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 mineralocorticoid receptor (MR) plays a role in congestive heart failure; however, the molecular mechanism(s) remains undefined. We hypothesized that interaction of the MR with a cardiac protein modulates the transcriptional activation function of the MR within the heart. We used the yeast two-hybrid technique to screen a human heart library and found an aldosterone-dependent interaction between the hMR and the cardiac myosin binding protein (cMBP-c). The EC(50) of the hMR-MBP-c interaction was approximately 80nM, and the cMBP-c did not interact with the glucocorticoid receptor (GR). The GST pull-down technique was used to confirm an interaction between the MR and the cMBP-c as well as the lack of interaction with the GR. Spironolactone partially blocked this interaction, further suggesting MR specificity. We also determined the cMBP-c binding site lies within the C-terminus of the MR. We propose that interaction of the MR with cMBP-c may play a role in cardiac remodeling.
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PMID:The myosin binding protein is a novel mineralocorticoid receptor binding partner. 1513 21

Human LKB1, also known as STK11, is a tumour-suppression protein that mediates important functions in cellular proliferation and polarization. It might constitute an important target in cancer therapy. In order to produce large amounts of recombinant protein for biochemical and functional studies, a full-length cDNA clone was subcloned and expressed in Escherichia coli and insect cells. Although fusion proteins corresponding to LKB1 with 6xHis, GST and MBP tags could be overexpressed in E. coli, only MBP-LKB1 was recovered in a soluble, but heavily degraded form. Further studies demonstrated that this protein was not functional. Subsequent expression in insect cells of LKB1 with 6xHis and GST tags yielded insoluble products also. However, when chaperones Hsp70 and its cofactors Hsp40 and Hsdj were co-expressed with GST-LKB1, a clear increase in the solubility of the final protein was obtained. Moreover, this soluble, purified recombinant GST-LKB1 demonstrated to be a phosphoprotein, with at least residue Ser325 phosphorylated. The purified protein was functionally active as being able to demonstrate autophosphorylation in the absence of any associated kinase.
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PMID:An efficient expression system for the production of functionally active human LKB1. 1560 22

Previously, we reported that anti-apoptotic Bfl-1 is converted to a pro-apoptotic protein following fusion at its N-terminus with green fluorescent protein (GFP) (GFP-Bfl-1). In this study, we performed a Bfl-1 deletion study in order to elucidate the underlying mechanism of GFP-Bfl-1-induced cell death. We found that the Bcl-2 homology (BH) domains in Bfl-1 are dispensable with respect to cell death and that GFP fusion with the 29 amino acids of the C-terminal region of Bfl-1 (GFP-BC) is sufficient to induce cell death. Moreover, when BC was fused with other tagging partners like GST or MBP, little cell death was observed, implying that the GFP region is as important as the BC region for GFP-BC-induced cell death. Further deletion analysis defined a region of GFP as a determinant of GFP-BC-induced cell death. Confocal microscopic analysis showed that GFP-chimeras containing the BC region of Bfl-1 are located mainly in mitochondria. The GFP-BC-induced cell death accompanied cellular caspase activation, and treatment with the pan-caspase inhibitor, Boc-D-FMK, partially inhibited GFP-BC-induced cell death. However, the over-expression of anti-apoptotic molecules, such as Bcl-x(L) and CrmA, did not block GFP-BC-induced cell death. In summary, GFP-BC induces cell death with caspase activation through mitochondria dependent process.
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PMID:C-terminal region of Bfl-1 induces cell death that accompanies caspase activation when fused with GFP. 1569 50

We used BIAcore to analyze the kinetics of interactions between CD81 and hepatitis C virus (HCV) envelope proteins. We immobilized different forms of HCV envelope proteins (E1E2, E2, and E2(661)) on the sensor and monitored their interaction with injected fusion proteins of CD81 large extracellular loop (CD81LEL) and glutathione-S-transferase (CD81LEL-GST) or maltose binding protein (CD81LEL-MBP). The difference between the GST and MBP fusion proteins was their multimeric and monomeric forms, respectively. The association rate constants between CD81LEL-GST or CD81LEL-MBP and the E1E2, E2 or E2(661) HCV envelope proteins were similar. However, the dissociation rate constants of CD81LEL-MBP were higher than those of CD81LEL-GST. Interestingly, the dissociation rate constant of CD81LEL-GST from E1E2 was much lower than from E2 or E2(661). The interaction between both forms of the CD81LEL fusion proteins and the HCV envelope proteins best-fitted the "heterogeneous ligand" model. This model implies that two kinds of interactions occur between envelope proteins and CD81LEL: one is strong, the other is weak. It also implies that the heterogeneity is likely due to the HCV envelope proteins, which are known to form non-covalently linked heterodimers and disulfide-linked aggregate.
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PMID:Kinetics of HCV envelope proteins' interaction with CD81 large extracellular loop. 1570 89

