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)

Transforming growth factor-beta (TGF-beta) is secreted by many cell types as part of a large latent complex composed of three subunits: TGF-beta, the TGF-beta propeptide, and the latent TGF-beta binding protein (LTBP). To interact with its cell surface receptors, TGF-beta must be released from the latent complex by disrupting noncovalent interactions between mature TGF-beta and its propeptide. Previously, we identified LTBP-1 and transglutaminase, a cross-linking enzyme, as reactants involved in the formation of TGF-beta. In this study, we demonstrate that LTBP-1 and large latent complex are substrates for transglutaminase. Furthermore, we show that the covalent association between LTBP-1 and the extracellular matrix is transglutaminase dependent, as little LTBP-1 is recovered from matrix digests prepared from cultures treated with transglutaminase inhibitors. Three polyclonal antisera to glutathione S-transferase fusion proteins containing amino, middle, or carboxyl regions of LTBP-1S were used to identify domains of LTBP-1 involved in cross-linking and formation of TGF-beta by transglutaminase. Antibodies to the amino and carboxyl regions of LTBP-1S abrogate TGF-beta generation by vascular cell cocultures or macrophages. However, only antibodies to the amino-terminal region of LTBP-1 block transglutaminase-dependent cross-linking of large latent complex or LTBP-1. To further identify transglutaminase-reactive domains within the amino-terminal region of LTBP-1S, mutants of LTBP-1S with deletions of either the amino-terminal 293 (deltaN293) or 441 (deltaN441) amino acids were expressed transiently in CHO cells. Analysis of the LTBP-1S content in matrices of transfected CHO cultures revealed that deltaN293 LTBP-1S was matrix associated via a transglutaminase-dependent reaction, whereas deltaN441 LTBP-1S was not. This suggests that residues 294-441 are critical to the transglutaminase reactivity of LTBP-1S.
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PMID:Latent transforming growth factor-beta binding protein domains involved in activation and transglutaminase-dependent cross-linking of latent transforming growth factor-beta. 906 Apr 78

Huntington's disease and six other neurodegenerative diseases are associated with abnormal gene products containing expanded polyglutamine (poly-Q; Qn) domains (n > or = 40). In the present work, we show that glutathione S-transferase (GST) fusion proteins containing a small, physiological-length poly-Q domain (GSTQ10) or a large, pathological-length poly-Q domain (GSTQ62) are excellent substrates of guinea pig liver (tissue) transglutaminase and that both GSTQ10 and GSTQ62 are activators of tissue transglutaminase-catalyzed hydroxaminolysis of N-alpha-carbobenzoxyglutaminylglycine. The present findings have implications for understanding the pathophysiological mechanisms of expanded CAG/poly-Q domain diseases.
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PMID:Polyglutamine domains are substrates of tissue transglutaminase: does transglutaminase play a role in expanded CAG/poly-Q neurodegenerative diseases? 920 40

Tissue transglutaminase (TGase II) catalyzes the posttranslational modification of proteins by transamidation of available glutamine residues and is also a guanosinetriphosphatase (GTPase) and adenosinetriphosphatase (ATPase). Based on its homology with factor XIIIA, an extracellular transglutaminase, the structure of TGase II is likely composed of an N-terminal beta-sandwich domain, an alpha/beta catalytic core, and two C-terminally located beta-barrels. Here we used a domain-deletion approach to identify the GTP and ATP hydrolytic domains of TGase II. Full-length TGase II and two domain-deletion mutants, one retaining the N-terminal beta-sandwich and core domains (betaSCore) and the other retaining only the core domain, were expressed as glutathione S-transferase (GST) fusion proteins and purified. GST-Full and GST-betaSCore exhibited calcium-dependent TGase activity, whereas GST-Core had no detectable TGase activity, indicating the beta-sandwich domain is required for TGase activity but the C-terminal beta-barrels are not. All three GST-TGase II fusion proteins were photoaffinity-labeled with [alpha-32P]-8-azidoGTP and were able to bind GTP-agarose. The GTPase activity of GST-betaSCore was equivalent to that of GST-Full, whereas the ATPase activity was approximately 40% higher than GST-Full. GST-Core had approximately 50% higher GTPase activity and approximately 75% higher ATPase activity than GST-Full. The GTPase and ATPase activities of each of the GST-TGase II fusion proteins were inhibited in a dose-dependent manner by both GTPgammaS and ATPgammaS. These results demonstrate that the GTP and ATP hydrolysis sites are localized within the core domain of TGase II and that neither the N-terminal beta-sandwich domain nor the C-terminal beta-barrels are required for either GTP or ATP hydrolysis. Taken together with previous work [Singh, U. S., Erickson, J. W., & Cerione, R. A. (1995) Biochemistry 34, 15863-15871; Lai, T.-S., Slaughter, T. F., Koropchak, C. M., Haroon, Z. A., & Greenberg, C. S. (1996) J. Biol. Chem. 271, 31191-31195] the results of this study indicate that the GTP and ATP hydrolysis sites are localized to a 5. 5 kDa (47 amino acid) region at the start of the core domain.
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PMID:The core domain of the tissue transglutaminase Gh hydrolyzes GTP and ATP. 930 55

