ABSTRACT
NRADD (neurotrophin receptor alike death domain protein) is a novel protein with transmembrane and cytoplasmic regions highly homologous to death receptors, particularly p75(NTR). However, the short N-terminal domain is unique. Expression of NRADD induced apoptosis in a number of cell lines. The apoptotic mechanism involved the activation of caspase-8 and execution of apoptosis without requiring mitochondrial components. The activation of this death receptor-like mechanism required the N-terminal domain, which is N-glycosylated and needed for subcellular targeting. Deletion of the N-terminal domain produced a dominant-negative form of NRADD that protected neurons and Schwann cells from a variety of endoplasmic reticulum (ER) stressors. NRADD may therefore be a necessary component for generating an ER-induced proapoptotic signal.
Subject(s)
Apoptosis/physiology , Endoplasmic Reticulum/metabolism , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Amino Acid Sequence , Animals , Animals, Newborn , Binding Sites/genetics , Caspases/metabolism , Cell Line , Cells, Cultured , Gene Expression , Gene Expression Regulation, Developmental , Glycosylation , HeLa Cells , Humans , JNK Mitogen-Activated Protein Kinases , Male , Membrane Glycoproteins/genetics , Membrane Proteins/genetics , Mice , Mitogen-Activated Protein Kinases/metabolism , Molecular Sequence Data , PC12 Cells , Rats , Receptors, Death Domain , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Signal Transduction , TransfectionABSTRACT
Glucose uptake and metabolism inhibit hypoxia-induced apoptosis in a variety of cell types, but the underlying molecular mechanisms remain poorly understood. In the present study, we explore hypoxia-mediated cell death pathways in Jurkat cells in the presence and absence of extracellular glucose. In the absence of extracellular glucose, hypoxia caused cytochrome c release, caspase 3 and poly(ADP-ribose)polymerase cleavage, and DNA fragmentation; this apoptotic response was blocked by the caspase 9 inhibitor z-LEHD-FMK. The presence of extracellular glucose during hypoxia prevented cytochrome c release and activation of caspase 9 but did not prevent apoptosis in Jurkat cells. In these conditions, overexpression of the caspase 8 inhibitor v-FLIP prevented hypoxia-mediated cell death. Thus hypoxia can stimulate two apoptotic pathways in Jurkat cells, one dependent on cytochrome c release from mitochondria that is prevented by glucose uptake and metabolism, and the other independent of cytochrome c release and resulting from activation of the death receptor pathway, which is accelerated by glucose uptake and metabolism.
Subject(s)
Apoptosis/physiology , Cell Hypoxia/physiology , DNA-Binding Proteins , Glucose/physiology , Apoptosis/drug effects , Blotting, Western , Caspase 9 , Caspase Inhibitors , Caspases/metabolism , Cell Hypoxia/drug effects , Cytochrome c Group/antagonists & inhibitors , Cytochrome c Group/metabolism , DNA Fragmentation , Enzyme Activation/physiology , Enzyme Inhibitors/pharmacology , Glucose/metabolism , Humans , Jurkat Cells , Mitochondria/physiology , Plant Proteins/biosynthesis , Poly(ADP-ribose) Polymerases/metabolism , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
A20, a TNF inducible gene, inhibits TNF-mediated apoptosis as well as NF-kappa B induced by this cytokine. Reporter assay experiments revealed that A20 is a very effective inhibitor of NF-kappa B signaling induced by TRAFs and several Map3 kinases, including NIK, MEKK1, COT, and TAK1. Similarly, the NF-kappa B inducing activity of TAX, an activator of the I kappa B kinase complex, is also abrogated by A20. Inhibition of NF-kappa B is specific as A20 has no effect on TNF-alpha-induced JNK activation. These results suggest that the molecular target of A20 is more distal to the receptor than TRAFs as previously proposed. A20 inhibits NF-kappa B-dependent transcription without a concomitant decrease in nuclear NF-kappa B DNA binding activity or nuclear translocation of p65. This apparent discrepancy between transcriptional readout and gel shift experiments is observed with a variety of stimuli, including expression of IKK beta. Therefore, in addition to the phosphorylation of I kappa B, another signal is needed for transcriptional activation of NF-kappa B. A20 inhibits this non-redundant signal. The observation that A20 associates with IKK alpha and is phosphorylated upon IKK beta co-expression may suggest that A20 interferes with some aspects of signalosome function.
