Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mouse Fas/APO-1 antigen represents a 45-kilodalton transmembrane receptor that initiates apoptosis by a poorly defined signaling mechanism. The cytoplasmic domain of Fas does not display any known enzymatic activities but is capable of interacting with a number of proteins that were identified recently using the yeast interactive cloning method. To investigate direct biochemical interactions from cellular lysates prepared from Fas-responsive cells, a series of recombinant glutathione S-transferase-mouse Fas fusion proteins representing different regions of the mouse Fas cytoplasmic domain was used. Polypeptides of 25, 50, and 70 kilodaltons were found to associate with the Fas intracellular domain, and this binding was stable in the presence of 1 M NaCl. These interactions were also detected using a mouse Fas fusion protein containing an Ile to Asn mutation, which is responsible for a lymphoproliferative disorder in certain strains of mice (lprcg). Furthermore, the binding of cellular proteins to Fas could be blocked upon incubation with a polyclonal antibody directed against the cytoplasmic domain of Fas. The strong association of cellular proteins with the cytoplasmic region implies that constitutive interactions may exist to regulate apoptotic signaling through the Fas antigen.
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PMID:Interactions of cellular polypeptides with the cytoplasmic domain of the mouse Fas antigen. 862 93

An expression vector, pLEF, has been used to produce the intracellular domain (IC) of the human CD95 (Fas/APO-1) apoptosis receptor as a glutathione S-transferase (GST) fusion protein in murine L929 fibroblasts. GST::CD95IC was affinity-purified in a single step using glutathione-Sepharose. Purification of GST::CD95IC from 32P-labelled L929 cells and cleavage with thrombin revealed that CD95IC was phosphorylated in vivo when produced as a GST fusion protein. Therefore, pLEF may facilitate the mapping of in vivo-modified sites of eukaryotic proteins.
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PMID:pLEF, a novel vector for expression of glutathione S-transferase fusion proteins in mammalian cells. 864 62

Sentrin is a ubiquitin-like molecule that has been shown to interact with the death domains of Fas and tumor necrosis factor receptor 1 (TNFR1), PML, Rad51, Rad52, and RanGAP1. We have reported previously that sentrin can be conjugated to other proteins in a manner analogous to protein ubiquitination (Kamitani, T., Nguyen, H. P., and Yeh, E. T. H. (1997) J. Biol. Chem. 272, 14001-14004). Furthermore, the conserved C-terminal Gly-Gly residues are required for sentrinization to occur. To identify enzymes which play a role in sentrinization, the yeast two-hybrid system was used to screen a human placenta cDNA library using sentrin as bait. A strong positive interacting clone was found to contain a cDNA insert encoding the ubiquitin-conjugating enzyme, Ubc9. The interaction between sentrin and Ubc9 required the ubiquitin domain and the C-terminal Gly-Gly residues of sentrin. This interaction appears to be specific because sentrin could only interact weakly with UbcH5B, but could not interact with HHR6B, UbcH6 nor E2-EPF. In vitro translated sentrin could be precipitated by a GST-Ubc9 fusion protein, but not by glutathione S-transferase. A beta-mercaptoethanol-sensitive Ubc9-sentrin conjugate could also be identified in the in vitro binding assay. Substitution of the conserved cysteine residue of Ubc9 by serine abolished the formation of the Ubc9-sentrin conjugate. Taken together, Ubc9 is a strong candidate to be the key conjugating enzyme in the sentrinization pathway.
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PMID:Preferential interaction of sentrin with a ubiquitin-conjugating enzyme, Ubc9. 935 68