Transglutaminase is a calcium-dependent enzyme which catalyzes amine incorporation and cross-linking of proteins. To isolate the amine acceptor protein substrates of transglutaminase in mammalian livers, a biotin-labeled primary amine substrate of transglutaminase, 5-(biotinamido) pentylamine, was used for biotin labeling of proteins in the liver extracts by endogenous transglutaminase activity. The biotin-labeled proteins were isolated and recovered by biotin-avidin-affinity chromatography. The obtained proteins were separated by SDS-PAGE. Proteins with molecular masses of 15, 24, 35, 40, 44, 93, and 134 kDa were the main components of labeled proteins in mouse liver extract. In rat and guinea pig liver extracts, 32-, 38-, 40-, 44-, and 134-kDa proteins and28-, 40-, 44-, 55-, 60-, 91-, and 134-kDa proteins were the main components of labeled proteins, respectively.Using amino-terminal amino acid sequence analyses and sequence homology searches, the 38-kDa protein from rat liver was identified as a subunit of glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and the 28-kDa protein from guinea pig liver was identified as a subunit of glutathione S-transferase (class theta) (EC 2.5.1.18). Both the glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle and glutathione S-transferase (class pi) from human placenta also could be amine acceptors in the amine incorporation catalyzed by guinea pig liver transglutaminase. These results suggest that these enzymes can be modified posttranslationally by cellular transglutaminase.
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PMID:Identification of amine acceptor protein substrates of transglutaminase in liver extracts: use of 5-(biotinamido) pentylamine as a probe. 970 18

Overexpression of 'tissue' transglutaminase (tTG) in the human neuroblastoma cells increases spontaneous apoptosis and renders these cells highly susceptible to death induced by various stimuli. We used immunoprecipitation to identify cellular proteins that interact specifically with tTG in SK-N-BE(2) -derived stable transfectants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that tTG binding proteins have molecular masses of 110, 50, 22, 14, and 12 kDa. Microsequencing and computer search analyses allowed us to identify these polypeptides as the beta-tubulin (50 kDa), the histone H2B (14 kDa), and two GST P1-1-truncated forms (22 and 12 kDa). The specificity of the interaction between tTG and these proteins was confirmed by competing tTG binding with purified enzyme and by detecting tTG in immunoprecipitates obtained using beta-tubulin or GST P1-1 mAb's. Here we demonstrate that the GST P1-1 acts as an efficient acyl donor as well as acceptor tTG substrate both in cells and in vitro. The tTG-catalyzed polymerization of GST P1-1 leads to its functional inactivation and is competitively inhibited by GSH. By contrast, the tTG-beta-tubulin interaction does not result in the cross-linking of this cytoskeletal protein, which suggests that microtubules act as the anchorage site for tTG and GST P1-1 interaction.
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PMID:Identification of 'tissue' transglutaminase binding proteins in neural cells committed to apoptosis. 997 24

Tissue transglutaminase(tTG) belongs to a class of transglutaminase family which is up-regulated in almost all cells apoptosis and is thought to be closely related to cell apoptosis. To investigate the mechanism of tTG in cell apoptosis, yeast two hybrid system was used to screen HeLa cDNA library. One of the 17 positive clones we have obtained encoded the glutamine-rich carboxyl terminus of TIAR, and this interaction between tTG and TIAR, which was finely regulated by Ca(2+), was proved in vitro by GST pull-down. These findings suggest that tTG might affect the function of TIAR by a calcium-dependent posttranslational modification and the interaction might possibly be involved in the regulation of cell apoptosis.
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PMID:[Screening of proteins interacting with tTG in HeLa cells]. 1276 11

Fluorescein and its analogs are among the best fluorophores to label proteins and the labeling generally involves chemical modification of a translated protein. Using this methodology, labeling at a specific position remains difficult. It is known that the guinea pig liver transglutaminase (TGase)-catalyzed enzymatic modification method can allow terminal-specific fluorophore labeling of a protein by monodansylcadaverine. However, native activity of the fluorescent protein has not been investigated so far, nor has direct comparison between the chemical modification and the TGase-catalyzed modification been attempted. Therefore, we compared the possibility of fluorescein labeling via chemical labeling and via TGase-catalyzed modification. The latter method was found to be very practical and overcame some of the problems associated with the specificity of the former; fluorescein was covalently attached only to the N- or C-terminal site of glutathione S-transferase when the reaction was catalyzed by TGase and the resulting labeled protein completely retained its native activity. The TGase-mediated labeling occurred not only at room temperature but also at 4 degrees C to the same extent, which is more desirable for preventing the inactivation of proteins.
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PMID:Transglutaminase-mediated N- and C-terminal fluorescein labeling of a protein can support the native activity of the modified protein. 1504 7

