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)

We previously reported that annexin V promoted the survival of cultured rat neocortical neurons. In an effort to elucidate the mechanism underlying this neurotrophic activity of annexin V, we have attempted to identify the target or binding proteins of annexin V in neuronal cells. Herein, we screened an embryonic day 17 rat brain cDNA library by western blot using glutathione S-transferase-annexin V fusion protein as a probe and then isolated four clones showing binding to annexin V in a Ca2+ - and phospholipid-dependent manner. Although these cDNAs encoded different polypeptides, all four polypeptides shared the unique feature of containing highly hydrophilic stretches with high Lys, Glu, and Ser contents. Deduced amino acid sequences of two clones showed high homology with human X-linked Helicase2 (XH2) and DNA (cytosine-5) methyltransferase (DMTase) sequences, whereas the other two were not related to any known peptide sequence. These results suggest that XH2 and DMTase are candidates for annexin V-binding proteins and thus may mediate the biological activity of annexin V.
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PMID:Molecular cloning and characterization of annexin V-binding proteins with highly hydrophilic peptide structure. 866 30

In order to understand the functional significance of HlyC-dependent acylation of the Escherichia coli hemolysin structural protein (HlyA), random as well as site-directed substitutions at the known regions of modification, i.e., those at lysine residues at amino acid positions 563 and 689 (HlyAK563 and HlyAK689, respectively), were isolated. Sixteen random hlyA mutations were identified on the basis of a screen for loss of immunoreactivity to the hemolysin-neutralizing D12 monoclonal antibody that reacts to only HlyC-activated HlyA. These substitutions occurred at the region from HlyAE684 to HlyAY696. A recombinant glutathione S-transferase-hemolysin gene fusion encoding glutathione S-transferase-HlyAS608-T725 residues reacts with monoclonal antibody when HlyC is coexpressed with the fusion protein. Therefore, at most only 12% of the total HlyA primary sequence is needed for HlyC-facilitated acylation at the HlyAK689 position, and this modification can occur in the absence of the proximal HlyAK563 acylation site. The cytolytic activities of these HlyA mutants against sheep erythrocytes and bovine and human lymphocyte cell lines (BL-3 and Raji cells, respectively) were analyzed. HlyAK563 and HlyAK689 substitutions displayed various degrees of loss of cytotoxicity that depended on the particular amino acid replacement. An HlyAK563C variant retained greater than 59 and 21% of its BL-3-lytic and erythrolytic activities, respectively, but was nearly inactive against Raji cells. An HlyA mutant with a K-to-E substitution at amino acid 689 (HlyAK689E) was essentially inactive against all three cell types, whereas an HlyAK689R substitution had a pattern of activity similar to that of the HlyAK563C mutant. Preceding the two in vitro acylated HlyA lysines are glycines that appear to be the only amino acids conserved in alignments of these regions among the RTX toxins. Remarkably, considering the retention of cytotoxic activity by some HlYAK689 mutants, each of three different substitutions at the HlyAG688 position was relatively inactive against all three cell types tested. This suggests that HlyAG688 plays a significant structural role in cytotoxic activity apart from its possible participation in an HlyC activation process which presumably requires recognition of pro-HlyA structures. The related RTX toxin, the Pasteurella haemolytica leukotoxin structural protein (LktA), can be activated in an E. coli recombinant background by HlyC. In amino acid sequence alignments, LktAK554 is equivalent to the HlyAK563 position but it has an asparagine (LktAN684) at the homologous HlyAK689 site. An LktAN684K substitution possesses wild-type leukotoxin activity against BL-3 cells and does not acquire hemolytic or Raji cell cytotoxic activity. Surprisingly, both LktAK554C and LktAK554T substitutions retain considerable BL-3 cytotoxicity (45 and 49%, respectively), indicating that there may be additional lysines within LktA that the HlyC activation mechanism is capable of acylating. Based on these results and a comparison of amino acid sequence alignments of 12 RTX toxins, a putative consensus structure of the RTX residues necessary for HlyC activation is hypothesized.
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PMID:Escherichia coli hemolysin mutants with altered target cell specificity. 875 37

