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)

Corneodesmosomes, the modified desmosomes of the uppermost layers of the epidermis, play an important role in corneocyte cohesion. Corneodesmosin is a secreted glycoprotein located in the corneodesmosomal core and covalently linked to the cornified envelope of corneocytes. Its glycine- and serine-rich NH(2)-terminal domain may fold to give structural motifs similar to the glycine loops described in epidermal cytokeratins and loricrin and proposed to display adhesive properties. A chimeric protein comprising human corneodesmosin linked to the transmembrane and cytoplasmic domains of mouse E-cadherin was expressed in mouse fibroblasts to test the ability of corneodesmosin to promote cell-cell adhesion. Classic aggregation assays indicated that corneodesmosin mediates homophilic cell aggregation. Moreover, Ca(2+) depletion showed a moderate effect on aggregation. To assess the involvement of the glycine loop domain in adhesion, full-length corneodesmosin, corneodesmosin lacking this domain, or this domain alone were expressed as glutathione S-transferase fusion proteins and tested for protein-protein interactions by overlay binding assays. The results confirmed that corneodesmosin presents homophilic interactions and indicated that its NH(2)-terminal glycine loop domain is sufficient but not strictly necessary to promote binding. Altogether, these results provide the first experimental evidence for the adhesive properties of corneodesmosin and for the involvement of its glycine loop domain in adhesion.
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PMID:Corneodesmosin, a component of epidermal corneocyte desmosomes, displays homophilic adhesive properties. 1173 86

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

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

The aim of this study was to test the protective immunity against Toxoplasma gondii in mice induced by a chimeric protein rSAG1/2. The PCR-amplified SAG2 fragment (558 bp) digested with the restriction enzyme XhoI was inserted into the XhoI site of plasmid pGEX-6p-1, termed pGexSAG2. The PCR-amplified SAG1 fragment (1,008 bp) digested with restriction enzymes EcoRI and XhoI was cloned into the EcoRI/ XhoI sites of a separate plasmid pGEX-6p-1, termed pGexSAG1. The SAG2 fragment (HindIII/HindIII) excised from pGexSAG2 was inserted into the HindIII site of pGexSAG1 and a chimeric vector constructed, pGexSAG1/2. The fusion proteins GST-SAG1/2, GST-SAG1 and GST-SAG2 were expressed in BL21 Star (DE3) Escherichia coli and purified by GSTrap FF columns. After removing the GST tag, the recombinant proteins rSAG1/2, rSAG1 and rSAG2 were independently collected and injected into different groups of mice to evaluate their protective capability. The highest proliferation of splenocytes stimulated with tachyzoite sonicate antigen (TsoAg) was observed in BALB/c mice which had received two intraperitoneal injections of rSAG1/2. Maximum production of IFN-gamma was also found in the culture supernatants of TsoAg-stimulated splenocytes from rSAG1/2-immunized mice. Finally, 73% (11/15) of mice immunized with rSAG1/2 survived at least 28 days after a lethal challenge of 1 x 10(3) live tachyzoites which killed all non-immunized mice within 10 days. Moderate survival rates were observed in mice immunized with either rSAG1 (60%) or rSAG2 (53%). These results show that the chimeric protein rSAG1/2 can elicit a Th1-associated protection against T. gondii infections in mice.
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PMID:Protective immunity against Toxoplasma gondii in mice induced by a chimeric protein rSAG1/2. 1460 77

