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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

munc18c is a critical protein involved in trafficking events associated with syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its interaction with syntaxin 4, fragments of munc18c were generated and their interaction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. In vitro protein-protein interaction studies were then used to confirm that the interaction between the proteins was direct. Full-length munc18c(1-592), munc18c(1-139) and munc18c(1-225), but not munc18c(226-592), munc18c(1-100), munc18c(43-139) or munc18c(66-139), interacted with the cytoplasmic portion of syntaxin 4, Stx4(2-273), as assessed by yeast two-hybrid assay of growth on nutritionally deficient media and by beta-galactosidase reporter induction. The N-terminal predicted helix-a-helix-b-helix-c region of syntaxin 4, Stx4(29-157), failed to interact with full-length munc18c(1-592), indicating that a larger portion of syntaxin 4 is necessary for the interaction. The yeast two-hybrid results were confirmed by protein-protein interaction studies between Stx4(2-273) and glutathione S-transferase fusion proteins of munc18c. Full-length munc18c(1-592), munc18c(1-139) and munc18c(1-225) interacted with Stx4(2-273) whereas munc18c(1-100) did not, consistent with the yeast two-hybrid data. These data thus identify a region of munc18c between residues 1 and 139 as a minimal domain for its interaction with syntaxin 4.
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PMID:Definition of a minimal munc18c domain that interacts with syntaxin 4. 1097 Jul 87

A glutathione S-transferase (GST) gene has been cloned from Schizosaccharomyces pombe for the first time. The nucleotide sequence determined was found to contain 2030 base pairs including an open reading frame of 229 amino acids that would encode a protein of a molecular mass of 27017 Da. The cloned GST gene was expressed and was found to function in S. pombe, Saccharomyces cerevisiae, and Escherichia coli. The plasmid pGT207 encoding the S. pombe GST gene appeared to be able to accelerate the growth of a wild type S. pombe culture. In a culture of S. pombe containing plasmid pGT207, the growth was inhibited less by mercuric chloride than in a culture with vector alone. The 1088 bp region upstream from the GST gene as well as the region encoding the N-terminal 14 amino acids was transferred into the promoterless beta-galactosidase gene of plasmid YEp357R to yield the fusion plasmid pYSH2000. beta-Galactosidase synthesis was induced by cadmium chloride, mercuric chloride, hydrogen peroxide, and menadione. It was also induced by high temperature. These results suggest that the cloned S. pombe GST gene is involved in the oxidative stress response.
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PMID:Characterization and regulation of glutathione S-transferase gene from Schizosaccharomyces pombe. 1151 61

Genetic labeling using recombinant retroviruses is a powerful strategy for the study of cell lineage in the liver. However, this type of vector is only able to infect dividing cells. The synthetic steroid cyproterone acetate (CPA) is mitogenic and carcinogenic in the adult rat liver. In this study, we used retroviral vectors carrying the nuclear targeted beta-galactosidase gene to selectively label and follow the fate of hepatocytes dividing on administration of CPA. Labeled cells as well as those in mitosis were preferentially located around the portal tract, whereas apoptotic bodies were predominant in the pericentral area. Labeled hepatocytes did not disappear after apoptosis, suggesting a preferential elimination of nontransduced cells. The presence of labeled binucleated hepatocytes showed the persistence of a binucleation process. Finally, we performed long-term analysis of labeled cells in transgenic animals tolerant for beta-galactosidase and treated with 2-acetylaminofluorene (2-AAF) to promote the growth of CPA-initiated hepatocytes. The presence of beta-galactosidase-positive hepatocyte clones showed that hepatocytes divided during treatment with 2-AAF. Only 3% of beta-galactosidase clones were positive for the placental form of glutathione S-transferase (GSTp), indicating the absence of a preferential appearance of preneoplastic foci in the population of beta-galactosidase-labeled hepatocytes. In conclusion, our results show that the mitogenic and tumor-initiating activities of CPA are directed toward different hepatocyte populations.
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PMID:In vivo cell lineage analysis in cyproterone acetate-treated rat liver using genetic labeling of hepatocytes. 1182

