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

---

Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The dihydrolipoyl acetyltransferase (E2) component of the mammalian pyruvate dehydrogenase complex forms a 60-subunit core in which E2's inner domain forms a dodecahedron shaped structure surrounded by its globular outer domains that are connected to each other and the inner domain by 2-3-kDa mobile hinge regions. Two of the outer domains are approximately 10 kDa lipoyl domains, an NH2-terminal one, E2L1, and, after the first hinge region a second one, E2L2. The pyruvate dehydrogenase kinase binds tightly to the lipoyl domain region of the oligomeric E2 core and phosphorylates and inactivates the pyruvate dehydrogenase (E1) component. We wished to determine whether lipoyl domain constructs prepared by recombinant techniques from a cDNA for human E2 could bind the bovine E1 kinase and, that being the case, to pursue which lipoyl domain the kinase binds. We also wished to gain insights into how a molecule of kinase tightly bound to the E2 core can rapidly phosphorylate 20-30 molecules of the pyruvate dehydrogenase (E1) component which are also bound to an outer domain of the E2 core. We prepared recombinant constructs consisting of the entire lipoyl domain region or the individual lipoyl domains with or without the intervening hinge region. Constructs were made and used both as free lipoyl domains and fused to glutathione S-transferase (GST). Using GSH-Sepharose to selectively bind GST constructs, tightly bound kinase was shown to rapidly transfer in a highly preferential way from intact E2 core to GST constructs containing the E2L2 domain rather than to ones containing only the E2L1 domain. GST-E2L2-kinase complexes could be eluted from GSH-Sepharose with glutathione. Delipoylation of E2L2 by treatment with lipoamidase eliminated kinase binding supporting a direct role of the lipoyl prosthetic group in this association. Transfer to and selective binding of the kinase by E2L2 but not E2L1 was also demonstrated with free constructs using a sucrose gradient procedure to separate the large E2 core from the various lipoyl domain constructs. E2L2 but not E2L1 increased the activity of resolved kinase by up to 43%. We conclude that the kinase selectively binds to the inner lipoyl domain of E2 subunits and that this association involves its lipoyl prosthetic group. We further suggest that transfer of tightly bound kinase between E2L2 domains occurs by a direct interchange mechanism without formation of free kinase (model presented).(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Binding of the pyruvate dehydrogenase kinase to recombinant constructs containing the inner lipoyl domain of the dihydrolipoyl acetyltransferase component. 782 13

The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex (PDC) associate to form a large inner core with a protruding structure composed of three globular domains connected by mobile linker regions. This exterior region of E2 includes two lipoyl domains which engage not only in the intermediate reactions of the complex but also have integral roles in the kinase-phosphatase regulatory interconversion of the pyruvate dehydrogenase (E1) component. To facilitate understanding of these roles, lipoyl domain constructs of the E2 component of human PDC were expressed as glutathione S-transferase (GST)-linked fusion proteins from plasmid inserts prepared by polymerase chain reaction procedures. The NH2-terminal lipoyl domain, E2L1, and the interior lipoyl domain, E2L2, are connected by a 30-amino-acid hinge region, H1. Constructs designed and expressed were E2L1(1-98), E2L1.H1(1-128), E2L2(120-233), E2H1.L2(98-233), and E2L1.H1.L2(1-233), where numbers in parentheses give the amino acid sequence for the portions of the E2 component incorporated into a construct. The domains were expressed in Escherichia coli with and without lipoate supplementation. GST constructs were purified to homogeneity by affinity chromatography and selectively released by thrombin treatment. Sequencing of insert DNAs and NH2-terminal sequencing confirmed that domains were produced as designed. Measurement of masses by electrospray mass spectrometry indicated that constructs with lipoylated, nonlipoylated, and octanoylated forms were produced when expression was with E. coli grown without lipoate supplementation and that fully lipoylated forms were produced upon lipoate supplementation. The lipoylation status was confirmed, following delipoylation with Enterococcus faecalis lipoamidase, by the expected decrease in mass and by the observation in native gel electrophoresis of a shift to a slower mobility (possibly less compact) form. Constructs were used in E1-catalyzed reductive-acetylation reaction in proportion to their degree of lipoylation and were effective substrates in a NADH-dependent dihydrolipoyl dehydrogenase reduction reaction. Thus, we have produced lipoyl domain constructs that can be employed in sorting the specific roles of E2L1 and E2L2 in facilitating catalytic and regulatory processes.
...
PMID:Recombinant expression and evaluation of the lipoyl domains of the dihydrolipoyl acetyltransferase component of the human pyruvate dehydrogenase complex. 786 52