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

Paget's disease is the most exaggerated example of bone remodeling with increased osteoclastic bone resorption followed by excessive bone formation. One of the earliest findings in our studies of Paget's disease is that pagetic osteoclast (OCL) precursors are hyper-responsive to 1,25-(OH)(2)D(3) and form OCL at concentrations of 1,25-(OH)(2)D(3) that are physiologic rather than pharmacologic. The increased responsivity to 1,25-(OH)(2)D(3) is not due to increased levels of the Vitamin D receptor (VDR) or to increased infinity of 1,25-(OH)(2)D(3) for VDR. We have recently shown using GST-VDR chimeric protein pull-down assays that TAF(II)-17, a member of the TAF(II)-D transcription complex, is increased in OCL precursors from patients with Paget's disease compared to normals. We further showed that TAF(II)-17 can enhance VDR mediated gene transcription and allow formation of the transcription complex at very low levels of 1,25-(OH)(2)D(3). In addition, coactivators of VDR including CPB300 and DRIP205 are also increased in OCL precursors from Paget's patients. These data suggest that the enhanced sensitivity of OCL precursors for 1,25-(OH)(2)D(3) in Paget's disease results from increased expression of coactivators of VDR and suggest that part of the pathophysiology underlying OCL formation in Paget's disease may result from enhanced expression of VDR coactivators.
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PMID:Paget's disease-a VDR coactivator disease? 1522 93

Viral protein R (Vpr) of human immunodeficiency virus type 1 has potent karyophilic properties, but details of the mechanism by which it enters the nucleus remain to be clarified. We reported previously that two regions, located between residues 17 and 34 (alphaH1) and between residues 46 and 74 (alphaH2), are indispensable for the nuclear localization of Vpr. Here, we reveal that a chimeric protein composed of the nuclear localization signal of Vpr, glutathione S-transferase, and green fluorescent protein was localized at the nuclear envelope and then entered the nucleus upon addition of importin-alpha. An in vitro transport assay using a series of derivatives of importin-alpha demonstrated that the carboxyl terminus was required for this nuclear import process. We also showed that Vpr interacts with importin-alpha through alphaH1 and alphaH2; only the interaction via alphaH1 is indispensable for the nuclear entry of Vpr. These observations indicate that importin-alpha functions as a mediator for the nuclear entry of Vpr.
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PMID:Importin-alpha promotes passage through the nuclear pore complex of human immunodeficiency virus type 1 Vpr. 1573 Dec 50

Enhanced green fluorescent protein (EGFP) from Aequorea victoria was fused to the C terminal region of protein ZZ, an artificial synthetic IgG Fc fragment binding protein derived from tandem repeats of the B domain of protein A. The ZZ-EGFP fusion protein was expressed in Escherichia coli with a His(6) tag and purified in high yield by one-step Ni(2+) chelating affinity chromatography. It was then used in the immunoblot analysis of GST and TNFalpha as well as in immunofluorescent assays of 293T cells transfected with IRF3, an interferon regulatory factor which localized in cytoplasm without virus infection. The fusion protein also performed effectively in FACS analysis of surface integrin beta3 subunit on 293 T cells. The chimeric protein bound various antibodies from different animal sources, directed against a variety of proteins. Thus, ZZ-EGFP showed broad promise in potential immunological applications.
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PMID:Application to immunoassays of the fusion protein between protein ZZ and enhanced green fluorescent protein. 1644 35

Specific monitoring of recombinant protein titer in DNA recombinant biotechnology traditionally has relied on SDS-PAGE, Western blotting, or bioactivity-based assays, but these are labor-intensive, time-consuming, and destructive and are not a good choice for the optimization of recombinant protein production. We describe a study in which enhanced green fluorescence protein (EGFP) was fused to the C terminus of a model protein glutathione S-transferase (GST) to optimize the chimeric protein production in Pichia pastoris by measurements of fluorescence of living cells in a 96-well microtiter plate using simple fluorescent scanning. Several common factors (e.g., time course of expression, effect of methanol concentration, frequency of methanol addition, medium pH) were tested using this strategy. Western blotting assay showed that the correct full-length GST-EGFP chimeric protein was expressed intracellularly in P. pastoris. The fluorescence intensity and GST bioactivity of cell extract yielded a direct correlation. The results show that the reported method provides an attractive platform for the optimization of recombinant protein production in vivo in real time as well as handling at least 96 samples in parallel.
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PMID:In vivo optimizing of intracellular production of heterologous protein in Pichia pastoris by fluorescent scanning. 1693 May 21


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