Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using a yeast two-hybrid screen of a B-cell cDNA library with an Epstein-Barr nuclear antigen 5 (EBNA5) molecule containing seven repeats of the W(1)W(2) domain as bait, we have isolated the EBNA5-interacting protein HAX-1. HAX-1 has previously been shown to associate with HS1, a protein specifically expressed in cells of the haematopoietic lineage, and is thought to be involved in signal transduction in B-cells. Immunofluorescence experiments showed that HAX-1 co-localized with the hsp60 protein that is associated with the mitochondria in the cell cytoplasm. Pull down experiments with a fusion protein between glutathione S-transferase and the seven copy repeat EBNA5 synthesized in bacteria and in yeast cells confirmed that HAX-1 can interact with EBNA5 in vitro. Conventionally, EBNA5 is regarded as a nuclear protein. However, we show here that the smallest EBNA5 species, composed of the unique Y domain and only one copy of the W(1)W(2) repeat domain, like HAX-1, co-localizes with the mitochondrial hsp60 protein in the B-cell cytoplasm. Furthermore, immunoprecipitation experiments demonstrate that the single repeat EBNA5 associates with HAX-1 in transfected B-lymphoblastoid cells.
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PMID:Epstein-Barr virus nuclear antigen 5 interacts with HAX-1, a possible component of the B-cell receptor signalling pathway. 1141 68

Zta has a dual role in the Epstein-Barr virus (EBV) lytic cycle, acting as a key regulator of EBV lytic gene expression and also being essential for lytic viral DNA replication. Zta's replication function is mediated in part through interactions with the core viral replication proteins. We now show interaction between Zta and the helicase (BBLF4) and map the binding region to within amino acids (aa) 22 to 86 of the Zta activation domain. In immunofluorescence assays, green fluorescent protein (GFP)-tagged BBLF4 localized to the cytoplasm of transfected cells. Cotransfection of Zta resulted in translocation of BBLF4-GFP into the nucleus indicating interaction between these two proteins. However, Zta with a deletion of aa 24 to 86 was unable to mediate nuclear translocation of BBLF4-GFP. Results obtained with Zta variants carrying deletions across the aa 24 to 86 region indicated more than one contact site for BBLF4 within this domain, and this was reinforced by the behavior of the four-point mutant Zta (m22/26,74/75), which was severely impaired for BBLF4 interaction. Binding of BBLF4 to Zta was confirmed using GST affinity assays. In both cotransfection-replication assays and replication assays performed in EBV-positive P3HR1 cells, the Zta (m22/26,74/75) mutant was replication defective. In Zta-transfected D98-HR1 cells, replication compartments could be detected by immunofluorescence staining using anti-BMRF1 monoclonal antibody. Cells transfected with Zta variants that were defective for helicase binding still formed replication compartments, but Zta was excluded from these compartments. These experiments reveal a role for the Zta-helicase interaction in targeting Zta to sites of viral DNA replication.
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PMID:Interaction with the Epstein-Barr virus helicase targets Zta to DNA replication compartments. 1150 24

Radiotherapy is the modality of choice for the treatment of nasopharyngeal carcinoma (NPC). However, systemic chemotherapy has recently been found to play an increasing role in the treatment of advanced or metastatic disease. The status of drug resistance gene expression that has crucial impact on chemotherapy has not been fully addressed for patients with NPC. In this study, we examined the expression of multidrug resistance 1 (MDR-1) and glutathione-S-transferase-Pi (GST-Pi) in primary, recurrent, and metastatic NPC using results of immunohistochemical examinations. The results were correlated with the expression of Epstein-Barr virus (EBV) latent protein, latent membrane protein 1 (LMP1), and clinicopathologic features, including stage, histopathologic types, and survival rates. MDR-1 protein expression was detected in 18 (12.6%) of 143 patients with primary NPC, 14 (32.6%) of 43 with recurrent NPC, and O (0%) of 20 with metastatic NPC, whereas 83 (58%) of 143 patients with primary NPC, 30 (69.8%) of 43 with recurrent NPC, and 13 (65%) of 20 with metastatic NPC expressed GST-Pi. EBV-LMP1 was expressed in 59 (41.3%) of 143 patients with primary NPC, 23 (53.5%) of 43 with recurrent NPC, and 9 (45%) of 20 with metastatic NPC. Simultaneous expression of MDR1 and GST-Pi was observed in 13 (72.2%) of 18 patients with primary NPC and 12 (85.7%) of 14 with recurrent NPC. The expression of LMP1 was detected in only 6 of the 13 patients with primary NPC and 6 of the 12 with recurrent NPC. We concluded that the expression of GST-Pi was more frequent in NPC tumor tissues than the expression of MDR-1. The expression of MDR-1 correlated with clinicopathologic features of primary NPC, including the histopathologic types and survival rates, but not with disease stage. The expression of GST-Pi did not correlate with clinicopathologic features. The expression of MDR-1 and GST-Pi did not correlate with expression of EBV-LMP1 for patients with NPC.
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PMID:Expression of multidrug resistance 1 and glutathione-S-transferase-Pi protein in nasopharyngeal carcinoma. 1172 64

