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)

It has been demonstrated that growth factors quiescin Q6 family was created by the fusion of the sulfhydryl oxidase fragment of the yeast essential for respiration and vegetative growth (ERV)1 prototype [an orthologue of hepatopoietin (HPO)] and thioredoxin (TRX)/disulfide isomerase domain during evolution. In this paper, our results demonstrated that two components of this composite protein, i.e., HPO and TRX, were involved in the same signal transduction and interacted physically in eukaryocyte. When HPO and TRX were cotransfected into COS7 cells, the activity of activator protein-1 (AP-1) and NF-kappaB was evidently enhanced compared with the transfection with HPO or TRX alone, at the same time, the phosphorylation of c-Jun was increased. They were colocalized in the cells. By Co-IP and GST pull-down experiments, we found that HPO could physically interact with TRX, which was also confirmed by yeast two-hybrid assay. By further investigation, we found both HPO and TRX were sensitive to cellular oxidative state. HPO dimer is in its natural state and could be reduced by dithiothreitol (DTT) in vitro and in vivo. Under the treatment of oxidants such as H(2)O(2) and diamide, the amount of HPO monomer was decreased significantly and assembled into dimer, and the free thiol in TRX was oxidized. HPO could transfer oxidizing equivalents to TRX via direct thiol-disulfide exchange in vitro, the redox state of TRX was also affected by HPO in vivo. Taken together, it was implicated that the oxidizing equivalents might flow from HPO to TRX and then to substrate protein by the dimerization of HPO, and its interaction with TRX finally activates the redox-sensitive transcription factor, suggesting a new redox signal pathway conducted by thiol-disulfide transformation in eukaryocytic cytoplasm.
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PMID:Direct association of hepatopoietin with thioredoxin constitutes a redox signal transduction in activation of AP-1/NF-kappaB. 1589 71

Human flotillin-1 (reggie-2), a major hydrophobic protein of biomembrane microdomain lipid rafts, was cloned and expressed in Escherichia coli with four different fusion tags (hexahistidine, glutathione S-transferase, NusA, and thioredoxin) to increase the yield. The best expressed flotillin-1 with thioredoxin tag was solubilized from inclusion bodies, first purified by immobilized metal affinity column under denaturing condition and direct refolded on column by decreasing urea gradient method. The thioredoxin tag was cleaved by thrombin, and the flotillin-1 protein was further purified by anion exchanger and gel filtration column. The purified protein was verified by denaturing gel electrophoresis and Western blot. The typical yield was 3.4 mg with purity above 98% from 1L culture medium. Using pull-down assay, the interaction of both the recombinant flotillin-1 and the native flotillin-1 from human erythrocyte membranes with c-Cbl-associated protein or neuroglobin was confirmed, which demonstrated that the recombinant proteins were functional active. This is the first report describing expression, purification, and characterization of active recombinant raft specific protein in large quantity and highly purity, which would facilitate further research such as X-ray crystallography.
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PMID:Expression, purification, and characterization of recombinant human flotillin-1 in Escherichia coli. 1593 99

Involvement of individual antioxidant proteins (AOXP) and antioxidants in the differentiation process has been already reported. A systematic search strategy for detecting differentially regulated AOXP in neuronal differentiation, however, has not been published so far. The aim of this study was to provide an analytical tool identifying AOXP and to generate a differentiation-related AOXP expressional pattern. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments: We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical (MALDI-TOF-TOF) identification of proteins to generate a map of AOXP. 16 AOXP were unambiguously determined in both cell lines; catalase, thioredoxin domain-containing protein 4 and hypothetical glutaredoxin/glutathione S-transferase C terminus-containing protein were detectable in the undifferentiated cells only. Five AOXP were observed in both, undifferentiated and differentiated cells and thioredoxin, thioredoxin-like protein p19, thioredoxin reductase 1, superoxide dismutases (Mn and Cu-Zn), glutathione synthetase, glutathione S-transferase P1 and Mu1 were detected in differentiated cells exclusively. Herein a differential expressional pattern is presented that reveals so far unpublished antioxidant principles involved in neuronal differentiation by a protein chemical approach, unambiguously identifying AOXP. This finding not only shows concomitant determination of AOXP but also serves as an analytical tool and forms the basis for design of future studies addressing AOXP and differentiation per se.
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PMID:The neuronal differentiation process involves a series of antioxidant proteins. 1598 80

