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)

Structural elucidation of posttranslationally modified peptides and proteins is of key importance in the understanding of an array of biological processes. Ubiquitination is a reversible modification that regulates many cellular functions. Consequences of ubiquitination depend on whether a single ubiquitin or polyubiquitin chain is added to the tagged protein. The lysine residue through which the polyubiquitin chain is formed is also critical for biological activity. Robust methods are therefore required to identify sites of ubiquitination modification, both in the target protein and in ubiquitin. Here, we demonstrate the suitability of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, in conjunction with activated ion electron capture dissociation (AI ECD) or infrared multiphoton dissociation (IRMPD), for the analysis of ubiquitinated proteins. Polyubiquitinated substrate protein GST-Ubc5 was generated in vitro. Tryptic digests of polyubiquitinated species contain modified peptides in which the ubiquitin C-terminal Gly-Gly residues are retained on the modified lysine residues. Direct infusion microelectrospray FT-ICR of the digest and comparison with an in silico digest enables identification of modified peptides and therefore sites of ubiquitination. Fifteen sites of ubiquitination were identified in GST-Ubc5 and four sites in ubiquitin. Assignments were confirmed by AI ECD or IRMPD. The Gly-Gly modification is stable and both tandem mass spectrometric techniques are suitable, providing extensive sequence coverage and retention of the modification on backbone fragments.
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PMID:Identification of sites of ubiquitination in proteins: a fourier transform ion cyclotron resonance mass spectrometry approach. 1557 50

A cDNA clone, designated CeCPI, encoding a novel phytocystatin was isolated from taro corms (Colocasia esculenta) using both degenerated primers/RT-PCR amplification and 5'-/3'-RACE extension. The full-length cDNA gene is 1,008 bp in size, encodes 206 amino acid residues, with a deduced molecular weight of 29 kDa. It contains a conserved reactive site motif Gln-Val-Val-Ser-Gly of cysteine protease inhibitors, and another consensus ARFAV sequence for phytocystatin. Sequence analysis revealed that CeCPI is phylogenetically closely related to Eudicots rather than to Monocots, despite taro belonging to Monocot. Recombinant GST-CeCPI fusion protein was overexpressed in Escherichia coli and its inhibitory activity against papain was identified on gelatin/SDS-PAGE. These results confirmed that recombinant CeCPI protein exhibited strong cysteine protease inhibitory activity. Investigation of its antifungal activity clearly revealed a toxic effect on the mycelium growth of phytopathogenic fungi, such as Sclerotium rolfsii Sacc. etc., at a concentration of 80 microg recombinant CeCPI/ ml. Moreover, mycelium growth was completely inhibited and the sclerotia lysed at a concentration of 150-200 microg/ml. Further studies have demonstrated that recombinant CeCPI is capable of acting against the endogenous cysteine proteinase in the fungal mycelium.
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PMID:Molecular cloning, recombinant gene expression, and antifungal activity of cystatin from taro (Colocasia esculenta cv. Kaosiung no. 1). 1564

The G-protein alpha subunit, alpha(13), regulates cell growth and differentiation through the monomeric Rho GTPase. Alpha(13) activates Rho through direct stimulation of the guanine nucleotide exchange factor p115RhoGEF, which contains a regulator of G-protein signaling homology domain (RH) in its N-terminus. Through its RH domain, p115RhoGEF also functions as a GAP for G alpha(13). The mechanism for the G alpha(13)/p115RhoGEF interaction is not well understood. Here, we determined specific alpha(13) residues important for its interaction with p115RhoGEF. GST-pulldowns and co-immunoprecipitation assays revealed that individually mutating alpha(13) residues Lys204, Glu229, or Arg232 to opposite charge residues disrupts the interaction of activated alpha(13) with the RH domain of p115RhoGEF or full-length p115RhoGEF. We further demonstrate that mutation of Glu229, and to a lesser extent Lys204 or Arg232, disrupts the ability of activated alpha(13) to induce the recruitment of p115RhoGEF to the plasma membrane (PM) and to activate Rho-mediated serum response element-luciferase gene transcription. Interestingly, an alpha(13) mutant where a conserved Gly was mutated to a Ser (G205S) retained its ability to bind to p115RhoGEF, induce p115RhoGEF recruitment to the PM, and activate Rho-dependent signaling, even though identical Gly to Ser mutations in other alpha disrupt their interaction with regulator of G-protein signaling (RGS) proteins. These results demonstrate that, whereas several features of a typical alpha/RGS interaction are preserved in the alpha(13)/p115RhoGEF interaction, there are also significant differences.
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PMID:Functional consequences of G alpha 13 mutations that disrupt interaction with p115RhoGEF. 1573 47