Self-association of viral proteins is important for many of their functions, including enzymatic, transcriptional, and transformational activities. Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) contains various numbers of W1W2 repeats and a unique carboxyl-terminal Y1Y2 domain. It was reported that EBNA-LP associates with a variety of cellular proteins and plays a critical role in EBV-induced transformation. We report here that EBNA-LP self-associates in vivo and the domain responsible for the homotypic association is a multifunctional domain mediating nuclear localization, nuclear matrix association, and EBNA-2-dependent coactivator function of the protein. Our conclusions are based on the following observations. (i) EBNA-LP interacts with itself or its derivatives in the yeast two-hybrid system. (ii) A purified chimeric protein consisting of glutathione S-transferase fused to EBNA-LP specifically formed complexes with EBNA-LP transiently expressed in COS-7 cells. (iii) When Flag epitope-tagged EBNA-LP with either one or two W1W2 repeats and EBNA-LP containing four W1W2 repeats were coexpressed in COS-7 cells, the latter was specifically coimmunoprecipitated with the former. (iv) Mutational analyses of EBNA-LP with deletion mutants revealed that the region between codons 19 and 39 (relative to the first amino acid residue of the W2 domain) is essential for self-association of the protein. The mapped region almost completely overlaps with CR2 and CR3, regions conserved among a subset of primate gamma-herpesviruses and critical for EBNA-2-dependent coactivator function. Amino acid substitutions in CR2 alone abolished the ability of the protein to self-interact. This laboratory previously reported that CR2 is also responsible for nuclear localization and nuclear matrix association (A. Yokoyama, Y. Kawaguchi, I. Kitabayashi, M. Ohki, and K. Hirai, Virology 279:401-413, 2001). (v) Sucrose gradient sedimentation showed that amino acid substitutions in CR2 reduced the ability of the protein to form protein complexes in B cells. These results suggest that self-association of EBNA-LP may be important for its various functions and interactions of the protein with multiple cellular proteins.
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PMID:Conserved region CR2 of Epstein-Barr virus nuclear antigen leader protein is a multifunctional domain that mediates self-association as well as nuclear localization and nuclear matrix association. 1177 78

The latent membrane protein 1 (LMP1) of Epstein-Barr virus causes cellular transformation and activates several intracellular signals, including NF-kappaB and c-Jun N-terminal kinase. Using yeast two-hybrid screening with the LMP1 C-terminal sequence as bait, we demonstrate that BRAM1 (bone morphogenetic protein receptor-associated molecule 1) is an LMP1-interacting protein. BRAM1 associates with LMP1, both in vitro and in vivo, as revealed by confocal microscopy, glutathione S-transferase pull-down, and co-immunoprecipitation assays. This association mainly involves the C-terminal half of BRAM1 comprising the MYND domain and the CTAR2 region of LMP1, which is critical in LMP1-mediated signaling pathways. We show that BRAM1 interferes with LMP1-mediated NF-kappaB activation but not the JNK signaling pathway. Because the CTAR2 region interacts with the tumor necrosis factor (TNF-alpha receptor-associated death domain protein, it is interesting to find that BRAM1 also interferes with NF-kappaB activation mediated by TNF-alpha. BRAM1 interferes LMP1-mediated and TNF-alpha-induced NF-kappaB activation by targeting IkappaBalpha molecules. Moreover, BRAM1 inhibits the resistance of LMP1-expressing cells to TNF-alpha-induced cytotoxicity. We therefore propose that the BRAM1 molecule associates with LMP1 and functions as a negative regulator of LMP1-mediated biological functions.
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PMID:Negative regulation of Epstein-Barr virus latent membrane protein 1-mediated functions by the bone morphogenetic protein receptor IA-binding protein, BRAM1. 1218 23