Pro-fs is a human immunodeficiency virus type 1 (HIV-l)-encoded putative selenoprotein, predicted by a theoretical analysis of the viral genome; it is potentially expressed by a -1 frameshift from the protease coding region. Pro-fs has significant sequence similarity to the DNA binding loop of nuclear factor kappa B (NF-kappaB), which is known to bind thioredoxin (Trx). We hypothesize that the putative HIV-1 pro-fs gene product functions by mimicry of NF-kappaB via binding to Trx. The hypothesis was tested in vitro by co-immunoprecipitation and GST-pull down assays, using a purified mutant pro-fs protein, in which the two potential selenocysteine residues were mutated to cysteines, in order to permit expression in bacteria. Both experiments showed that pro-fs binds to human wild type Trx (Trx-wt) with high affinity. Mutation of the two conserved cysteine residues in the Trx active site redox center to serine (Ser) (Trx-CS) weakened but failed to abolish the interaction. In pro-fs-transfected 293T cells, using confocal microscopy and fluorescence resonance energy transfer (FRET), we have observed that pro-fs localizes in cell nuclei and forms oligomers. Upon stimulation by phorbol 12-myristate 13-acetate (PMA), Trx translocates into cell nuclei. Significant FRET efficiency was detected in the nuclei of PMA-stimulated 293T cells co-expressing fluorescence-tagged pro-fs and Trx-wt or Trx-CS. These results indicate that in living cells the double cysteine mutant of pro-fs binds to both Trx and Trx-CS with high affinity, suggesting that Trx-pro-fs binding is a structurally-specific interaction, involving more of the Trx molecule than just its active site cysteine residues. These results establish the capacity for functional mimicry of the Trx binding ability of the NF-kappaB/Rel family of transcription factors by the putative HIV-1 pro-fs protein.
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PMID:An HIV-1 encoded peptide mimics the DNA binding loop of NF-kappaB and binds thioredoxin with high affinity. 1605 58

Glutathione transferases (GSTs) are a superfamily of enzymes that play a vital functional role in the cellular detoxification process. They catalyze the conjugation of the thiol group of glutathione (GSH) to the electrophilic groups of a wide range of hydrophobic substrates, leading to an easier removal of the latter from the cells. The kappa class is the least studied one among various classes within the superfamily. We report here the expression, purification, and crystal structure of human kappa class GST (hGSTK), which has been determined by the multiple-isomorphous replacement method and refined to 1.93 A resolution. The overall structure of hGSTK is similar to the recently reported structure of kappa class GST from rat mitochondrion. Each subunit of the dimeric hGSTK contains a thioredoxin (TRX)-like domain and a helical domain. A molecule of glutathione sulfinate, an oxidized product of GSH, is found to bind at the G site of each monomer. One oxygen atom of the sulfino group of GSF forms a hydrogen bond with the hydroxyl group of the catalytic residue Ser16. The TRX-like domain of hGSTK shares 19% sequence identity and structure similarity with human theta class GST, suggesting that the kappa class of GST is more closely related to the theta class enzyme within the GST superfamily. The structure of the TRX-like domain of hGSTK is also similar to that of glutathione peroxidase (GPx), implying an evolutionary relationship between GST and GPx.
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PMID:Thioredoxin-like domain of human kappa class glutathione transferase reveals sequence homology and structure similarity to the theta class enzyme. 1608 49

In an effort to better understand the Ah receptor nuclear translocator (Arnt)-dependent signaling mechanisms, we employed a phage display system to identify Arnt-interacting peptides. Human liver cDNA library was utilized to screen for Arnt-interacting peptides using an Arnt construct fused to thioredoxin (TH-ArntCDelta418). Two clones, namely Ainp1 and Ainp2 (Arnt-interacting peptide), were identified and subsequently Ainp2 was further characterized. Ainp2 interacts with TH-ArntCDelta418 in the GST pull-down and mammalian two-hybrid assays. Northern blot results revealed that Ainp2 is predominantly expressed in human liver. The putative full-length Ainp2 cDNA sequence was subsequently cloned using RACE PCR. Endogenous expression of Ainp2 was found in Jurkat cells at the mRNA and protein levels. Results from the transient transfection studies using a DRE-driven reporter plasmid and the real-time QPCR experiments examining the endogenous CYP1A1 expression showed that Ainp2 enhances the 3-methylchloranthrene-induced activity in HepG2 cells, suggesting that Ainp2 plays a role in the Arnt-dependent function
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PMID:A novel Arnt-interacting protein Ainp2 enhances the aryl hydrocarbon receptor signalling. 1611 50

Sieve tubes are comprised of sieve elements, enucleated cells that are incapable of RNA and protein synthesis. The proteins in sieve elements are supplied from the neighboring companion cells through plasmodesmata. In rice plants, it was unclear whether or not all proteins produced in companion cells had the same distribution pattern in the sieve element-companion cell complex. In this study, the distribution pattern of four proteins, beta-glucuronidase (GUS), green fluorescent protein (GFP), thioredoxin h (TRXh) and glutathione S-transferase (GST) were analyzed. The foreign proteins GUS and GFP were expressed in transgenic rice plants under the control of the TRXh gene promoter (PTRXh), a companion cell-specific promoter. Analysis of leaf cross-sections of PTRXh-GUS and PTRXh-GFP plants indicated high accumulation of GUS and GFP, respectively, in companion cells rather than in sieve elements. GUS and GFP were also detected in phloem sap collected from leaf sheaths of the transgenic rice plants, suggesting these proteins could enter sieve elements. Relative amounts of GFP and endogenous phloem proteins, TRXh and GST, in phloem sap and total leaf extracts were compared. Compared to TRXh and GST, GFP content was higher in total leaf extracts, but lower in phloem sap, suggesting that GFP accumulated mainly in companion cells rather than in sieve elements. On the other hand, TRXh and GST appeared to accumulate in sieve elements rather than in companion cells. These results indicate the evidence for differential distribution of proteins between sieve elements and companion cells in rice plants.
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PMID:Differential distribution of proteins expressed in companion cells in the sieve element-companion cell complex of rice plants. 1612 Jun 85