Cervical cancers evolve from lesions generated by genital human papillomaviruses (HPV). "Low-risk" genital HPVs cause benign proliferations whereas "high-risk" types have the potential to progress into cancer. High-risk HPV E6 oncoproteins interact with the ubiquitin ligase E6AP and target several cellular proteins, including p53 and proteins of the MAGI family, towards ubiquitin-mediated degradation. E6AP, like other E6 binding proteins such as E6BP, IRF-3 and paxillin, interacts with E6 via a consensus leucine-charged motif. Here we have investigated the kinetics of the interactions of a 15-mer peptide containing the LxxvarphiLsh motif of E6AP with E6. For this we have developed a Biacore assay based on antibody-capture on the sensor surface of GST- and/or MBP-E6AP peptide constructs followed by E6 protein injection. Our experiments show that E6 oncoproteins from four major high-risk (16, 18, 33 and 58) HPV types bind to E6AP with equilibrium dissociation constants in the low micromolar range. The kinetic dissociation parameters of these interactions are remarkably similar. On the other hand, low-risk HPV 11 E6 does not interact with E6AP even at relatively high concentrations. We also show that the two zinc-binding domains of E6 are required for E6AP recognition. Finally, we have analysed the binding properties of site-directed mutants of the E6AP-derived peptide. We demonstrate the importance for binding of conserved aliphatic side-chains and the moderate role of the global negative charge of the peptide. This work provides the first quantitative data on an HPV E6-mediated interaction, which support the current models of E6AP-mediated degradation.
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PMID:Kinetic analysis of the interactions of human papillomavirus E6 oncoproteins with the ubiquitin ligase E6AP using surface plasmon resonance. 1589 Feb 4

In proteomics research, generation of recombinant proteins in their native, soluble form with large quantity is often a challenging task. To tackle the expression difficulties, different expression vectors with distinct affinity fusion tags, i.e. pET-43.1a (N-utilization substance A tag), pMAL-cRI (maltose binding protein tag) (MBP tag), pGEX-4T-2 (glutathione S-transferase tag), and pET-15b (hexahistidine tag) were compared for their effects on the productivity and solubility, which were assessed by SDS-PAGE and immunoblotting, of the integrin betaA domain. The incubation temperatures were tested for its effects on these parameters. Our data suggested that MBP tag enhanced the yield and solubility of the betaA domain protein, which can also be recognized using an anti-CD18 antibody, at room temperature incubation. Thus, the nature of fusion partner chosen for expression in bacteria and its incubation temperature would significantly affect the yield and solubility of the recombinant target protein.
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PMID:Increased solubility of integrin betaA domain using maltose-binding protein as a fusion tag. 1680 Jul 94

Mycothiol ligase (MshC) is a key enzyme in the biosynthesis of mycothiol, a small molecular weight thiol found in Mycobacteria spp. and other actinomycetes. Mycothiol plays a fundamental role in these organisms by helping to provide protection from the effects of reactive oxygen species and electrophiles, including many antibiotics. It has recently been demonstrated that the MshC gene and more generally the production of mycothiol are essential to Mycobacterium tuberculosis, indicating that MshC may represent a novel target for new classes of antituberculars. Because MshC cannot be expressed heterologously in Escherichia coli and isolation from Mycobacterium smegmatis is impractical, we have optimized the E. coli-M. smegmatis shuttle vector pACE for cloning and recombinant expression of MshC (under control of an acetamidase-inducible promoter). To improve expression levels and simplify purification, we further constructed three N-terminal-MshC fusion proteins where N-terminal tags included the B1 domain of streptococcal protein G (to give GB1-MshC), glutathione-S-transferase (to give GST-MshC) and maltose binding protein (to give MBP-MshC), for expression in M. smegmatis. By expressing all three fusion proteins in a mutant strain of M. smegmatis mc(2)155, namely I64 L205P MshC M. smegmatis which lacks mycothiol ligase activity, we demonstrate in vivo mycothiol ligase activity for each construct. Recombinant GST-MshC and MBP-MshC were isolated in one step by affinity chromatography in a yield of 0.7 and 1.2 mg fusion protein/L and exhibited specific activities of 9 nmolmin(-1)mg(-1) and 25 nmolmin(-1)mg(-1), respectively.
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PMID:Cloning, expression and rapid purification of active recombinant mycothiol ligase as B1 immunoglobulin binding domain of streptococcal protein G, glutathione-S-transferase and maltose binding protein fusion proteins in Mycobacterium smegmatis. 1690 86