We previously identified a novel src- and ras-suppressed gene, 322, encoding a mitogenic regulatory function (Lin, X., Nelson, P. J., Frankfort, B., Tombler, E., Johnson, R., and Gelman, I. H. (1995) Mol. Cell. Biol. 15, 2754-2762). Here, we characterize the 322 gene product as an in vivo and in vitro substrate of protein kinase C (PKC). Hence, we named this product SSeCKS (pronounced essex) for Src Suppressed C Kinase Substrate. Rabbit polyclonal sera raised against glutathione S-transferase (GST)-SSeCKS recognized a myristylated 280/290-kDa doublet in Rat-6 fibroblasts. SSeCKS levels in src- and ras-transformed Rat-6 cells were 15- and 8-fold less, respectively, than those in untransformed cells. Short-term addition of phorbol ester resulted in a 5-fold increase in SSeCKS phosphorylation which was inhibited by bis-indolylmaleimide. In vitro phosphorylation of GST-SSeCKS by purified rabbit brain PKC-alpha was enhanced by phosphatidylserine and blocked by excess PKC pseudosubstrate inhibitor peptide. GST-SSeCKS bound purified PKC-alpha or PKC from Rat-6 lysates in a phosphatidylserine-dependent manner. Four SSeCKS domains containing Lys/Arg-rich motifs similar to the PKC phosphorylation site in MARCKS were phosphorylated in vitro by PKC. Immunofluorescence analysis showed SSeCKS present throughout the cytoplasm with enrichment in podosomes and at the cell edge. Short-term addition of phorbol esters caused the movement of SSeCKS from plasma membrane sites to the perinucleus coincident with a loss of actin stress fibers. These data suggest a role for SSeCKS in the control of cellular cytoskeletal architecture.
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PMID:A novel src- and ras-suppressed protein kinase C substrate associated with cytoskeletal architecture. 891 Apr 68

The equine herpesvirus 1 (EHV-1) immediate-early (IE) phosphoprotein is essential for the activation of transcription from viral early and late promoters and regulates transcription from its own promoter. The EHV-1 EICP22 protein, a homolog of ICP22 of herpes simplex virus, increased the in vitro DNA binding activity of the IE protein for sequences in the IE, early, and late promoters. The EICP22 protein affected the rate as well as the extent of the IE protein binding to promoter DNA sequences. To study the DNA binding activity of the IE protein, Trp493, Gln495, Asn496, and Lys498 of the WLQN region, which is directly involved in DNA binding, were replaced with Ser (IEW493S), Glu (IEQ495E), Ile (IEN496I), and Glu (IEK498E), respectively. Gel shift assays revealed that the glutathione S-transferase (GST)-IEQ495E(407-615) and GST-IEK498E(407-615) proteins failed to bind to the IE promoter, indicating that the Gln and Lys residues are important for the DNA binding activity. In the presence of the GST-EICP22 protein, DNA binding activity of the GST-IEQ495E(407-615) protein was restored, suggesting that the EICP22 protein cooperates with the IE protein to regulate EHV-1 gene expression. Transient-transfection assays also showed that the EICP22 protein allowed the IEQ495E mutant to be functional as a transactivator. These results are unique and may represent an important role for the EICP22 protein in EHV-1 gene regulation.
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PMID:The ICP22 protein of equine herpesvirus 1 cooperates with the IE protein to regulate viral gene expression. 899 19

Previously we demonstrated that nonvisual arrestins exhibit a high affinity interaction with clathrin, consistent with an adaptor function in the internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this report we show that a short sequence of highly conserved residues within the globular clathrin terminal domain is responsible for arrestin binding. Limited proteolysis of clathrin cages results in the release of terminal domains and concomitant abrogation of arrestin binding. The nonvisual arrestins, beta-arrestin and arrestin3, but not visual arrestin, bind specifically to a glutathione S-transferase-clathrin terminal domain fusion protein. Deletion analysis and alanine scanning mutagenesis localize the binding site to residues 89-100 of the clathrin heavy chain and indicate that residues 1-100 can function as an independent arrestin binding domain. Site-directed mutagenesis identifies an invariant glutamine (Glu-89) and two highly conserved lysines (Lys-96 and Lys-98) as residues critical for arrestin binding, complementing hydrophobic and acidic residues in arrestin3 which have been implicated in clathrin binding (Krupnick, J. G., Goodman, O. B., Jr., Keen, J. H., and Benovic, J. L. (1997) J. Biol. Chem. 272, 15011-15016). Despite exhibiting high affinity clathrin binding, arrestins do not induce coat assembly. The terminal domain is oriented toward the plasma membrane in coated pits, and its binding of both arrestins and AP-2 suggests that this domain is the anchor responsible for adaptor-receptor recruitment to the coated pit.
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PMID:Arrestin/clathrin interaction. Localization of the arrestin binding locus to the clathrin terminal domain. 916 77