The Bcl-2 family member Bcl-xL has often been correlated with apoptosis resistance. We have shown recently that in peripheral human T cells resistance to CD95-mediated apoptosis is characterized by a lack of caspase-8 recruitment to the CD95 death-inducing signaling complex (DISC) and by increased expression of Bcl-xL (Peter, M. E., Kischkel, F. C., Scheuerpflug, C. G., Medema, J. P., Debatin, K.-M., and Krammer, P. H. (1997) Eur. J. Immunol. 27, 1207-1212). This raises the possibility that Bcl-xL directly prevents caspase-8 activation by the DISC. To test this hypothesis a cell line in which CD95 signaling was inhibited by overexpression of Bcl-xL was used. In these MCF7-Fas-bcl-xL cells Bcl-xL had no effect on the recruitment of caspase-8 to the DISC. It did not affect the activity of the DISC nor the generation of the caspase-8 active subunits p18 and p10. In contrast, cleavage of a typical substrate for caspase-3-like proteases, poly(ADP-ribose) polymerase, was inhibited in comparison with the control-transfected CD95-sensitive MCF7-Fas cells. To test whether Bcl-xL would inhibit active caspase-8 subunits in the cytoplasm, a number of immunoprecipitation experiments were performed. Using monoclonal antibodies directed against different domains of caspase-8, anti-Bcl-xL antibodies, or fusion proteins of glutathione S-transferase with different domains of caspase-8, no evidence for a direct or indirect physical interaction between caspase-8 and Bcl-xL was found. Moreover, overexpression of Bcl-xL did not inhibit the activity of the caspase-8 active subunits p18/p10. Therefore, in this cell line that has become resistant to CD95-induced apoptosis due to overexpression of Bcl-xL, Bcl-xL acts independently and downstream of caspase-8.
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PMID:Bcl-xL acts downstream of caspase-8 activation by the CD95 death-inducing signaling complex. 945 59

Fas (Apo-1, CD95), a member of the TNFR family, is expressed on a variety of cell types and transduces an apoptotic signal. Since Fas does not possess known enzymatic activities, proteins that interact with the cytoplasmic domain of Fas regulate the death signal. Several proteins have been identified, primarily using the yeast two-hybrid system, that associate with the death domain of Fas. One of these proteins, FADD/MORT1, can be phosphorylated, although the kinase that is responsible has not been identified. Furthermore, direct signaling connections between Fas and its known activation of sphingomyelinase or NF-kappaB have not been made, suggesting that other proteins may associate with Fas. In this study, a series of glutathione S-transferase fusion proteins was constructed that contained the cytoplasmic domain of murine Fas. These proteins were used to search for additional proteins that associate with Fas. Novel proteins, including kinases, were identified that associated specifically with the membrane-proximal, cytoplasmic tail of Fas but not with the death domain. One of these kinases phosphorylates FADD/MORT1. Moreover, the membrane-proximal region of Fas itself was phosphorylated by one of the associating kinases. These findings suggest that, similar to the Fas-related p55 TNFR, the membrane-proximal region of Fas likely participates in signaling.
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PMID:Phosphorylation of FADD/MORT1 and Fas by kinases that associate with the membrane-proximal cytoplasmic domain of Fas. 959 Feb 35

We have identified and characterized a cDNA encoding human Fas associated factor 1 (hFAF1) cDNA and a shorter form of hFAF1 cDNA [hFAF1(s)] with a 456 bp internal in-frame deletion from a human HeLa cDNA library. The nucleotide sequences of hFAF1 and hFAF1(s) were identical except for the deletion. GST-hFAF1 fusion protein bound to the in vitro translation product of Fas. The N-terminal region (amino acid 1 approximately 201) including the upstream ubiquitin homology domain of hFAF1 could bind with the death domain of Fas unlike that of qFAF1 whose binding region with Fas could not be determined. However hFAF1 did not bind to the death domain of Fas mutant, lpr(cg). hFAF1 was expressed abundantly in testis, skeletal muscle, and heart as 2.8 kb mRNA. Polyclonal antibody against hFAF1 detected 74 kD protein, a deduced protein size from the ORF and 40 kD protein in some cell lines.
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PMID:Identification and characterization of human Fas associated factor 1, hFAF1. 1046 85