In an effort to gain further insight into the conformational and topographical requirements for recognition by the N-terminal SH2 domain of protein tyrosine phosphatase SHP-1, we synthesized a series of linear and cyclic peptides derived from the sequence surrounding phosphotyrosine 2267 in the receptor tyrosine kinase Ros (EGLNpYMVL). A molecular modeling approach was used to suggest peptide modifications sterically compatible with the N-SH2-peptide binding groove and possibly enhanced binding affinities compared to the parent peptide. The potencies of the synthesized compounds were evaluated by assaying their ability to stimulate phosphatase activity as well as by their binding affinities to the GST-fused N-SH2 domain of SHP-1. In the series of linear peptides, structural modifications of Ros pY2267 in positions pY + 1 to pY + 3 by amino acid residues structurally related to Phe, for example l-erythro/threo-Abu(betaPh) (5a, 5b), yielded ligands with increased binding affinity. The incorporation of d-amino acid residues at pY + 1 and pY + 3 led to inactive peptides. The replacement of Phe in both pY + 1 and pY + 3 by Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) was also not tolerated due to steric hindrance. Cyclic peptides (13, 14) that were linked via residues in positions pY - 1 (Lys) and pY + 2 (Asp/Glu) and contained a Gly residue in the bridging unit displayed much lower potencies for the stimulation of SHP-1 activity but increased binding affinities compared to Ros pY2267. They partially competed with Ros pY2267 in the activation assay. Such cyclic structures may serve as scaffolds for competitive SHP-1 inhibitor design targeting N-SH2 domain-protein interactions that block SHP-1 activation.
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PMID:Design and biological evaluation of linear and cyclic phosphopeptide ligands of the N-terminal SH2 domain of protein tyrosine phosphatase SHP-1. 1574 95

Signal Transducers and Activators of Transcription (STATs) are transcription factors shown to be activated by G protein-coupled receptors. In the present study, we demonstrate that acute morphine or [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) exposure of COS-7 cells transiently transfected with the micro-opioid receptor and STAT5A, leads to receptor-dependent tyrosine phosphorylation of STAT5A. Activation of HEK293 cells, stably expressing the micro-opioid receptor with micro-opioid agonists results in the transcriptional activation of a STAT-responsive reporter gene. Pertussis toxin has no effect on the level of STAT5A phosphorylation, while the Src inhibitor PP1 abolishes opioid-dependent STAT5A phosphorylation. All three opioid receptor subtypes -micro, delta and kappa- share the conserved motif YXXL (amino-acids 336-339 for the micro-opioid receptor), known to be critical for STAT5A/5B binding. Co-immunoprecipitation and pull-down experiments using a GST-carboxyl-terminal tail of the micro-opioid receptor and rat brain, or COS-7 cell cytosolic extracts, demonstrate the direct binding of STAT5A to this region. Mutation of the Y336 to alanine does not prevent STAT5A binding, whereas deletion of the entire putative STAT5A binding site YXXL abolishes STAT5A interaction to the carboxyl-terminal tail of the micro-opioid receptor. Collectively, our results demonstrate the association of STAT5A with the micro-opioid receptor and reveal novel signalling pathways in the regulation of transcription by the micro-opioid receptor.
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PMID:STAT5A interacts with and is phosphorylated upon activation of the mu-opioid receptor. 1585 95