A vector has been constructed to allow genetic fusions of guest antigens via a hinge domain to the C terminus of the highly immunogenic C fragment of tetanus toxin. A fusion has been constructed with the gene encoding the protective 28-kDa glutathione S-transferase (EC 2.5.1.18) from Schistosoma mansoni. The recombinant vector has been electroporated into the nonvirulent Salmonella typhimurium aroA live vaccine strain SL3261. The corresponding chimeric protein is stably expressed in a soluble form in Salmonella as evaluated by Western blotting with fragment C and glutathione S-transferase antisera. Mice immunized intravenously with a single dose of the live recombinant bacteria elicit antibodies to both fragment C and glutathione S-transferase as detected by enzyme-linked immunosorbent assays. Furthermore, all of the mice were solidly protected when challenged with lethal doses of either tetanus toxin or the virulent Salmonella typhimurium strain C5. Mice have also elicited antibodies to fragment C and glutathione S-transferase after oral immunization. It may be that a live trivalent vaccine against typhoid, tetanus, and schistosomiasis is feasible.
...
PMID:Construction, expression, and immunogenicity of the Schistosoma mansoni P28 glutathione S-transferase as a genetic fusion to tetanus toxin fragment C in a live Aro attenuated vaccine strain of Salmonella. 797 44

The protein tyrosine kinase p72syk (Syk) is expressed in a variety of hematopoietic cell types, including B cells, thymocytes, mast cells and others. Both the activity and phosphotyrosine content of this enzyme increase in these cells in response to engagement of the appropriate cell surface receptors. Herein, we describe the cloning of murine Syk and its expression in Sf9 cells as a catalytically active protein. Full-length Syk and a catalytically active 42.5 kDa carboxyl terminal fragment were also expressed as glutathione S-transferase fusion proteins. Comparative reverse phase HPLC and 40% alkaline gel analysis of tryptic digests of phosphorylated Syk demonstrated that all of the major sites of autophosphorylation were also present in GST-Syk and all but one were contained in the 42.5 kDa fragment. The sites of autophosphorylation were identified using a combination of Edman sequencing and mass spectrometric analysis. Ten sites were identified. One site is located in the amino terminal half of the molecule between the two tandem Src homology 2 (SH2) domains. Five sites are located in the hinge region located between the carboxyl terminal SH2 domain and the kinase domain. Two sites lie in the kinase domain within the catalytic loop and two near the extreme carboxyl terminus. Sequences of phosphorylation sites located within the hinge region predict that Syk serves as a docking site for other SH2 domain-containing proteins. Consistent with this prediction, autophosphorylated Syk efficiently binds the carboxyl terminal SH2 domain of phospholipase C-gamma 1.
...
PMID:Identification of the major sites of autophosphorylation of the murine protein-tyrosine kinase Syk. 904 38

Activating signal cointegrator 1 (ASC-1) harbors an autonomous transactivation domain that contains a putative zinc finger motif which provides binding sites for basal transcription factors TBP and TFIIA, transcription integrators steroid receptor coactivator 1 (SRC-1) and CBP-p300, and nuclear receptors, as demonstrated by the glutathione S-transferase pull-down assays and the yeast two-hybrid tests. The ASC-1 binding sites involve the hinge domain but not the C-terminal AF2 core domain of nuclear receptors. Nonetheless, ASC-1 appears to require the AF2-dependent factors to function (i.e., CBP-p300 and SRC-1), as suggested by the ability of ASC-1 to coactivate nuclear receptors, either alone or in cooperation with SRC-1 and p300, as well as its inability to coactivate a mutant receptor lacking the AF2 core domain. By using indirect immunofluorescence, we further show that ASC-1, a nuclear protein, is localized to the cytoplasm under conditions of serum deprivation but is retained in the nucleus when it is serum starved in the presence of ligand or coexpressed CBP or SRC-1. These results suggest that ASC-1 is a novel coactivator molecule of nuclear receptors which functions in conjunction with CBP-p300 and SRC-1 and may play an important role in establishing distinct coactivator complexes under different cellular conditions.
...
PMID:Activating signal cointegrator 1, a novel transcription coactivator of nuclear receptors, and its cytosolic localization under conditions of serum deprivation. 1045 79