Subject(s)
Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Plant Proteins , Proteins/metabolism , Proteins/physiology , Active Transport, Cell Nucleus , Cell Line , Cytoplasm/metabolism , DNA-Binding Proteins , Dose-Response Relationship, Drug , Enzyme Activation , Humans , I-kappa B Proteins/metabolism , Immunohistochemistry , Intracellular Signaling Peptides and Proteins , Luciferases/metabolism , Mitogen-Activated Protein Kinase 8 , Nuclear Proteins , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Plasmids/metabolism , Precipitin Tests , Protein Binding , Signal Transduction , Transcription Factor RelA , Transcription, Genetic , Transcriptional Activation , Transfection , Tumor Necrosis Factor alpha-Induced Protein 3 , Tumor Necrosis Factor-alpha/metabolism , p38 Mitogen-Activated Protein KinasesABSTRACT
The death domain superfamily, composed of the death domain (DD), death effector domain (DED) and caspase recruitment domain (CARD) families of proteins, plays a pivotal role in signaling events that regulate apoptosis. This review compares and contrasts the ten superfamily members with known structures. In particular, the two heterodimerization modes described to date, the CARD-CARD interaction between human Apaf-1 and procaspase 9, and the DD-DD interaction between Drosophila Pelle and Tube, are examined. The dimerization modes are strikingly different and, importantly, are not mutually exclusive. In fact, a trimer can be formed using both interactions.
Subject(s)
Carrier Proteins/physiology , Amino Acid Sequence , Animals , Apoptosis/physiology , Carrier Proteins/genetics , Carrier Proteins/metabolism , Drosophila , Models, Molecular , Molecular Sequence Data , Sequence Homology, Amino Acid , Signal Transduction/physiologyABSTRACT
The p75 neurotrophin receptor (p75(NTR)) belongs to the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In some cells derived from neuronal tissues it causes cell death through a poorly characterized pathway. We developed a neuronal system using conditionally immortalized striatal neurons, in which the expression of p75(NTR) is inducibly controlled by the ecdysone receptor. In these cells p75(NTR) induces apoptosis through its death domain in a nerve growth factor-independent manner. Caspases 9, 6, and 3 are activated by receptor expression indicating the activation of the common effector pathway of apoptosis. Cell death is blocked by a dominant negative form of caspase 9 and Bcl-X(L) consistent with a pathway that involves mitochondria. Significantly, the viral flice inhibitory protein E8 protects from p75(NTR)-induced cell death indicating that death effector domains are involved. A p75(NTR) construct with a deleted death domain dominantly interferes with p75(NTR) signaling, implying that receptor multimerization is required. However, in contrast to the other receptors of the family, p75(NTR)-mediated apoptosis does not involve the adaptor proteins Fas-associated death domain protein or tumor necrosis factor-associated death domain protein, and the apical caspase 8 is not activated. We conclude that p75(NTR) signals apoptosis by similar mechanisms as other death receptors but uses different adaptors and apical caspases.