The cDNA and protein structures of Xenopus metaxin 3, along with those of Xenopus metaxins 1 and 2, have been characterized. A protein of 309 amino acid residues is encoded by X. laevis metaxin 3 (XMTX3) cDNA. In comparison, the cDNA of X. laevis metaxin 1 (XMTX1) specifies a protein of 320 residues, while the metaxin 2 cDNAs of X. laevis (XMTX2) and X. tropicalis (SMTX2) both specify proteins of 274 amino acids. Aligning the amino acid sequences of XMTX3 and XMTX1 showed 39% identities; 22% identities were found for XMTX3 and XMTX2. However, 55% amino acid identities were revealed in aligning the XMTX3 and zebrafish metaxin 3 sequences. The construction of a phylogenetic tree gave further evidence for the existence of three distinct groups of metaxin genes and their common ancestry. Two conserved protein domains are present in each of the Xenopus metaxins: a glutathione S-transferase (GST) domain and a thioredoxin-like domain. The protein secondary structure predicted for the Xenopus metaxins is dominated by regions of alpha helix which alternate with regions that are neither alpha helix nor beta strand.
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PMID:Characterization of the cDNA and amino acid sequences of Xenopus Metaxin 3, and relationship to Xenopus Metaxins 1 and 2. 1614 84

Despite the availability of numerous gene fusion systems, recombinant protein expression in Escherichia coli remains difficult. Establishing the best fusion partner for difficult-to-express proteins remains empirical. To determine which fusion tags are best suited for difficult-to-express proteins, a comparative analysis of the newly described SUMO fusion system with a variety of commonly used fusion systems was completed. For this study, three model proteins, enhanced green fluorescent protein (eGFP), matrix metalloprotease-13 (MMP13), and myostatin (growth differentiating factor-8, GDF8), were fused to the C termini of maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and SUMO tags. These constructs were expressed in E. coli and evaluated for expression and solubility. As expected, the fusion tags varied in their ability to produce tractable quantities of soluble eGFP, MMP13, and GDF8. SUMO and NUS A fusions enhanced expression and solubility of recombinant proteins most dramatically. The ease at which SUMO and NUS A fusion tags were removed from their partner proteins was then determined. SUMO fusions are cleaved by the natural SUMO protease, while an AcTEV protease site had to be engineered between NUS A and its partner protein. A kinetic analysis showed that the SUMO and AcTEV proteases had similar KM values, but SUMO protease had a 25-fold higher kcat than AcTEV protease, indicating a more catalytically efficient enzyme. Taken together, these results demonstrate that SUMO is superior to commonly used fusion tags in enhancing expression and solubility with the distinction of generating recombinant protein with native sequences.
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PMID:Comparison of SUMO fusion technology with traditional gene fusion systems: enhanced expression and solubility with SUMO. 1632 73

Using 2-DE of total cell protein extracts, we compared soluble proteins from murine melanoma lines Tm1 and Tm5 with proteins from the nontumoral cell melan-a from which they were derived. Seventy-one of the 452 spots (average) detected with CBB were differentially accumulated, i.e., increased or decreased twofold. Forty-four spots were identified by PMF/MALDI-TOF, 15 with increased and 29 with decreased protein levels. SAGE showed that 17/34 (50%) of the differentially accumulated proteins, pI range 4-7, presented similar differences at the mRNA level. Major reductions in protein were observed in tumor cells of proteins that degrade reactive oxygen species (ROS). Decreases of > or = twofold in GST, superoxide dismutase, aldehyde dehydrogenase, thioredoxin, peroxiredoxin 2, and peroxiredoxin 6 protein were observed. SAGE indicated the reduction of other proteins involved in ROS degradation. As expected, the accumulation of exogenous peroxides was significantly higher in the tumor cells while the levels of glutathionylation were two times lower in the tumor cells compared to melan-a. The differential accumulation of proteins involved in oncogene/tumor suppressor pathways was observed. Melanoma cells can favor survival pathways activated by ROS by inhibiting p53 pathways and activation of Ras and c-myc pathways.
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PMID:Proteomic and SAGE profiling of murine melanoma progression indicates the reduction of proteins responsible for ROS degradation. 1642 58


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