Mammalian transglutaminase (TGase) catalyzes covalent cross-linking of peptide-bound lysine residues or incorporation of primary amines to limited glutamine residues in substrate proteins. Using an unbiased M13 phage display random peptide library, we developed a screening system to elucidate primary structures surrounding reactive glutamine residue(s) that are preferred by TGase. Screening was performed by selecting phage clones expressing peptides that incorporated biotin-labeled primary amine by the catalytic reactions of TGase 2 and activated Factor XIII (Factor XIIIa). We identified several amino acid sequences that were preferred as glutamine donor substrates, most of which have a marked tendency for individual TGases: TGase 2, QxPphiD(P), QxPphi, and QxxphiDP; Factor XIIIa, QxxphixWP (where x and phi represent a non-conserved and a hydrophobic amino acid, respectively). We further confirmed that the sequences were favored for transamidation using modified glutathione S-transferase (GST) for recombinant peptide-GST fusion proteins. Most of the fusion proteins exhibited a considerable increase in incorporation of primary amines over that of modified GST alone. Furthermore, we identified the amino acid sequences that demonstrated higher specificity and inhibitory activity in the cross-linking reactions by TGase 2 and Factor XIIIa.
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PMID:Screening for the preferred substrate sequence of transglutaminase using a phage-displayed peptide library: identification of peptide substrates for TGASE 2 and Factor XIIIA. 1663 49

The factors affecting enzymatic protein immobilization with microbial transglutaminase (MTG) were explored. As model proteins, enhanced green fluorescent protein (EGFP) and glutathione S-transferase (GST) were chosen and tagged with a neutral Gln-donor substrate peptide for MTG (Leu-Leu-Gln-Gly, LLQG-tag) at their C-terminus. To create a specific surface, displaying reactive Lys residues, to be cross-linked with the Gln residue in the LLQG-tag of target proteins by MTG catalysis, a polystyrene surface was physically coated with beta-casein. Both recombinant proteins were immobilized onto the beta-casein-coated surface only in the presence of active MTG, indicating that those proteins were enzymatically immobilized to the surface. MTG-mediated protein immobilization markedly depends on the pH and ionic strength of the reaction media. The optimal pH range of MTG-mediated immobilization of both recombinant proteins was around 5, at which point the MTG-catalyzed reaction in aqueous solution is not normally preferred. By utilizing a pH-dependent change in EGFP fluorescence, we found that the apparent pH at the surface is likely to be lower than bulk pH, this difference is not attributed to an optimal pH shift in MTG-mediated immobilization. On the other hand, lower yields of protein immobilization at higher ionic strength suggest that electrostatic interaction is a key factor governing MTG catalysis at a solid surface. The results of this study indicate that, in enzymatic catalysis at a solid surface, the concentration of substrates at the surface can enhance the catalytic efficiency, and this could alter the pH dependence of enzymatic catalysis.
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PMID:Exploring enzymatic catalysis at a solid surface: a case study with transglutaminase-mediated protein immobilization. 1752 Jan 45

To understand molecular responses of crustacean hemocytes to virus infection, we applied 2-DE proteomics approach to investigate altered proteins in hemocytes of Penaeus vannamei during Taura syndrome virus (TSV) infection. At 24 h postinfection, quantitative intensity analysis and nano-LC-ESI-MS/MS revealed 11 forms of 8 proteins that were significantly up-regulated, whereas 9 forms of 5 proteins were significantly down-regulated in the infected shrimps. These altered proteins play important roles in host defense (hemocyanin, catalase, carboxylesterase, transglutaminase, and glutathione transferase), signal transduction (14-3-3 zeta), carbohydrate metabolism (acetylglucosamine pyrophosphorylase), cellular structure and integrity (beta-tubulin, beta-actin, tropomyosin, and myosin), and ER-stress response (protein disulfide isomerase). Semiquantitative RT-PCR and Western blot analysis confirmed the upregulation of 14-3-3 at both mRNA and protein levels. Interestingly, several altered protein spots were identified as fragments of hemocyanin. Mass spectrometric analysis showed that the hemocyanin spots at acidic and basic regions represented the C- and N-terminal hemocyanin fragments, respectively. As three-quarters of C-terminal fragments were up-regulated, whereas two-thirds of N-terminal hemocyanin fragments were down-regulated, we therefore hypothesize that C- and N-terminal hemocyanin fragments may have differential roles in hemocytes. Further investigation of these data may lead to better understanding of the molecular responses of crustacean hemocytes to TSV infection.
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PMID:Proteomic analysis of differentially expressed proteins in Penaeus vannamei hemocytes upon Taura syndrome virus infection. 1772 5


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