Subject(s)
Apoptosis , Receptors, Nerve Growth Factor/metabolism , Signal Transduction , Caspase 3 , Caspase 6 , Caspase 9 , Caspases/metabolism , Cell Death , Cell Line , DNA Fragmentation , Dimerization , Enzyme Activation , Humans , Immunoblotting , In Situ Nick-End Labeling , Mutation , Neurons/metabolism , Precipitin Tests , Protein Structure, Tertiary , Receptor, Nerve Growth Factor/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
Apoptosis is mediated by a highly regulated signal transduction cascade that eventually leads to precisely directed cell death. The death-inducing signaling complex (DISC), composed of Fas, FADD, and caspase-8, is an apical signaling complex that mediates receptor-induced apoptosis. We have docked the experimentally determined structures of the Fas and FADD death domains into a model of a partial DISC signaling complex. The arrangement of Fas and FADD was determined using the interaction modes of the two heterodimer crystal structures determined to date, Pelle/Tube and Apaf-1/procaspase-9. The proposed model reveals that both interactions can be accommodated in a single multimeric complex. Importantly, the model is consistent with reported site-directed mutagenesis data indicating residues throughout the domain are critical for function. These results imply that members of the death domain superfamily have the potential for multivalent interactions, offering novel possibilities for regulation of apoptotic signaling.
Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis/physiology , Carrier Proteins/metabolism , Caspases/metabolism , fas Receptor/metabolism , Amino Acid Sequence , Animals , Carrier Proteins/chemistry , Caspase 8 , Caspase 9 , Caspases/chemistry , Dimerization , Fas-Associated Death Domain Protein , Humans , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Sequence Homology, Amino Acid , fas Receptor/chemistryABSTRACT
Src-like adapter protein (SLAP) was identified as a signaling molecule in a yeast two-hybrid system using the cytoplasmic domain of EphA2, a receptor protein tyrosine kinase (Pandey et al., 1995. Characterization of a novel Src-like adapter protein that associates with the Eck receptor tyrosine kinase. J. Biol. Chem. 270, 19201-19204). It is very similar to members of the Src family of cytoplasmic tyrosine kinases in that it contains very homologous SH3 and SH2 domains (Abram and Courtneidge, 2000. Src family tyrosine kinases and growth factor signaling. Exp. Cell. Res. 254, 1-13.). However, instead of a kinase domain at the C-terminus, it contains a unique C-terminal region. In order to exclude the possibility that an alternative form exists, we have isolated genomic clones containing the murine Slap gene as well as the human SLA gene. The coding regions of murine Slap and human SLA genes contain seven exons and six introns. Absence of any kinase domain in the genomic region confirm its designation as an adapter protein. Additionally, we have cloned and sequenced approximately 2.6 kb of the region 5' to the initiator methionine of the murine Slap gene. When subcloned upstream of a luciferase gene, this fragment increased the transcriptional activity about 6-fold in a human Jurkat T cell line and approximately 52-fold in a murine T cell line indicating that this region contains promoter elements that dictate SLAP expression. We have also cloned the promoter region of the human SLA gene. Since SLAP is transcriptionally regulated by retinoic acid and by activation of B cells, the cloning of its promoter region will permit a detailed analysis of the elements required for its transcriptional regulation.
Subject(s)
Adaptor Proteins, Signal Transducing , Promoter Regions, Genetic , Proto-Oncogene Proteins pp60(c-src)/genetics , Animals , Base Sequence , Cells, Cultured , Cloning, Molecular , Expressed Sequence Tags , Humans , Jurkat Cells , Luciferases/genetics , Luciferases/metabolism , Mice , Molecular Sequence Data , Proto-Oncogene Proteins pp60(c-src)/metabolism , T-Lymphocytes/physiologyABSTRACT
The availability of large amounts of sequence data has made it possible to identify death receptors by homology. Because the genome has not been analyzed completely, a few additional members of this family probably will be identified in the next few years. Rapid progress also has been made recently on the signaling mechanisms used by the death receptors. Considerable conservation of the intracellular signaling mechanisms is seen between different receptors suggesting that it is unlikely that new elements will be added to the molecular framework of death receptor signaling. The analysis of signaling mechanisms has exposed the complexity and multiplicity of cellular responses on death receptor activation. It is not surprising, therefore, that understanding the biological function the death receptors lags behind their characterization at the molecular level. In particular, the role of death receptors in many disease states, such as myocardial disease, remains to be elucidated. (38) This complexity in death-receptor function has constrained their potential for pharmacological manipulation. In most cases it is not sufficient to simply activate a specific death receptor. Manipulation of only one of the multiple responses induced by the receptor is desirable. Currently, no solutions to this challenge have been applied. The exception to this conundrum may be TRAIL. Injection of recombinant TRAIL has few side effects in animal studies and combination therapies that use TRAIL as a radiation sensitizer show early promise.
