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)

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is a phosphoprotein suggested to play important roles in EBV-induced immortalization. Earlier studies have shown that the major site of phosphorylation of EBNA-LP by cellular kinase(s) is a serine residue at position 35 (Ser-35) and that the phosphorylation of Ser-35 is critical for regulation of the coactivator function of EBNA-LP (Yokoyama et al., J Virol 75, 5119-5128, 2001). In the present study, we have attempted to identify protein kinase(s) responsible for the phosphorylation of EBNA-LP at Ser-35. A purified chimeric protein consisting of glutathione S-transferase (GST) fused to a domain of EBNA-LP containing Ser-35 was found to be specifically phosphorylated by purified cdc2 in vitro, while GST fused to a mutated domain of EBNA-LP in which Ser-35 was replaced with alanine was not. In addition, overexpression of cdc2 in mammalian cells caused a significant increase in the phosphorylation of EBNA-LP, while this increased phosphorylation was eliminated if Ser-35 of EBNA-LP was replaced with alanine. These results indicate that the cellular protein kinase cdc2 mediates the phosphorylation of EBNA-LP at Ser-35. Recently, we reported that cdc2 and conserved protein kinases encoded by herpesviruses phosphorylate the same amino acid residue of target proteins (Kawaguchi et al., J Virol 77, 2359-2368, 2003). Consistent with this, the EBV-encoded conserved protein kinase BGLF4 specifically mediated the phosphorylation of EBNA-LP at Ser-35. These results indicate that the coactivator function of EBNA-LP can be regulated by the activity of these cellular and viral protein kinases.
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PMID:Identification of protein kinases responsible for phosphorylation of Epstein-Barr virus nuclear antigen leader protein at serine-35, which regulates its coactivator function. 1464 19

Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is critical for EBV immortalization of infected B lymphocytes and can coactivate the EBV LMP1 promoter with EBNA2. EBNA3C amino acids 365 to 545 are necessary and sufficient for coactivation and are required for SUMO-1 and SUMO-3 interaction. We found that EBNA3C but not EBNA3CDelta343-545 colocalized with SUMO-1 in nuclear bodies and was modified by SUMO-2, SUMO-3, and SUMO-1. EBNA3C amino acids 545 to 628 and amino acids 30 to 365 were also required for EBNA3C sumolation and nuclear body localization but were dispensable for coactivation, indicating that EBNA3C sumolation is not required for coactivation. Furthermore, EBNA3C amino acids 476 to 992 potently coactivated with EBNA2 but EBNA3C amino acids 516 to 922 lacked activity, indicating that amino acids 476 to 515 are critical for coactivation. EBNA3C amino acids 476 to 515 include DDDVIEV(507-513), which are similar to SUMO-1 EEDVIEV(84-90). EBNA3C m1 and m2 point mutations, DDD(507-509) mutated to AAA and DVIEVID(509-513) mutated to AVIAVIA, respectively, diminished SUMO-1 and SUMO-3 interaction in directed yeast two-hybrid and glutathione S-transferase pulldown assays. Furthermore, EBNA3C m1 and m2 did not coactivate the LMP1 promoter with EBNA2. Overexpression of wild-type SUMO-1, SUMO-3, and the SUMO-conjugating enzyme UBC9 coactivated the LMP1 promoter with EBNA2. Since EBNA2 activation is dependent on p300/CBP, the possible effect of EBNA3C on p300-mediated transcription was assayed. EBNA3C potentiated transcription of p300 fused to a heterologous DNA binding domain, whereas EBNA3C m1 and m2 did not. All of these data are consistent with a model in which EBNA3C upregulates EBNA2-mediated gene activation by binding to a sumolated repressor and inhibiting repressive effects on p300/CBP and other transcription factor(s) at EBNA2-regulated promoters.
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PMID:EBNA3C coactivation with EBNA2 requires a SUMO homology domain. 1467 Nov 18