In attempting to produce the intracellular portion of human Fas (IC175 - 319) as a GST-fusion protein we found that expression of GST-IC175 - 319, but not GST alone or GST-IC231 - 298 (containing the Fas death domain), rapidly caused the death of host E. coli cells. Expression of GST-IC175 - 319 with a single amino acid substitution (V238N) corresponding to the mouse lprcg mutation, or E245A, which abolishes the ability of Fas to self-associate, did not kill bacteria. Deletional analysis identified a 20-amino acids region (Asp210 - Lys230) as essential for the killing activity, and introduction of a single amino acid substitution (T225P) in this 20 amino acid region markedly decreased the ability of Fas- IC175 - 319 to cause bacterial death. These data indicate that Fas can deliver a death signal in prokaryotic organisms by a means that shares some features with eukaryotic cells, and raise the possibility that certain mechanisms leading to programmed cell death may be conserved from bacteria to mammalian cells.
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PMID:Bacterial death induced by expression of the intracellular portion of human Fas. 1046 55

Previous reports have indicated that apoptosis is selectively decreased in enzyme-altered foci (EAF) in the livers of rats treated with a carcinogen. Here we have investigated the effects of an anti-Fas antibody (anti-Fas Ab) on EAF cells in vitro. Hepatocytes were isolated from rats treated repeatedly with diethylnitrosamine (DEN), whose livers contained glutathione S-transferase P (GST-P)-positive EAF. Subsequently, primary cultures of GST-P-positive and GST-P-negative hepatocytes were established and exposed to anti-Fas Ab. Anti-Fas Ab (4 microg/ml) preferentially induced apoptosis in GST-P-negative cells. Furthermore, GST-P-positive cells were shown to be resistant to p53-mediated apoptosis. We conclude that EAF hepatocytes are resistant to Fas-mediated apoptosis in vitro. This lack of response may explain the selective decrease in apoptosis in EAF.
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PMID:Fas-mediated apoptosis is attenuated in preneoplastic GST-P-positive hepatocytes isolated from diethylnitrosamine-treated rats. 1069 23

Sentrin is a ubiquitin-like protein that can covalently modify cellular proteins, and is a Fas binding protein that protects cells against anti-Fas induced cell death. However, the mechanism by which sentrin exerts its effect upon Fas-mediated apoptosis is not well known. Thus, this study examined the interaction of sentrin with Daxx. Sentrin interacted with Daxx but not with FADD when analyzed by yeast two-hybrid assay. In vitro translated Daxx bound to GST-sentrin fusion protein. FLAG-sentrin fusion protein was also coimmunoprecipitated with Daxx in BOSC23 cells. Also, Daxx interacted with Ubc9, an essential protein as a key conjugating enzyme. Amino acids 625-740 of Daxx, known as Fas binding region, was also mapped as sentrin and Ubc9 binding region. Colocalization of Fas, sentrin, and Ubc9 binding regions suggests the importance of that region upon the regulation of Daxx. Our data also demonstrated that sentrin could homooligomerize by protein-protein interaction.
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PMID:Interaction of Daxx, a Fas binding protein, with sentrin and Ubc9. 1111 9

The transcription factor NF-kappaB regulates a wide set of genes involved in the establishment of many cellular processes that control cell activation, proliferation, and apoptosis. IkappaB inhibitory subunits integrate NF-kappaB activation signals through phosphorylation and ubiquitination of its N-terminal domain. Using the two-hybrid system in yeast, we searched for IkappaB-alpha N-terminal domain interactors and therefore potential NF-kappaB regulators. An interaction of IkappaB-alpha with the mitochondrial ATP/ADP translocator ANT was detected in yeast and confirmed in glutathione S-transferase pull-down assays and co-precipitation experiments in transfected cells. Subcellular cell fractionation, resistance to proteinase K treatment, and electron microscopy experiments demonstrated the presence of IkappaB-alpha and associated p65 NF-kappaB in the mitochondrial intermembrane space. IkappaB-alpha.NF-kappaB appeared to be released from mitochondria upon the induction of apoptosis by engagement of the Fas receptor. These data suggest that the mitochondrial IkappaB-alpha.NF-kappaB pool participates in the regulation of apoptosis.
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PMID:Ikappa b-alpha, the NF-kappa B inhibitory subunit, interacts with ANT, the mitochondrial ATP/ADP translocator. 1128 11


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