Subject(s)
Apoptosis/physiology , Cardiovascular Diseases/physiopathology , Membrane Glycoproteins/physiology , Receptors, Tumor Necrosis Factor/physiology , Signal Transduction/physiology , Tumor Necrosis Factor-alpha/physiology , Apoptosis Regulatory Proteins , Humans , TNF-Related Apoptosis-Inducing LigandABSTRACT
We report the deduced amino acid sequences of two alternately spliced isoforms, designated DEFCAP-L and -S, that differ in 44 amino acids and encode a novel member of the mammalian Ced-4 family of apoptosis proteins. Similar to the other mammalian Ced-4 proteins (Apaf-1 and Nod1), DEFCAP contains a caspase recruitment domain (CARD) and a putative nucleotide binding domain, signified by a consensus Walker's A box (P-loop) and B box (Mg(2+)-binding site). Like Nod1, but different from Apaf-1, DEFCAP contains a putative regulatory domain containing multiple leucine-rich repeats (LRR). However, a distinguishing feature of the primary sequence of DEFCAP is that DEFCAP contains at its NH(2) terminus a pyrin-like motif and a proline-rich sequence, possibly involved in protein-protein interactions with Src homology domain 3-containing proteins. By using in vitro coimmunoprecipitation experiments, both long and short isoforms were capable of strongly interacting with caspase-2 and exhibited a weaker interaction with caspase-9. Transient overexpression of full-length DEFCAP-L, but not DEFCAP-S, in breast adenocarcinoma cells MCF7 resulted in significant levels of apoptosis. In vitro death assays with transient overexpression of deletion constructs of both isoforms using beta-galactosidase as a reporter gene in MCF7 cells suggest the following: 1) the nucleotide binding domain may act as a negative regulator of the killing activity of DEFCAP; 2) the LRR/CARD represents a putative constitutively active inducer of apoptosis; 3) the killing activity of LRR/CARD is inhibitable by benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone and to a lesser extent by Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone; and 4) the CARD is critical for killing activity of DEFCAP. These results suggest that DEFCAP is a novel member of the mammalian Ced-4 family of proteins capable of inducing apoptosis, and understanding its regulation may elucidate the complex nature of the mammalian apoptosis-promoting machinery.
Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis , Caenorhabditis elegans Proteins , Calcium-Binding Proteins/genetics , Carrier Proteins/genetics , Helminth Proteins/genetics , Protein Isoforms/genetics , Amino Acid Sequence , Apoptosis Regulatory Proteins , Base Sequence , Calcium-Binding Proteins/chemistry , Carrier Proteins/chemistry , Cell Line , Chromosome Mapping , Cloning, Molecular , DNA, Complementary , Helminth Proteins/chemistry , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , NLR Proteins , RNA, Messenger/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
Expansion of a polyglutamine sequence in the N terminus of huntingtin is the gain-of-function event that causes Huntington's disease. This mutation affects primarily the medium-size spiny neurons of the striatum. Huntingtin is expressed in many neuronal and non-neuronal cell types, implying a more general function for the wild-type protein. Here we report that wild-type huntingtin acts by protecting CNS cells from a variety of apoptotic stimuli, including serum withdrawal, death receptors, and pro-apoptotic Bcl-2 homologs. This protection may take place at the level of caspase-9 activation. The full-length protein also modulates the toxicity of the poly-Q expansion. Cells expressing full-length mutant protein are susceptible to fewer death stimuli than cells expressing truncated mutant huntingtin.