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with cancers in immunocompromised populations. Antiviral drugs targeted against lytic viral replication have limited efficacy in these disease settings. EBV infection of peripheral blood mononuclear cells induces growth proliferation and the EBV latency Epstein-Barr virus-encoded nuclear antigen (EBNA)2 transcriptional transactivator (TAT) is essential for this response. EBNA2 targets the cellular DNA-binding protein CBF1 to mimic activated Notch signaling. A 10-aa peptide from the CBF1 interaction domain of EBNA2 was synthesized as a fusion with the protein transduction domain of HIV-1 TAT. The EBNA2-TAT peptide blocked EBNA2-CBF1 interaction in an in vitro GST affinity assay and labeling with fluorescein confirmed that the EBNA2-TAT peptide efficiently entered cultured B cells. Neither EBNA2-TAT, nor a mutant peptide with a 2-aa substitution that was unable to block the EBNA2-CBF1 interaction, significantly affected the growth of non-EBNA2-expressing EBV(-) B cells or Burkitt's lymphoma Akata cells. However, treatment of an EBV-immortalized lymphoblastoid cell line with the EBNA2-TAT peptide stopped cell growth and reduced cell viability. RT-PCR analyses of gene expression in the peptide-treated lymphoblastoid cell line cultures revealed that EBNA2-TAT treatment down-regulated the EBNA2-responsive viral LMP1 and LMP2 genes and cellular CD23, intercellular adhesion molecule 1, BATF, and Cdk1 genes while up-regulating expression of the cyclin-dependent kinase inhibitor p21. EBV-induced outgrowth of B cells from cultured peripheral blood mononuclear cells was also blocked in a dose-responsive manner by the EBNA2-TAT peptide. This study suggests that cell-permeable EBNA2 peptides may have potential as novel anti-EBV therapeutics.
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PMID:Inhibition of Epstein-Barr virus-induced growth proliferation by a nuclear antigen EBNA2-TAT peptide. 1507 Jul 68

Epstein-Barr virus (EBV) expresses an immediate-early protein, Rta, to activate the transcription of EBV lytic genes and the lytic cycle. This work identifies Ubc9 and PIAS1 as binding partners of Rta in a yeast two-hybrid screen. These bindings are verified by glutathione S-transferase pull-down assay, coimmunoprecipitation, and confocal microscopy. The interactions appear to cause Rta sumoylation, because not only can Rta be sumoylated in vitro but also sumoylated Rta can be detected in P3HR1 cells following lytic induction and in 293T cells after transfecting plasmids that express Rta and SUMO-1. Moreover, PIAS1 stimulates conjugation of SUMO-1 to Rta, thus acting as an E3 ligase. Furthermore, transfecting plasmids that express Ubc9, PIAS1, and SUMO-1 increases the capacity of Rta to transactivate the promoter that includes an Rta response element, indicating that the modification by SUMO-1 increases the transactivation activity of Rta. This study reveals that Rta is sumoylated at the Lys-19, Lys-213, and Lys-517 residues and that SUMO-1 conjugation at the Lys-19 residue is crucial for enhancing the transactivation activity of Rta. These results indicate that sumoylation of Rta may be important in EBV lytic activation.
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PMID:Post-translational modification of Rta of Epstein-Barr virus by SUMO-1. 1522 20

The DNA-binding protein recombination signal-binding protein-Jk (RBP-J) plays a key role in transcriptional regulation by targeting the intracellular domain of Notch (NIC) and the Epstein-Barr virus nuclear antigen 2 (EBNA2) to specific promoters. In the absence of the Notch signaling, RBP-J acts as a transcriptional suppressor through recruiting co-suppressors such as histone deacetylase (HDAC). KyoT2 is a LIM domain protein that suppresses the RBP-J-mediated transcriptional activation. In the current study, we show that the polycomb group (PcG) protein HPC2, which functions as a transcriptional suppressor, is a candidate of KyoT2-binding proteins. To confirm the physical and functional interaction between KyoT2 and HPC2, we carried out yeast two-hybrid, GST-pull down, co-immunoprecipitation, as well as mammalian two-hybrid assays. Our results showed HPC2 and KyoT2 interacted both in vitro and in vivo, probably through the C-terminal fragment of HPC2 and LIM domains of KyoT2. In addition, we also found that overexpression of HPC2, not only inhibited transactivation of a RBP-J-dependent promoter by NIC, but also transactivation by RBP-J-VP16, a constitutively active form of RBP-J. Taken together, our results suggested that KyoT2 might inhibit the RBP-J-mediated transactivation through NIC by recruiting co-suppressors such as HPC2.
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PMID:The PcG protein HPC2 inhibits RBP-J-mediated transcription by interacting with LIM protein KyoT2. 1571 Apr 17

