of the c-Jun N-terminal kinase (JNK) and the transcription factor NF-kappaB through its DD. A point mutation in MyD88, MyD88-lpr (F56N), which prevents dimerization of the DD, also blocks induction of these activities. MyD88-induced NF-kappaB activation is inhibited by the dominant negative versions of TRAF6 and IRAK, which also inhibit IL-1-induced NF-kappaB activation. Overexpression of MyD88-lpr or MyD88-Toll (expressing only the Toll domain) acted to inhibit IL-1-induced NF-kappaB and JNK activation in a 293 cell line overexpressing the IL-1RI. MyD88 coimmunoprecipitates with the IL-1R signaling complex in an IL-1-dependent manner.
Subject(s)
Antigens, Differentiation , Interleukin-1/metabolism , Mitogen-Activated Protein Kinases , Proteins/metabolism , Receptors, Immunologic , Signal Transduction , Adaptor Proteins, Signal Transducing , Animals , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Line , Dimerization , Enzyme Activation , Humans , JNK Mitogen-Activated Protein Kinases , Mice , Myeloid Differentiation Factor 88 , NF-kappa B/metabolism , Recombinant Proteins/metabolismABSTRACT
Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-alpha undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-alpha) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high.
Subject(s)
Apoptosis , Liver/pathology , Membrane Glycoproteins/metabolism , Protein Processing, Post-Translational , Amino Acid Sequence , Animals , Down-Regulation , Fas Ligand Protein , Humans , Mice , Mice, Inbred BALB C , Models, Biological , Molecular Sequence Data , Protein Binding , Receptors, Tumor Necrosis Factor/metabolism , Solubility , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Two receptors for TRAIL, designated TRAIL-R2 and TRAIL-R3, have been identified. Both are members of the tumor necrosis factor receptor family. TRAIL-R2 is structurally similar to the death-domain-containing receptor TRAIL-R1 (DR-4), and is capable of inducing apoptosis. In contrast, TRAIL-R3 does not promote cell death. TRAIL-R3 is highly glycosylated and is membrane bound via a putative phosphatidylinositol anchor. The extended structure of TRAIL-R3 is due to the presence of multiple threonine-, alanine-, proline- and glutamine-rich repeats (TAPE repeats). TRAIL-R2 shows a broad tissue distribution, whereas the expression of TRAIL-R3 is restricted to peripheral blood lymphocytes (PBLs) and skeletal muscle. All three TRAIL receptors bind TRAIL with similar affinity, suggesting a complex regulation of TRAIL-mediated signals.
Subject(s)
Membrane Glycoproteins/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Tumor Necrosis Factor-alpha/metabolism , Amino Acid Sequence , Apoptosis , Apoptosis Regulatory Proteins , Cell Line , Chromosome Mapping , Cloning, Molecular , GPI-Linked Proteins , Humans , Kinetics , Lymphocytes/metabolism , Molecular Sequence Data , Muscle, Skeletal/metabolism , Polymerase Chain Reaction , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor/chemistry , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor, Member 10c , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Sequence Tagged Sites , Signal Transduction , TNF-Related Apoptosis-Inducing Ligand , Transfection , Tumor Necrosis Factor Decoy ReceptorsABSTRACT
The widely expressed protein Fas is a member of the tumour necrosis factor receptor family which can trigger apoptosis. However, Fas surface expression does not necessarily render cells susceptible to Fas ligand-induced death signals, indicating that inhibitors of the apoptosis-signalling pathway must exist. Here we report the characterization of an inhibitor of apoptosis, designated FLIP (for FLICE-inhibitory protein), which is predominantly expressed in muscle and lymphoid tissues. The short form, FLIPs, contains two death effector domains and is structurally related to the viral FLIP inhibitors of apoptosis, whereas the long form, FLIP(L), contains in addition a caspase-like domain in which the active-centre cysteine residue is substituted by a tyrosine residue. FLIPs and FLIP(L) interact with the adaptor protein FADD and the protease FLICE, and potently inhibit apoptosis induced by all known human death receptors. FLIP(L) is expressed during the early stage of T-cell activation, but disappears when T cells become susceptible to Fas ligand-mediated apoptosis. High levels of FLIP(L) protein are also detectable in melanoma cell lines and malignant melanoma tumours. Thus FLIP may be implicated in tissue homeostasis as an important regulator of apoptosis.
Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis , Carrier Proteins/physiology , Caspases , Intracellular Signaling Peptides and Proteins , Amino Acid Sequence , Animals , CASP8 and FADD-Like Apoptosis Regulating Protein , Carrier Proteins/genetics , Carrier Proteins/metabolism , Caspase 8 , Caspase 9 , Cells, Cultured , Chromosomes, Human, Pair 2 , Cloning, Molecular , Cysteine Endopeptidases/metabolism , Fas-Associated Death Domain Protein , Humans , Lymphocyte Activation , Melanoma/metabolism , Molecular Sequence Data , Sequence Homology, Amino Acid , T-Lymphocytes/cytology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tumor Cells, Cultured , fas Receptor/metabolismABSTRACT
The death-inducing receptor Fas is activated when cross-linked by the type II membrane protein Fas ligand (FasL). When human soluble FasL (sFasL, containing the extracellular portion) was expressed in human embryo kidney 293 cells, the three N-linked glycans of each FasL monomer were found to be essential for efficient secretion. Based on the structure of the closely related lymphotoxin alpha-tumor necrosis factor receptor I complex, a molecular model of the FasL homotrimer bound to three Fas molecules was generated using knowledge-based protein modeling methods. Point mutations of amino acid residues predicted to affect the receptor-ligand interaction were introduced at three sites. The F275L mutant, mimicking the loss of function murine gld mutation, exhibited a high propensity for aggregation and was unable to bind to Fas. Mutants P206R, P206D, and P206F displayed reduced cytotoxicity toward Fas-positive cells with a concomitant decrease in the binding affinity for the recombinant Fas-immunoglobulin Fc fusion proteins. Although the cytotoxic activity of mutant Y218D was unaltered, mutant Y218R was inactive, correlating with the prediction that Tyr-218 of FasL interacts with a cluster of three basic amino acid side chains of Fas. Interestingly, mutant Y218F could induce apoptosis in murine, but not human cells.
Subject(s)
Membrane Glycoproteins/metabolism , fas Receptor/metabolism , Amino Acid Sequence , Animals , Binding Sites , Chromatography, Gel , Enzyme-Linked Immunosorbent Assay , Fas Ligand Protein , Glycosylation , Humans , Jurkat Cells , Ligands , Membrane Glycoproteins/genetics , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis , Proline/metabolism , Sequence Alignment , Solubility , Species Specificity , Tyrosine/metabolismABSTRACT
Viruses have evolved many distinct strategies to avoid the host's apoptotic response. Here we describe a new family of viral inhibitors (v-FLIPs) which interfere with apoptosis signalled through death receptors and which are present in several gamma-herpesviruses (including Kaposi's-sarcoma-associated human herpesvirus-8), as well as in the tumorigenic human molluscipoxvirus. v-FLIPs contain two death-effector domains which interact with the adaptor protein FADD, and this inhibits the recruitment and activation of the protease FLICE by the CD95 death receptor. Cells expressing v-FLIPs are protected against apoptosis induced by CD95 or by the related death receptors TRAMP and TRAIL-R. The herpesvirus saimiri FLIP is detected late during the lytic viral replication cycle, at a time when host cells are partially protected from CD95-ligand-mediated apoptosis. Protection of virus-infected cells against death-receptor-induced apoptosis may lead to higher virus production and contribute to the persistence and oncogenicity of several FLIP-encoding viruses.
Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis , Caspases , Cysteine Endopeptidases/metabolism , Gammaherpesvirinae/physiology , Viral Proteins/physiology , Amino Acid Sequence , Animals , Apoptosis Regulatory Proteins , Carrier Proteins/metabolism , Caspase 8 , Caspase 9 , Cell Line , Cell Transformation, Viral , Fas-Associated Death Domain Protein , Gammaherpesvirinae/genetics , Herpesvirus 2, Saimiriine/physiology , Humans , Membrane Glycoproteins/metabolism , Mice , Molecular Sequence Data , Receptors, Tumor Necrosis Factor/metabolism , Receptors, Tumor Necrosis Factor, Member 25 , Sequence Homology, Amino Acid , Signal Transduction , TNF-Related Apoptosis-Inducing Ligand , Tumor Necrosis Factor-alpha/metabolism , Virus Replication , fas Receptor/metabolismABSTRACT
A novel member of the tumor necrosis factor (TNF) receptor family, designated TRAMP, has been identified. The structural organization of the 393 amino acid long human TRAMP is most homologous to TNF receptor 1. TRAMP is abundantly expressed on thymocytes and lymphocytes. Its extracellular domain is composed of four cysteine-rich domains, and the cytoplasmic region contains a death domain known to signal apoptosis. Overexpression of TRAMP leads to two major responses, NF-kappaB activation and apoptosis. TRAMP-induced cell death is inhibited by an inhibitor of ICE-like proteases, but not by Bcl-2. In addition, TRAMP does not appear to interact with any of the known apoptosis-inducing ligands of the TNF family.