Subject(s)
Apoptosis/physiology , Caspases/metabolism , Nerve Tissue Proteins/genetics , Neurons/cytology , Neurons/enzymology , Nuclear Proteins/genetics , Animals , Caspase 3 , Caspase 9 , Cell Line, Transformed , Cell Survival/physiology , Cerebral Cortex/cytology , Corpus Striatum/cytology , Gene Expression Regulation, Enzymologic , In Situ Nick-End Labeling , Mutagenesis/physiology , Neurons/chemistry , Promoter Regions, Genetic/physiology , Proto-Oncogene Proteins c-bcl-2/genetics , Transfection , bcl-X ProteinABSTRACT
By searching the expressed sequence tag database, a novel murine tumor necrosis factor receptor designated TNFRSF19 was identified. TNFRSF19 cDNA encodes a putative membrane protein of 348 amino acids with one incomplete and two complete cysteine-rich motifs within its extracellular region and a large cytoplasmic domain. TNFRSF19 mRNA can be detected in most murine tissues examined, particularly in brain, reproductive organs, and late developmental stages of murine embryo, but not in tissues of the immune system. The cell surface expression of the ligand of TNFRSF19 is highly restricted. Of 22 human and murine cell lines examined by FACS analysis, only Raji (B cell lymphoma cell line), GM847 (fibroblast cell line), 293 (embryonic kidney cell line), and K562 (chronic myeloid leukemia) were positive. TNFRSF19 did not bind newly cloned TNF ligands, including TWEAK (HGMW-approved symbol TNFSF12), VEGI/TL1 (HGMW-approved symbol TNFSF15), TL6/endokine (HGMW-approved symbol TNFSF18), APRIL (HGMW-approved symbol TNFSF13), OPGL (HGMW-approved symbol TNFSF11), LIGHT (HGMW-approved symbol TNFSF14), or BAFF/THANK (HGMW-approved symbol TNFSF13B) by enzyme-linked immunosorbent assay and FACS analyses. Overexpression of TNFRSF19 transduced neither apoptotic signaling nor signals leading to NF-kappaB induction. Taken together with the data that the TNFRSF19 extracellular domain-immunoglobulin fusion protein did not affect the allogeneic mixed lymphocyte reaction, our data indicate that TNFRSF19 is not involved in the modulation of immune responses.
Subject(s)
Genes , Mice/genetics , Multigene Family , Receptors, Tumor Necrosis Factor/genetics , Amino Acid Sequence , Animals , Apoptosis , Cell Line , Embryonic and Fetal Development/genetics , Enzyme-Linked Immunosorbent Assay , Expressed Sequence Tags , Fetal Proteins/biosynthesis , Fetal Proteins/genetics , Flow Cytometry , Humans , Immunity/genetics , Ligands , Lymphocyte Culture Test, Mixed , Molecular Sequence Data , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Organ Specificity , Protein Binding , Protein Structure, Tertiary , Receptors, Tumor Necrosis Factor/biosynthesis , Receptors, Tumor Necrosis Factor/physiology , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , Transcription, Genetic , Transfection , Tumor Cells, CulturedABSTRACT
In this report, we describe the cloning and characterization of Boo, a novel anti-apoptotic member of the Bcl-2 family. The expression of Boo was highly restricted to the ovary and epididymis implicating it in the control of ovarian atresia and sperm maturation. Boo contains the conserved BH1 and BH2 domains, but lacks the BH3 motif. Like Bcl-2, Boo possesses a hydrophobic C-terminus and localizes to intracellular membranes. Boo also has an N-terminal region with strong homology to the BH4 domain found to be important for the function of some anti-apoptotic Bcl-2 homologues. Chromosomal localization analysis assigned Boo to murine chromosome 9 at band d9. Boo inhibits apoptosis, homodimerizes or heterodimerizes with some death-promoting and -suppressing Bcl-2 family members. More importantly, Boo interacts with Apaf-1 and forms a multimeric protein complex with Apaf-1 and caspase-9. Bak and Bik, two pro-apoptotic homologues disrupt the association of Boo and Apaf-1. Furthermore, Boo binds to three distinct regions of Apaf-1. These results demonstrate the evolutionarily conserved nature of the mechanisms of apoptosis. Like Ced-9, the mammalian homologues Boo and Bcl-xL interact with the human counterpart of Ced-4, Apaf-1, and thereby regulate apoptosis.