In the accompanying paper (Chung, J.-K., Sekiya, F., Kang, H.-S., Lee, C., Han, J.-S., Kim, S. R., Bae, Y. S., Morris, A. J., and Rhee, S. G. (1997) J. Biol. Chem. 272, 15980-15985), synaptojanin is identified as a protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2). Here, the purification from rat brain cytosol of another PLD-inhibitory protein that is immunologically distinct from synaptojanin is described, and this protein is identified as clathrin assembly protein 3 (AP3) by peptide sequencing and immunoblot analysis. AP3 binds both inositol hexakisphosphate and preassembled clathrin cages with high affinity. However, neither inositol hexakisphosphate binding nor clathrin cage binding affected the ability of AP3 to inhibit PLD. AP3 also binds to PI(4,5)P2 with low affinity. But the PI(4,5)P2 binding was not responsible for PLD inhibition, because the potency and efficacy of AP3 as an inhibitor of PLD were similar in the absence and presence of PI(4,5)P2. A bacterially expressed fusion protein, glutathione S-transferase-AP3 (GST-AP3), also inhibited PLD with a potency equal to that of brain AP3. The inhibitory effect of AP3 appeared to be the result of direct interaction between AP3 and PLD because PLD bound GST-AP3 in an in vitro binding assay. Using GST fusion proteins containing various AP3 sequences, we found that the sequence extending from residues Pro-290 to Lys-320 of AP3 is critical for both inhibition of and binding to PLD. The fact that AP3 is a synapse-specific protein indicates that the AP3-dependent inhibition of PLD might play a regulatory role that is restricted to the rapid cycling of synaptic vesicles.
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PMID:Inhibition of phospholipase D by clathrin assembly protein 3 (AP3). 918 1

Chlamydia trachomatis is an obligate intracellular pathogen, long recognized as an agent of blinding eye disease and more recently as a common sexually transmitted infection. Recently, two eukaryotic histone H1-like proteins, designated Hc1 and Hc2, have been identified in Chlamydia. Expression of Hc1 in recombinant Escherichia coli produces chromatin condensation similar to nucleoid condensation observed late in the parasite's own life cycle. In contrast, chromatin decondensation, observed during the early life cycle, accompanies down-regulation and nondetection of Hc1 and Hc2 among internalized organisms. We reasoned that the early upstream open reading frame (EUO) gene product might play a role in Hc1 degradation and nucleoid decondensation since it is expressed very early in the chlamydial life cycle. To explore this possibility, we fused the EUO coding region between amino acids 4 and 177 from C. trachomatis serovar Lz with glutathione S-transferase (GST) and examined the effects of fusion protein on Hc1 in vitro. The purified fusion protein was able to digest Hc1 completely within 1 h at 37 degrees C. However, GST alone exhibited no Hc1-specific proteolytic activity. The chlamydial EUO-GST gene product also cleaves very-lysine-rich calf thymus histone H1 and chicken erythrocyte histone H5 but displays no measurable activity towards core histones H2A, H2B, H3, and H4 or chlamydial RNA polymerase alpha-subunit. This proteolytic activity appears sensitive to the serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) and aspartic protease inhibitor pepstatin but resistant to high temperature and other broad-spectrum protease inhibitors. The proteolytic activity specified by the EUO-GST fusion product selectively digested the C-terminal portion of chlamydial Hc1, the domain involved in DNA binding, while leaving the N terminus intact. At a molar equivalent ratio of 1:1 between Hc1 and DNA, the EUO gene product cleaves Hc1 complexed to DNA and this cleavage appears sufficient to initiate dissociation of DNA-Hc1 complexes. However, at a higher molar equivalent ratio of Hc1/DNA (10:1), there is partial protection conferred upon Hc1 to an extent that prevents dissociation of DNA-Hc1 complexes.
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PMID:The chlamydial EUO gene encodes a histone H1-specific protease. 929 54

A 1.7-kilobase pair segment from the conjugative transfer region of plasmid R388 DNA was cloned and sequenced. It contained trwD, a gene essential for plasmid R388 conjugation, for expression of the conjugative W-pilus and for sensitivity to phage PRD1. The deduced amino acid sequence of TrwD showed homology to the PulE/VirB11 superfamily of potential ATPases involved in various types of transport processes. A fusion of trwD with the glutathione S-transferase (GST) was constructed, and the resulting fusion protein was purified from overproducing bacteria. Factor Xa hydrolysis of GST-TrwD and further purification rendered TrwD protein with more than 95% purity. Antibodies raised against TrwD localized it both in the soluble fraction and in the outer membrane of Escherichia coli. TrwD is probably a peripheral outer membrane protein because it could be solubilized by increasing salt concentration to 0.5 M NaCl in the lysis buffer. Both purified GST-TrwD and TrwD could hydrolize ATP. ATPase activity increased 2-fold in the presence of detergent-phospholipid mixed micelles. To study the importance of the nucleotide-binding site, Walker box A (GXXGXGK(T/S)), present in TrwD, the conserved lysine residue was replaced by glutamine. The mutant protein, expressed and purified under the same conditions as the wild type, did not exhibit ATPase activity. TrwD(K203Q) was not able to complement the mutation in trwD of the R388 mutant plasmid, suggesting the essentiality of the ATPase activity of the protein in the conjugative process. Furthermore, the dominant character of this mutation suggested that GST-TrwD(K432Q) was still able to interact either with itself or with other component(s) of the conjugative machinery.
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PMID:TrwD, a protein encoded by the IncW plasmid R388, displays an ATP hydrolase activity essential for bacterial conjugation. 932 77