Hsp90 association with glucocorticoid receptors (GRs) is required for steroid binding. We recently reported that seven amino acids (547-553) overlapping the amino-terminal end of the rat GR ligand-binding domain are necessary for hsp90 binding, and consequently steroid binding. The role of a LXXLL motif at the COOH terminus of this sequence has now been analyzed by determining the properties of Leu to Ser mutations in full-length GR and glutathione S-transferase chimeras. Surprisingly, these mutations decreased steroid binding capacity without altering receptor levels, steroid binding affinity, or hsp90 binding. Single mutations in the context of the full-length receptor did not affect the transcriptional activity but the double mutant (L550S/L553S) was virtually inactive. This biological inactivity was found to be due to an increased rate of steroid dissociation from the activated mutant complex. These results, coupled with those from trypsin digestion studies, suggest a model in which the GR ligand-binding domain is viewed as having a "hinged pocket," with the hinge being in the region of the trypsin digestion site at Arg(651). The pocket would normally be kept shut via the intramolecular interactions of the LXXLL motif at amino acids 550-554 acting as a hydrophobic clasp.
...
PMID:The seven amino acids (547-553) of rat glucocorticoid receptor required for steroid and hsp90 binding contain a functionally independent LXXLL motif that is critical for steroid binding. 1059 51

Estrogen receptors (ERs) are ligand-activated transcription factors that regulate gene expression and cell growth. Two ERs now have been identified: ERalpha and the more recently discovered ERbeta. The physiological function of ERbeta remains unclear, but evidence from vascular injury studies and from ERbeta knockout mice suggests that ERbeta may be involved in the regulation of cellular proliferation. Here we show a direct and specific interaction between ERbeta and the cell cycle mitotic spindle assembly checkpoint protein, MAD2 (mitosis arrest-deficient 2). The ERbeta-MAD2 interaction was identified by screening of a yeast two-hybrid system vascular endothelial cell library with ERbeta and confirmed with glutathione S-transferase-fusion protein interaction studies. In contrast, ERalpha did not interact with MAD2 in either the two-hybrid system or in the protein-protein interaction experiments. Amino acids 173-208 in the hinge region of ERbeta were sufficient to mediate the interaction with MAD2 in the two-hybrid system and in glutathione S-transferase-fusion protein studies. These data identify a link between ERbeta and MAD2 of potential importance to regulation of the cell cycle and support a function of ERbeta distinct from the established role of ERs as transcription factors.
...
PMID:Specific association of estrogen receptor beta with the cell cycle spindle assembly checkpoint protein, MAD2. 1070 29

A zebrafish Ftz-F1 homologue, zFF1A (zebrafish Ff1a or Nr5a2, a member of nuclear receptor superfamily) and its C-terminally truncated variant (zFF1B) were previously identified. Due to lack of the identity box (I-box) and activation function 2 (AF-2) domain, zFF1B lacks transactivation function and fails to synergize with estrogen receptor (ER) in regulating promoters. It was speculated that the I-box might be involved in the zFF1A/ER interaction. In the present study, the function of the I-box was examined. In the absence of the I-box or with an altered heptad 9, the AF-2 of zFF1A was not functional, either in the presence or absence of ER. The GST pull-down assay showed that zFF1A and its mutants exerted similar physical contacts with ER-LBD, suggesting that the "dimerization" domain (I-box) is essential for the transcriptional activity of zFF1A. Moreover, nuclear receptor coactivator selectively activated zFF1 with the I-box but exerted no effect on zFF1B, indicating that the I-box is able to interact with the coactivators. By deletion study and analysis of the identified domains in GAL4-DNA binding domain, other regions of zFF1A critical for its AF were also delineated. Consistent with the mutation analysis, AF-2 was active only in the presence of the I-box. We also identified a novel AF domain (AF-3) located in the hinge region (amino acids 155-267), although the activity of AF-3 was inhibited by its flanking region. We suggest that the D and E regions of zFF1A possess both positive and negative transactivation functions, and interdomain "cross-talk" may confer the full transcriptional activity of the protein.
...
PMID:A zebrafish ftz-F1 (Fushi tarazu factor 1) homologue requires multiple subdomains in the D and E regions for its transcriptional activity. 1074 75