The nucleocapsid (N) protein of SARS coronavirus (SARS_CoV) is a major structural component of virions, which appears to be a multifunctional protein involved in viral RNA replication and translation. Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is related to the pre-mRNA splicing in the nucleus and translation regulation in the cytoplasm. In this report, based on the relevant biophysical and biochemical assays, the nucleocapsid protein of SARS_CoV (SARS_N) was discovered to exhibit high binding affinity to human hnRNP A1. GST pull-down results clearly demonstrated that SARS_N protein could directly and specifically bind to human hnRNP A1 in vitro. Yeast two-hybrid assays further indicated in vivo that such binding relates to the fragment (aa 161-210) of SARS_N and the Gly-rich domain (aa 203-320) of hnRNP A1. Moreover, kinetic analyses by surface plasmon resonance (SPR) technology revealed that SARS_N protein has a specific binding affinity against human hnRNP A1 with K(D) at 0.35 +/- 0.02 microM (k(on) = 5.83 +/- 0.42 x 10(3) M(-1)s(-1) and k(off) = 2.06 +/- 0.12 x 10(-3)s(-1)). It is suggested that both SARS_N and hnRNP A1 proteins are possibly within the SARS_CoV replication/transcription complex and SARS_N/human hnRNP A1 interaction might function in the regulation of SARS_CoV RNA synthesis. In addition, the determined results showed that SARS_N protein has only one binding domain for interacting with human hnRNP A1, which is different from the mouse hepatitis virus (MHV) binding case where the nucleocapsid protein of MHV (MHV_N) was found to have two binding domains involved in the MHV_N/hnRNP A1 interaction, thereby suggesting that SARS_N protein might carry out a different binding mode to bind to human hnRNP A1 for its further function performance in comparison with MHV_N.
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PMID:The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1. 1586

Lipid phosphate phosphohydrolase-3 (LPP3) is a cell surface protein that exhibits ectoenzyme activity. Previously, we identified human LPP3 in a functional assay of angiogenesis and showed that the Arg-Gly-Asp (RGD) motif in the proposed second extracellular domain interacts with a subset of integrins to mediate cell-cell adhesion. In contrast to the RGD domain of human LPP3, murine Lpp3 contains a variant sequence, Arg-Gly-Glu (RGE). Whether the RGE motif of murine Lpp3 mediates cell-cell interaction has not been studied. In this report, we test the hypothesis that the cell adhesion function of the LPP3 protein is conserved across mouse and human. A glutathione S-transferase (GST) fusion protein of the proposed second extracellular loop of the murine Lpp3 sequence (GST-mLpp3-RGE) promoted attachment of cells in a long-term cell adhesion assay. GST-mLpp3-RGE interacted with alpha(5)beta(1) and alpha(v)beta(3) integrins in a solid-phase ELISA, while a mutant control, GST-hLPP3-RAD, did not. Long-term adhesion of endothelial cells to GST-mLpp3-RGE induced phosphorylation of FAK, SHC, and CAS, whereas adhesion to GST-hLPP3-RAD failed to do so. Upon long-term adhesion both the GST-hLPP3-RGD and GST-mLpp3-RGE substrates bound to the alpha(5)beta(1) integrin of FRT-alpha(5)(+) cells, an interaction that was inhibited by an anti-alpha(5) integrin antibody. In addition, a cell aggregation assay showed that the intact mLpp3-RGE protein interacts with alpha(5)beta(1) and alpha(v)beta(3) integrins expressed by adjacent cells, an interaction that can be blocked by GRGDSP peptides and anti-LPP3-RGD antibodies. These data, together with the known importance of integrins in angiogenesis, provide a mechanism for the function of LPP3 in cell-cell interactions in both human and mouse.
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PMID:Murine lipid phosphate phosphohydrolase-3 acts as a cell-associated integrin ligand. 1609 22