Functional and structural studies require gene overexpression and purification of soluble proteins. We wanted to express proteins from the psychrophilic bacterium Vibrio salmonicida in Escherichia coli, but encountered solubility problems. To improve the solubility of the proteins, we compared the effects of six N-terminal fusion proteins (Gb1, Z, thioredoxin, GST, MBP and NusA) and an N-terminal His6-tag. The selected test set included five proteins from the fish pathogen V. salmonicida and two related products from the mesophilic human pathogen Vibrio cholerae. We tested the expression in two different expression strains and at three different temperatures (16, 23 and 37 degrees C). His6-tag was the least effective tag, and these vector constructs were also difficult to transform. MBP and NusA performed best, expressing soluble proteins with all fusion partners in at least one of the cell types. In some cases MBP, GST and thioredoxin fusions resulted in products of incorrect size. The effect of temperature is complex: in most cases level of expression increased with temperature, whereas the effect on solubility was opposite. We found no clear connection between the preferred expression temperature of the protein and the temperature of the original host organism's natural habitat.
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PMID:Comparative expression study to increase the solubility of cold adapted Vibrio proteins in Escherichia coli. 1706 34

The amino-terminal domains (ATDs) of N-methyl-d-aspartate (NMDA) receptors contain binding sites for modulators and may serve as potential drug targets in neurological diseases. Here, three fusion tags (6xHis-, GST-, and MBP-) were fused to the ATD of NMDA receptor NR2B subunit (ATD2B) and expressed in Escherichia coli. Each tag's ability to confer enhanced solubility to ATD2B was assessed. Soluble ATD2B was successfully obtained as a MBP fusion protein. Dynamic light scattering revealed the protein (1mg/ml) exists as monodispersed species at 25 degrees C. Functional studies using circular dichroism showed that the soluble MBP-ATD2B bound ifenprodil in a dose-dependent manner. The dissociation constants obtained for ifenprodil were similar in the absence (64nM) and presence (116nM) of saturating concentration of maltose. Moreover, the yield of soluble MBP-ATD2B is 18 times higher than the refolded 6xHis-ATD2B. We have reported a systematic comparison of three different affinity tagging strategies and identified a rapid and efficient method to obtain large amount of ATD2B recombinant protein for biochemical and structural studies.
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PMID:Improving solubility of NR2B amino-terminal domain of N-methyl-d-aspartate receptor expressed in Escherichia coli. 1770 1

Adeno-associated virus (AAV) is a nonpathogenic single-stranded human parvovirus which usually requires the presence of a "helper" virus for strong DNA replication. In addition to adeno- and herpes viruses, human papillomavirus (HPV) can serve as an AAV helper. We recently published that HPV type 16 (HPV-16) E1 protein contributes significantly as an individual helper gene for AAV-2 DNA replication and transcription. As Rep78 and E1 are the corresponding DNA helicase/replication proteins of AAV and HPV, respectively, and Rep78 and E1 have a degree of homology, we assayed whether these two proteins interact physically. The full length proteins were purified from bacteria as GST-E1 and MBP-Rep78 and used in five assays to observe Rep78-E1 interactions. All five assays (pull-down, coimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), chemical cross-linking, and ATPase activity) provided evidence consistent with Rep78-E1 interaction. Most intriguing, an overall decrease in ATPase activity was observed when both proteins were present together. These data strongly suggest that E1 and Rep78 interact and that this interaction modulates at least some of their individual biochemical functions. This study adds to our understanding of AAV-HPV interaction biology, E1's modulation of Rep78 biochemistry, Rep78's modulation of E1 biochemistry and provides initial clues which may lead to the underlying mechanism of HPV E1 helper function for AAV DNA replication.
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PMID:AAV-2 Rep78 and HPV-16 E1 interact in vitro, modulating their ATPase activity. 1809 9


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