Retinoid receptor-related testis-associated receptor (RTR)/germ cell nuclear factor is a nuclear orphan receptor that plays an important role in the control of gene expression during early embryonic development and gametogenesis. It has been shown to repress transcriptional activation. In this study, we further characterize this repressor function. We demonstrate that RTR can suppress the transcriptional activation induced by the estrogen receptor related-receptor alpha1 through its response element. The latter is at least in part due to competition for binding to the same response element. In addition, RTR inhibits basal transcriptional activation, indicating that it functions as an active repressor. Mammalian two-hybrid analyses showed that RTR interacts with the co-repressor nuclear co-repressor (N-CoR) but is unable to interact with the co-repressor SMRT or RIP140. Pull-down analyses with glutathione S-transferase-RTR fusion protein demonstrated that RTR physically interacts with N-CoR in vitro, suggesting a potential role for N-CoR in the transcriptional repression by RTR. To identify the regions in RTR essential for the binding of RTR to N-CoR, the effect of various deletion and point mutations on this interaction was examined. This analysis revealed that this interaction requires the hinge domain, helix 3 as well as the helix 12 region of RTR. The residues Ser(246)-Tyr(247) in the hinge domain, Lys(318) in helix 3, and Lys(489)-Thr(490) in helix 12 are identified as being critical in this interaction. Our results demonstrate that RTR can function as an active transcriptional repressor and that this repression can be mediated through interactions with the co-repressor N-CoR. We show that this interaction exhibits several characteristics unique to RTR. Through its repressor function, RTR can suppress the induction of transcriptional activation by other nuclear receptors. These repressor activities may provide important mechanisms by which RTR regulates gene expression during development and gametogenesis.
...
PMID:Characterization of the repressor function of the nuclear orphan receptor retinoid receptor-related testis-associated receptor/germ cell nuclear factor. 1094 Mar 6

Antibodies against the Trypanosoma cruzi ribosomal P2beta protein (TcP2beta) have been associated with the chronic cardiac pathology of Chagas' disease in humans. Using synthetic peptides spanning the entire TcP2beta molecule, we investigated their epitope recognition by antibodies from mice chronically infected with T. cruzi and from mice immunized with two recombinant TcP2betas. We found clear differences in epitope recognition between antibodies from T. cruzi-infected mice and mice immunized with two different recombinant TcP2betas associated with different schedules of immunization. Major epitopes recognized by antibodies from mice immunized with recombinant glutathione S-transferase (GST) or histidine (Hist) fusion TcP2beta (GST-TcP2beta or Hist-TcP2beta) are located in the central and hinge regions of the molecule. Nevertheless, mice immunized with Hist-TcP2beta were also able to elicit antibodies against the TcP2beta C terminus, a region which is highly conserved in both T. cruzi and mammal ribosomal P proteins. Strikingly, antibodies from infected animals recognized only the TcP2beta C terminus. By using these antisera with distinct profiles of epitope recognition, it could be shown that only C terminus-specific antibodies were able to increase the beating frequency of cardiomyocytes from neonatal rats in vitro by selective stimulation of the beta1-adrenergic receptor. Thus, antibodies against the TcP2beta C terminus elicited in the absence of infection are able to modulate a functional activity of host cells through a molecular mimicry mechanism.
...
PMID:Modulation of cardiocyte functional activity by antibodies against trypanosoma cruzi ribosomal P2 protein C terminus. 1094 33


1 2 3 4 Next >>

---