Earlier studies have shown that translation elongation factor 1delta (EF-1delta) is hyperphosphorylated in various mammalian cells infected with representative alpha-, beta-, and gammaherpesviruses and that the modification is mediated by conserved viral protein kinases encoded by herpesviruses, including UL13 of herpes simplex virus type 1 (HSV-1), UL97 of human cytomegalovirus, and BGLF4 of Epstein-Barr virus (EBV). In the present study, we attempted to identify the site in EF-1delta associated with the hyperphosphorylation by the herpesvirus protein kinases. Our results are as follows: (i) not only in infected cells but also in uninfected cells, replacement of the serine residue at position 133 (Ser-133) of EF-1delta by alanine precluded the posttranslational processing of EF-1delta, which corresponds to the hyperphosphorylation. (ii) A purified chimeric protein consisting of maltose binding protein (MBP) fused to a domain of EF-1delta containing Ser-133 (MBP-EFWt) is specifically phosphorylated in in vitro kinase assays by purified recombinant UL13 fused to glutathione S-transferase (GST) expressed in the baculovirus system. In contrast, the level of phosphorylation by the recombinant UL13 of MBP-EFWt carrying an alanine replacement of Ser-133 (MBP-EFS133A) was greatly impaired. (iii) MBP-EFWt is also specifically phosphorylated in vitro by purified recombinant BGLF4 fused to GST expressed in the baculovirus system, and the level of phosphorylation of MBP-EFS133A by the recombinant BGLF4 was greatly reduced. (iv) The sequence flanking Ser-133 of EF-1delta completely matches the consensus phosphorylation site for a cellular protein kinase, cdc2, and in vitro kinase assays revealed that purified cdc2 phosphorylates Ser-133 of EF-1delta. (v) As observed with EF-1delta, the casein kinase II beta subunit (CKIIbeta) was specifically phosphorylated by UL13 in vitro, while the level of phosphorylation of CKIIbeta by UL13 was greatly diminished when a serine residue at position 209, which has been reported to be phosphorylated by cdc2, was replaced with alanine. These results indicate that the conserved protein kinases encoded by herpesviruses and a cellular protein kinase, cdc2, have the ability to target the same amino acid residues for phosphorylation. Our results raise the possibility that the viral protein kinases mimic cdc2 in infected cells.
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PMID:Conserved protein kinases encoded by herpesviruses and cellular protein kinase cdc2 target the same phosphorylation site in eukaryotic elongation factor 1delta. 1255 73

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) consists of W1W2 repeats and a unique C-terminal Y1Y2 domain and plays a critical role in EBV-induced transformation. To identify the cellular proteins associating with EBNA-LP, we performed a yeast two-hybrid screen using EBNA-LP cDNA containing a single W1W2 domain as bait and an EBV-transformed human peripheral blood lymphocyte cDNA library as the source of cellular genes. Our results were as follows. (i) A cDNA in the positive yeast colony was found to encode a cellular protein, human oestrogen-related receptor 1 (hERR1), which is a constitutive transcriptional activator of the various types of oestrogen response elements. (ii) A purified chimeric protein consisting of glutathione S-transferase (GST) fused to hERR1 specifically formed complexes with EBNA-LPs containing one (EBNA-LPR1), two (EBNA-LPR2) or four W1W2 repeats (EBNA-LPR4) transiently expressed in COS-7 cells. Reciprocally, GST fused to EBNA-LPR1 or EBNA-LPR2 pulled down hERR1 transiently expressed in COS-7 cells. (iii) Mutational analyses of EBNA-LP revealed that the Y2 domain of EBNA-LP is responsible for the interaction with hERR1 and two leucines in the Y2 domain (Leu-78 and -82), which are conserved among a subset of primate gammaherpesviruses, are interactive sites for hERR1. So far, it has been reported that the only domain of EBNA-LP critical for EBV-induced transformation is the Y1Y2 domain. Potential roles of hERR1 in EBV-induced transformation are discussed.
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PMID:Physical interaction of Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) with human oestrogen-related receptor 1 (hERR1): hERR1 interacts with a conserved domain of EBNA-LP that is critical for EBV-induced B-cell immortalization. 1256 May 63