The kidney and several other thyroid hormone-responsive tissues contain a NADP-regulated thyroid hormone (TH)-binding protein (THBP), with an apparent molecular mass of 36 kDa on SDS-PAGE, responsible for most of the intracellular high-affinity T3 and T4 binding. THBP was purified to homogeneity from human kidney cytosol and used to generate proteolytic peptides. Microsequencing of four peptides revealed identity to amino acid sequences deduced from a human cDNA homolog to a cDNA encoding kangaroo mu-crystallin. This protein is a major structural kangaroo lens protein with no known function in other species. A full-sized cDNA (TH5.9) was isolated by 5'- and 3'-rapid amplification of cDNA ends using a human brain cDNA library and gene-specific PCR primers, confirming identity to the previously cloned human cDNA. The TH5.9 cDNA encodes a 314-residue protein (theoretical mol wt = 33,775) with significant homologies (40 to 60%) with two bacterial enzymes: lysine cyclodeaminase and ornithine cyclodeaminase. The TH5.9 cDNA was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Purified GST fusion protein, but not GST, bound T3 specifically with high affinity [dissociation constant (Kd) = 0.5 nM] in the presence of NADPH, and was labeled by UV-driven cross-linking of underivatized [(125)I]T3. T3 binding and photoaffinity labeling of GST fusion protein were activated by NADPH [activation constant (K[act]) = 10(-8) M], but not by NADH. The expressed protein displays the appropriate binding properties, indicating that TH5.9 cDNA encodes the NADP-regulated THBP characterized in human tissues.
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PMID:Purification, molecular cloning, and functional expression of the human nicodinamide-adenine dinucleotide phosphate-regulated thyroid hormone-binding protein. 932 54

Escherichia coli-expressed chicken-liver glutathione S-transferase, cGSTA1-1, displays high ethacrynic acid (EA)-conjugating activity. Molecular modelling of cGSTA1-1 with EA in the substrate binding site reveals that the side chain of Phe-111 protrudes into the substrate binding site and possibly interacts with EA. Replacement of Phe-111 with alanine resulted in an enzyme (F111A mutant) with a 4.5-fold increase in EA-conjugating activity (9.2 mmol/min per mg), and an incremental Gibbs free energy (DeltaDeltaG) of 4.0 kJ/mol lower than that of the wild-type cGSTA1-1. Two other amino acid residues that possibly interact with EA are Ser-208 and Lys-15. Substitution of Ser-208 with methionine generated a cGSTA1-1(F111AS208M) double mutant that has low EA-conjugating activity (2.0 mmol/min per mg) and an incremental Gibbs free energy of +3.9 kJ/mol greater than the cGSTA1-1(F111A) single mutant. The cGSTA1-1(F111A) mutant, with an additional Lys-15-to-leucine substitution, lost 90% of the EA-conjugating activity (0.55 mmol/min per mg). The Km values of the cGSTA1-1(F111A) and cGSTA1-1(F111AK15L) mutants for EA are nearly identical. The wild-type cGSTA2-2 isoenzyme has a low EA-conjugating activity (0.56 mmol/min per mg). The kcat of this reaction can be increased 2. 5-fold by substituting Arg-15 and Glu-104 with lysine and glycine respectively. The KmEA of the cGSTA2-2(R15KE104G) double mutant is nearly identical with that of the wild-type enzyme. Another double mutant, cGSTA2-2(E104GL208S), has a KmEA that is 3.3-fold lower and a kcat that is 1.8-fold higher than that of the wild-type enzyme. These results, taken together, illustrate the interactions of Lys-15 and Ser-208 on cGSTA1-1 with EA.
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PMID:Characterization of chicken-liver glutathione S-transferase (GST) A1-1 and A2-2 isoenzymes and their site-directed mutants heterologously expressed in Escherichia coli: identification of Lys-15 and Ser-208 on cGSTA1-1 as residues interacting with ethacrynic acid. 935 34


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