Subject(s)
Apoptosis/physiology , Proteins/physiology , Proto-Oncogene Proteins c-bcl-2/physiology , Amino Acid Sequence , Animals , Apoptotic Protease-Activating Factor 1 , Caspase 9 , Caspases/chemistry , Caspases/physiology , Cell Line , Chromosome Mapping , Cloning, Molecular , Conserved Sequence , Dimerization , Female , Gene Expression , Humans , In Situ Hybridization, Fluorescence , Macromolecular Substances , Male , Mice , Molecular Sequence Data , Ovary/cytology , Ovary/metabolism , Proteins/chemistry , Proto-Oncogene Proteins c-bcl-2/chemistry , Proto-Oncogene Proteins c-bcl-2/genetics , Sequence Homology, Amino Acid , Spermatozoa/growth & development , Spermatozoa/metabolismABSTRACT
Caspases are a family of cysteine proteases related to interleukin-1 converting enzyme (ICE) and represent the effector arm of the cell death pathway. The zymogen form of all caspases is composed of a prodomain plus large and small catalytic subunits. Herein we report the characterization of a novel caspase, MICE (for mini-ICE), also designated caspase-14, that possesses an unusually short prodomain and is highly expressed in embryonic tissues but absent from all adult tissues examined. In contrast to the other short prodomain caspases (caspase-3, caspase-6, and caspase-7), MICE preferentially associates with large prodomain caspases, including caspase-1, caspase-2, caspase-4, caspase-8, and caspase-10. Also unlike the other short prodomain caspases, MICE was not processed by multiple death stimuli including activation of members of the tumor necrosis factor receptor family and expression of proapoptotic members of the bcl-2 family. Surprisingly, however, overexpression of MICE itself induced apoptosis in MCF7 human breast cancer cells, which was attenuated by traditional caspase inhibitors.
Subject(s)
Caspases/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Amino Acid Sequence , Animals , Caspase 14 , Caspases/metabolism , Cell Line , Cloning, Molecular , DNA, Complementary , Humans , Mice , Molecular Sequence Data , Sequence Homology, Amino Acid , Signal Transduction , Substrate SpecificityABSTRACT
Tumor nectosis factor (TNF) receptors are key players in inflammation and immune regulation. A new member of this family, termed death receptor-6 (DR6), has been identified. Like other death receptors, DR6 is a type I transmembrane receptor, possesses four extracellular cysteine-rich motifs and a cytoplasmic death domain. DR6 is expressed in most human tissues and abundant transcript was detected in heart, brain, placenta, pancreas, thymus, lymph node and several non-lymphoid cancer cell lines. DR6 interacts with TRADD, which has previously been shown to associate with TNFR1. Furthermore, ectopic expression of DR6 in mammalian cells induces apoptosis and activation of both NF-kappaB and JNK.
Subject(s)
Mitogen-Activated Protein Kinases , Receptors, Tumor Necrosis Factor/metabolism , Amino Acid Sequence , Apoptosis/physiology , Base Sequence , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cloning, Molecular , Humans , JNK Mitogen-Activated Protein Kinases , Molecular Sequence Data , NF-kappa B/metabolism , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/physiology , Sequence Homology, Amino Acid , Tumor Cells, CulturedABSTRACT
The pivotal discovery that the death proteases caspase 8 (FLICE) and caspase 10 (Mch4/FLICE2) are recruited to the CD-95 and tumor necrosis factor receptor-1 signaling complexes suggested a mechanism used by these cytotoxic receptors to initiate apoptosis. In this report, we describe the cloning and characterization of I-FLICE, a novel inhibitor of tumor necrosis factor receptor-1- and CD-95-induced apoptosis. The overall architecture of I-FLICE is strikingly similar to that of FLICE and Mch4/FLICE2. However, I-FLICE lacks both a catalytic active site and residues that form the substrate binding pocket, in keeping with its dominant negative inhibitory function. I-FLICE is the first example of a catalytically inert caspase that can inhibit apoptosis.