Here we provide evidence that EBNA2 is methylated in vivo and that methylation of EBNA2 is a prerequisite for binding to SMN. We present SMN as a novel binding partner of EBNA2 by showing that EBNA2 colocalizes with SMN in nuclear gems and that both proteins can be coimmunoprecipitated from cellular extract. Furthermore, in vitro methylation of either wild-type EBNA2 or a glutathione S-transferase-EBNA2 fusion protein encompassing the arginine-glycine (RG) repeat element is necessary for in vitro binding to the Tudor domain of SMN. The recently shown functional cooperation of SMN and EBNA2 in transcriptional activation and the previous observation of a severely reduced transformation potential yet strongly enhanced transcriptional activity of an EBNA2 mutant lacking the RG repeat indicate that binding of SMN to EBNA2 is a critical step in B-cell transformation by Epstein-Barr virus.
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PMID:Epstein-Barr virus nuclear antigen 2 binds via its methylated arginine-glycine repeat to the survival motor neuron protein. 1266 8

Epstein-Barr virus (EBV) carrying lymphoblastoid cells of normal origin express the full program of all 9 virus-encoded, growth transformation associated proteins. They have an intact p53 pathway as a rule. This raises the question of whether any of the viral proteins impair the pathway functionally. Using a yeast 2-hybrid system, we have shown that EBNA-5 but not the other EBNAs interacts with the p14ARF protein, a regulator of the p53 pathway. The interaction was confirmed in vitro using a GST pull-down assay. Moreover, expression of EBNA-5 increased the survival of p14ARF-transfected cells. EBV infection of resting B cells induced the expression of p14ARF mRNA without increased level of the protein. A fraction of the p14ARF localized to the nucleoli but the bulk of the protein accumulated in nuclear but extranucleolar inclusions. Formation of the extranucleolar inclusions led to complete relocalization of EBNA-5 from nucleoplasm to these structures. The inclusions also contained p53 and HDM2, and were surrounded by PML bodies and proteasomes, which suggests that these inclusions could be targets for proteasome dependent protein degradation.
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PMID:EBV-encoded EBNA-5 associates with P14ARF in extranucleolar inclusions and prolongs the survival of P14ARF-expressing cells. 1274 Sep 13

The Epstein-Barr virus (EBV) protein EB2 (also called Mta, SM, or BMLF1) has properties in common with mRNA export factors and is essential for the production of EBV infectious virions. However, to date no RNA-binding motif essential for EB2-mediated mRNA export has been located in the protein. We show here by Northwestern blot analysis that the EB2 protein purified from mammalian cells binds directly to RNA. Furthermore, using overlapping glutathione S-transferase (GST)-EB2 peptides, we have, by RNA electrophoretic mobility shift assays (REMSAs) and Northwestern blotting, located an RNA-binding motif in a 33-amino acid segment of EB2 that has structural features of the arginine-rich RNA-binding motifs (ARMs) also found in many RNA-binding proteins. A synthetic peptide (called Da), which contains this EB2 ARM, bound RNA in REMSA. A GST-Da fusion protein also bound RNA in REMSA without apparent RNA sequence specificity, because approximately 10 GST-Da molecules bound at multiple sites on a 180-nucleotide RNA fragment. Importantly, a short deletion in the ARM region impaired both EB2 binding to RNA in vivo and in vitro and EB2-mediated mRNA export without affecting the shuttling of EB2 between the nucleus and the cytoplasm. Moreover, ectopic expression of ARM-deleted EB2 did not rescue the production of infectious virions by 293 cells carrying an EBVDeltaEB2 genome, which suggests that the binding of EB2 to RNA plays an essential role in the EBV productive cycle.
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PMID:A region of the Epstein-Barr virus (EBV) mRNA export factor EB2 containing an arginine-rich motif mediates direct binding to RNA. 1285 28


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