Latency of Epstein-Barr virus (EBV) is maintained by the transmembrane protein latent membrane protein (LMP) 2A, which mimics the B-cell receptor (BCR) and perturbs BCR signaling. LMP2A contains a cytoplasmic N-terminal domain composed of 119 amino acids, which provides signals that are responsible for the association with various signal molecules, resulting in negative regulation of B-cell signaling and the EBV lytic cycle. In the present study, to obtain N-terminal domain of LMP2A (LMP2A NTD, 13 kDa) in Escherichia coli for structural analysis, a strategy for obtaining the unfused form of LMP2A NTD without any fusion partners was proposed. Recombinant LMP2A NTD has previously been expressed using the GST fusion system in E. coli [Virology 268 (2000) 178, J. Virol. 71 (1997) 4752, Mol. Cell. Biol. 20 (2000) 8526]. However, we were unable to obtain untagged LMP2A NTD from this construct because of rapid proteolysis by thrombin. To overcome the proteolysis by thrombin, C-terminal His-tagged LMP2A NTD and intein-fused LMP2A NTD were prepared. As a result, LMP2A NTD without a fusion partner could be successfully obtained using non-enzymatic cleavage. The secondary structure of the recombinant LMP2A NTD was analyzed using circular dichroism. In aqueous solution, LMP2A NTD adopts an unordered structure, which was not affected by varying pH and salt concentration. In addition, any secondary structural components of LMP2A NTD were not induced in the membrane-mimicking environments, suggesting that LMP2A NTD may intrinsically have a random coil-like structure. The biological activity of recombinant LMP2A NTD was monitored by chemical shift perturbation in HSQC spectra of LMP2A NTD with or without WW domains, which result supports that the structural change induced by WW domains is restricted within narrow region.
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PMID:Expression and characterization of N-terminal domain of Epstein-Barr virus latent membrane protein 2A in Escherichia coli. 1580 16

A yeast two-hybrid screen using EBNA3C as bait revealed an interaction between this Epstein-Barr virus (EBV)-encoded nuclear protein and the C8 (alpha7) subunit of the human 20S proteasome. The interaction was confirmed by glutathione S-transferase (GST) pull-down experiments and these also revealed that the related proteins EBNA3A and EBNA3B can bind similarly to C8/alpha7. The interaction between these viral proteins and GST-C8/alpha7 was shown to be significantly more robust than the previously reported interaction between C8/alpha7 and the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). Co-immunoprecipitation of the EBNA3 proteins with C8/alpha7 was also demonstrated after transfection of expression vectors into B cells. Consistent with this ability to bind directly to an alpha-subunit of the 20S proteasome, EBNAs 3A, 3B and 3C were all degraded in vitro by purified 20S proteasomes. However, surprisingly, no sign of proteasome-mediated turnover of these latent viral proteins in EBV-immortalized B cells could be detected, even in the presence of gamma interferon. In actively proliferating lymphoblastoid cell lines, EBNAs 3A, 3B and 3C appear to be remarkably stable, with no evidence of either de novo synthesis or proteasome-mediated degradation.
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PMID:Epstein-Barr virus EBNA3 proteins bind to the C8/alpha7 subunit of the 20S proteasome and are degraded by 20S proteasomes in vitro, but are very stable in latently infected B cells. 1583 37