Subject(s)
Antigens, CD/chemistry , Apoptosis , Carrier Proteins/metabolism , Caspases , Intracellular Signaling Peptides and Proteins , Receptors, Tumor Necrosis Factor/chemistry , fas Receptor/metabolism , Adult , Amino Acid Sequence , CASP8 and FADD-Like Apoptosis Regulating Protein , Carrier Proteins/chemistry , Carrier Proteins/genetics , Caspase 10 , Caspase 8 , Caspase 9 , Catalysis , Cell Line , Cloning, Molecular , Cysteine Endopeptidases/metabolism , Humans , Molecular Sequence Data , Receptors, Tumor Necrosis Factor, Type I , Tissue DistributionABSTRACT
Molluscum contagiosum virus proteins MC159 and MC160 and the equine herpesvirus 2 protein E8 share substantial homology to the death effector domain present in the adaptor molecule Fas-associated death domain protein (FADD) and the initiating death protease FADD-like interleukin-1beta-converting enzyme (FLICE) (caspase-8). FADD and FLICE participate in generating the death signal from both tumor necrosis factor receptor-1 (TNFR-1) and the CD-95 receptor. The flow of death signals from TNFR-1 occurs through the adaptor molecule tumor necrosis factor receptor-associated death domain protein (TRADD) to FADD to FLICE, whereas for CD-95 the receptor directly communicates with FADD and then FLICE. MC159 and E8 inhibited both TNFR-1- and CD-95-induced apoptosis as well as killing mediated by overexpression of the downstream adaptors TRADD and FADD. Neither viral molecule, however, inhibited FLICE-induced killing, consistent with an inhibitory action upstream of the active death protease. These data suggest the existence of a novel strategy employed by viruses to attenuate host immune killing mechanisms. Given that bovine herpesvirus 4 protein E1.1 and Kaposi's sarcoma associated-herpesvirus protein K13 also possess significant homology to the viral inhibitory molecules MC159, MC160, and E8, it may be that this class of proteins is used ubiquitously by viruses to evade host defense.
Subject(s)
Adaptor Proteins, Signal Transducing , Antigens, CD/metabolism , Apoptosis/drug effects , Carrier Proteins/metabolism , Caspases , Membrane Glycoproteins/metabolism , Receptors, Tumor Necrosis Factor/metabolism , fas Receptor/metabolism , Amino Acid Sequence , Animals , Antigens, CD/chemistry , Carrier Proteins/chemistry , Caspase 8 , Caspase 9 , Cattle , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Fas Ligand Protein , Fas-Associated Death Domain Protein , Interleukin-1/metabolism , Membrane Glycoproteins/chemistry , Molecular Sequence Data , Proteins/chemistry , Proteins/metabolism , Receptors, Tumor Necrosis Factor/chemistry , Receptors, Tumor Necrosis Factor, Type I , Sequence Alignment , TNF Receptor-Associated Factor 1 , fas Receptor/chemistryABSTRACT
The pivotal discovery that Fas-associated death domain protein (FADD) interleukin-1beta-converting enzyme (FLICE)/MACH was recruited to the CD95 signaling complex by virtue of its ability to bind the adapter molecule FADD established that this protease has a role in initiating the death pathway (Boldin, M. P., Goncharov, T. M. , Goltsev, Y. V., and Wallach, D. (1996) Cell 85, 803-815; Muzio, M., Chinnaiyan, A. M., Kischkel, K. C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996) Cell 85, 817-827). In this report, we describe the cloning and characterization of a new member of the caspase family, a homologue of FLICE/MACH, and Mch4. Since the overall architecture and function of this molecule is similar to that of FLICE, it has been designated FLICE2. Importantly, the carboxyl-terminal half of the small catalytic subunit that includes amino acids predicted to be involved in substrate binding is distinct. We show that the pro-domain of FLICE2 encodes a functional death effector domain that binds to the corresponding domain in the adapter molecule FADD. Consistent with this finding, FLICE2 is recruited to both the CD95 and p55 tumor necrosis factor receptor signaling complexes in a FADD-dependent manner. A functional role for FLICE2 is suggested by the finding that an active site mutant of FLICE2 inhibits CD95 and tumor necrosis factor receptor-mediated apoptosis. FLICE2 is therefore involved in CD95 and p55 signal transduction.