Glycine transporters are members of the Na+/Cl- dependent transporter gene family and play crucial roles in regulating inhibitory as well as excitatory neurotransmission. In this report we show that calcium elevation in spinal cord synaptosomes decreases the levels of glycine transporter, GlyT1, N-terminal immunoreactivity, and that this decrease can be blocked by calpain inhibitor. Sequencing of GST fusion proteins containing the N-terminal domains of GlyT1A and B splice variants cleaved with rat recombinant calpain identified calpain cleavage sites after glycine 17 in GlyT1B and N-terminally of the first conserved arginine residue in both GlyT1A and GlyT1B. Expression in HEK293 cells revealed that truncation of the N-terminus of GlyT1 results in significant inhibition of glycine uptake. A syntaxin1A GST fusion protein was able to pull-down N-terminally deleted GlyT1, indicating that calpain cleavage does not eliminate syntaxin1A binding. These results suggest that calpain cleavage may regulate the transport activity/turnover of GlyT1 in vivo by cleaving its N-terminal domain.
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PMID:Calpain sensitive regions in the N-terminal cytoplasmic domains of glycine transporters GlyT1A and GlyT1B. 1629 1

O-methyltransferases (OMTs) catalyze the transfer of a methyl group from S-adenosine-L-methionine to a hydroxyl group of an acceptor molecule to form methyl ether derivatives and can modify the basic backbone of a secondary metabolite. A new O-methyltransferase, SOMT-9, was cloned from Glycine max and found to encode a protein whose molecular weight is 27-kDa. SOMT-9 was expressed as a GST-fusion protein in Escherichia coli and several compounds such as caffeic acid, esculetin, narigenin, kaempferol, quercetin, and luteolin were tested as putative substrates of SOMT-9. HPLC and NMR results showed that SOMT-9 transfers a methyl group to the 3'-OH group of substrates having ortho-hydroxyl groups. SOMT-9 showed the highest affinity for quercetin, suggesting that SOMT-9 uses a flavonoid as a substrate. Based on its molecular weight and substrate specificity, SOMT-9 belongs to a new class of OMT and is likely to be involved in the biosynthesis of isorhamnetin.
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PMID:Characterization of an O-methyltransferase from soybean. 1677 24

Dimeric glutathione S-transferases (GSTs) are pharmacological targets for several diseases, including cancer. Isoform specificity has been difficult to achieve due to their overlapping substrate selectivity. Here we demonstrate the utility of bivalent GST inhibitors and their optimization via combinatorial linker design. A combinatorial library with dipeptide linkers emanating symmetrically from a central scaffold (bis-3,5-aminomethyl benzoic acid, AMAB) to connect two ethacrynic acid moieties was prepared and decoded via iterative deconvolution, against the isoforms GSTA1-1 and GSTP1-1. The library yielded high affinity GSTA1-1 selective inhibitors (70-120-fold selectivity) and with stoichiometry of one inhibitor: one GSTA1-1 dimer. Saturation Transfer Difference (STD) NMR with one of these inhibitors, with linker structure (Asp-Gly-AMAB-Gly-Asp) and K(D) = 42 nM for GSTA1-1, demonstrates that the Asp-Gly linker interacts tightly with GSTA1-1, but not P1-1. H/D exchange mass spectrometry was used to map the protein binding site and indicates that peptides within the intersubunit cleft and in the substrate binding site are protected by inhibitor from solvent exchange. A model is proposed for the binding orientation of the inhibitor, which is consistent with electrostatic complementarity between the protein cleft and inhibitor linker as the source of isoform selectivity and high affinity. The results demonstrate the utility of combinatorial, or "irrational", linker design for optimizing bivalent inhibitors.
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PMID:Optimization of bivalent glutathione S-transferase inhibitors by combinatorial linker design. 1680 28


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