Epstein-Barr virus (EBV) expresses an immediate-early protein, Rta, to activate the viral lytic cycle. This study identifies PIASxalpha and PIASxbeta as binding partners of Rta in a yeast two-hybrid screen and demonstrates the binding of Rta to PIASxalpha and PIASxbeta in vitro by GST pull-down analysis. Coimmunoprecipitation experiments and indirect immunofluorescence analysis show that Rta interacts and colocalizes with PIASxalpha and PIASxbeta in the nucleus. These interactions seem to enhance Rta sumoylation as transfecting plasmids expressing PIASxalpha, PIASxbeta, Ubc9, or SUMO-1 increase the capacity of Rta to transactivate a promoter that contains an Rta-response element and the promoters of p21 and BNLF1 in transient transfection assay. This study also finds that Rta sumoylation is preferentially enhanced by PIASxbeta, which could be attributed to the fact that PIASxbeta, compared to PIASxalpha, has a strong affinity to Rta, suggesting that affinity of a SUMO E3 ligase to its target protein influences the function of protein sumoylation.
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PMID:Sumoylation of Rta of Epstein-Barr virus is preferentially enhanced by PIASxbeta. 1646 Aug 27

Latent membrane protein 1 (LMP1), an oncogenic protein encoded by Epstein-Barr virus (EBV), has been verified to be phosphorylated in vitro by protein casein kinase 2 (CK2). In this study, we characterized the phosphorylation of the carboxyl terminus of LMP1 fused with glutathione-S-transferase (GST-LMP1c) and the FLAG-epitope-tagged LMP1 (F-LMP1) proteins expressed in HEK293T cells. Using a combination of chemical modification and tandem mass spectrometry, we detected the phosphorylation of a tryptic peptide, 191-223 amino acids, in both GST-LMP1c catalysed by CK2 and F-LMP1-expressing cell lines. Serine residues at positions 211 and 215 were determined to be the substrates of CK2 in vitro. Most importantly, the S215 phosphorylation was also detected in F-LMP1-expressing human cell lines. The phosphorylation of S215, which is located in the carboxyl-terminus activation region 1 of LMP1, provides a new insight for investigating the role and modulation of the phosphorylation of LMP1.
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PMID:Identification of a new in vivo phosphorylation site in the cytoplasmic carboxyl terminus of EBV-LMP1 by tandem mass spectrometry. 1687 69

BHRF1, an early gene product of Epstein-Barr virus (EBV), is structurally and functionally homologous to Bcl-2, a cellular anti-apoptotic protein. BHRF1 has been shown to protect cells from apoptosis induced by numerous external stimuli. Nasopharyngeal carcinoma is an epithelial cancer associated closely with EBV infection. Specific proteins that might interact with and modulate the BHRF1 anti-apoptotic activity in normal epithelial cells are of interest. Therefore, a cDNA library derived from normal human foreskin keratinocytes was screened by the yeast two-hybrid system and a cellular gene encoding human vaccinia virus B1R kinase-related kinase 2 (VRK2) was isolated. Interaction between the cellular VRK2 and viral BHRF1 proteins was further demonstrated by glutathione S-transferase pull-down assays, confocal laser-scanning microscopy and co-immunoprecipitation. Analyses of VRK2-deletion mutants revealed that a 108 aa fragment at the C terminus was important for VRK2 to interact with BHRF1. For BHRF1, aa 1-18 and 89-142 were crucial in interacting with VRK2 and these two regions are counterparts of Bcl-2 homology domains 4 and 1. Overexpressed VRK2 alone showed a modest effect in anti-apoptosis and appeared to enhance cell survival in the presence of BHRF1. However, this enhancement was not observed when VRK2 was co-expressed with Bcl-2. The results indicate that human VRK2 interacts specifically with EBV BHRF1 and that the interaction is involved in protecting cells from apoptosis.
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PMID:Human cellular protein VRK2 interacts specifically with Epstein-Barr virus BHRF1, a homologue of Bcl-2, and enhances cell survival. 1696 44


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