Subject(s)
Adaptor Proteins, Signal Transducing , Antigens, CD/physiology , Apoptosis , Carrier Proteins/physiology , Caspases , Cysteine Endopeptidases/physiology , Receptors, Tumor Necrosis Factor/physiology , fas Receptor/physiology , Amino Acid Sequence , Base Sequence , Caenorhabditis elegans Proteins , Caspase 10 , Cloning, Molecular , Fas-Associated Death Domain Protein , Gene Expression , Helminth Proteins/chemistry , Humans , Molecular Sequence Data , Protein Binding , Receptors, Tumor Necrosis Factor, Type I , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , Tissue DistributionABSTRACT
A20, a novel zinc finger protein, is an inhibitor of tumor necrosis factor-induced apoptosis. The mechanism by which A20 exerts its protective effect is currently unknown. Several isoforms of the 14-3-3 proteins were found to interact with A20 in a yeast two-hybrid screen. A20 bound several 14-3-3 isoforms in vitro. Moreover, transfected A20 was found to preferentially bind the endogenous eta14-3-3 isoform, whereas the beta/zeta isoforms co-immunoprecipitated much less efficiently, and epsilon14-3-3 had an intermediate affinity. Importantly, c-Raf, a previously described 14-3-3-interacting protein, also preferentially bound the eta isoform. The cellular localization and subcellular fractionation of A20 was dramatically altered by co-transfected 14-3-3, providing the first experimental evidence for the notion that 14-3-3 can function as a chaperone. Furthermore, c-Raf and A20 co-immunoprecipitated in a 14-3-3-dependent manner, suggesting that 14-3-3 can function as a bridging or adapter molecule.
Subject(s)
Molecular Chaperones/metabolism , Proteins/metabolism , Tyrosine 3-Monooxygenase , 14-3-3 Proteins , Amino Acid Sequence , DNA-Binding Proteins , Humans , Immunologic Techniques , Intracellular Signaling Peptides and Proteins , Macromolecular Substances , Molecular Sequence Data , Nuclear Proteins , Peptides/chemistry , Peptides/immunology , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-raf , Recombinant Proteins , Saccharomyces cerevisiae , Signal Transduction , Solubility , Tumor Necrosis Factor alpha-Induced Protein 3 , Zinc FingersABSTRACT
The developmentally regulated sea urchin early histone gene repeat (SUEHGR) from Strongylocentrotus purpuratus was isolated as chromatin by nucleoprotein hybridization. This technique is a novel method to isolate specific sequences as chromatin. Because the purification scheme is based only on the gene sequence and is independent of other physical properties such as protein composition and transcriptional activity, we were able to isolate the same gene in different functional states. Gene size chromatin fragments were solubilized by restriction endonuclease digestion of cell nuclei. Using T7 gene 6 exonuclease, the 3'termini of the fragments were exposed and then hybridized in solution to a biotinylated oligonucleotide complementary to one end of the SUEHGR fragment. The hybrids were bound to an Avidin D matrix. DTT cleavage of the biotin linker yielded a chromatin fraction greater than 700 fold enriched in SUEHGR. Overall yields were between 2% and 15%. The purity of the isolated material was independently measured to be greater than 80%. The homogeneous native structure of the inactive genes was preserved as shown by electron microscopy and micrococcal nuclease digestion of the purified SUEHGR. Minor heterogeneity was observed for the purified active genes by micrococcal nuclease digestion but the main features of the active chromatin were preserved during isolation. This isolation offers the first opportunity to study the structure of an RNA polymerase II gene at different stages of the cell cycle and development.