The activation of the pleomorphic adenoma gene 1 (PLAG1) is the most frequent gain-of-function mutation found in pleomorphic adenomas of the salivary glands. To gain more insight into the regulation of PLAG1 function, we searched for PLAG1-interacting proteins. Using the yeast two-hybrid system, we identified karyopherin alpha2 as a PLAG1-interacting protein. Physical interaction between PLAG1 and karyopherin alpha2 was confirmed by an in vitro glutathione S-transferase pull-down assay. Karyopherin alpha2 escorts proteins into the nucleus via interaction with a nuclear localization sequence (NLS) composed of short stretches of basic amino acids. Two putative NLSs were identified in PLAG1. The predicted NLS1 (KRKR) was essential for physical interaction with karyopherin alpha2 in glutathione S-transferase pull-down assay, and its mutation resulted in decreased nuclear import of PLAG1. Moreover, NLS1 was able to drive the nuclear import of the cytoplasmic protein beta-galactosidase. In contrast, predicted NLS2 of PLAG1 (KPRK) was not involved in karyopherin alpha2 binding nor in its nuclear import. The residual nuclear import of PLAG1 after mutation of the NLS1 was assigned to the zinc finger domain of PLAG1. These observations indicate that the nuclear import of PLAG1 is governed by its zinc finger domain and by NLS1, a karyopherin alpha2 recognition site.
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PMID:Identification of a karyopherin alpha 2 recognition site in PLAG1, which functions as a nuclear localization signal. 1188 54

A second glutathione S-transferase gene (GST II) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe. The nucleotide sequence determined contains 1908 bp including an open reading frame of 230 amino acids that would encode a protein of a molecular mass of 26843.4 Da. The amino acid sequence of the putative GST II is very homologous with that of the previously isolated GST gene (GST I) located in the same chromosome III of S. pombe. The cloned GST II gene produces the functional GST in S. pombe, and it gives much higher GST in the stationary phase than in the exponential phase. Regulation of the GST II gene was studied using the GST II-lacZ fusion. The synthesis of beta-galactosidase from the fusion plasmid is greatly enhanced by the treatments with oxidative stresses such as menadione and mercuric chloride. It is also induced by o-dinitrobenzene, one of the GST substrates. NO-generating S-nitroso-N-acetylpenicillamine has a weak induction effect on the expression of GST II gene. These results indicate that the S. pombe GST II gene is involved in the oxidative stress response and detoxification. However, physiological meaning on the existence of the two similar GST genes in S. pombe remains unknown yet.
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PMID:A second stress-inducible glutathione S-transferase gene from Schizosaccharomyces pombe. 1199 10

Cyclophilin 40 (CyP40) is a tetratricopeptide repeat (TPR)-containing immunophilin and a modulator of steroid receptor function through its binding to heat shock protein 90 (Hsp90). Critical to this binding are the carboxyl-terminal MEEVD motif of Hsp90 and the TPR domain of CyP40. Two different models of the CyP40-MEEVD peptide interaction were used as the basis for a comprehensive mutational analysis of the Hsp90-interacting domain of CyP40. Using a carboxyl-terminal CyP40 construct as template, 24 amino acids from the TPR and flanking acidic and basic domains were individually mutated by site-directed mutagenesis, and the mutants were coexpressed in yeast with a carboxyl-terminal Hsp90beta construct and qualitatively assessed for binding using a beta-galactosidase filter assay. For quantitative assessment, mutants were expressed as glutathione S-transferase fusion proteins and assayed for binding to carboxyl-terminal Hsp90beta using conventional pulldown and enzyme-linked immunosorbent assay microtiter plate assays. Collectively, the models predict that the following TPR residues help define a binding groove for the MEEVD peptide: Lys-227, Asn-231, Phe-234, Ser-274, Asn-278, Lys-308, and Arg-312. Mutational analysis identified five of these residues (Lys-227, Asn-231, Asn-278, Lys-308, and Arg-312) as essential for Hsp90 binding. The other two residues (Phe-234 and Ser-274) and another three TPR domain residues not definitively associated with the binding groove (Leu-284, Lys-285, and Asp-329) are required for efficient Hsp90 binding. These data confirm the critical importance of the MEEVD binding groove in CyP40 for Hsp90 recognition and reveal that additional charged and hydrophobic residues within the CyP40 TPR domain are required for Hsp90 binding.
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PMID:A structure-based mutational analysis of cyclophilin 40 identifies key residues in the core tetratricopeptide repeat domain that mediate binding to Hsp90. 1214 16

A third gene encoding glutathione S-transferase (GSTIII) was cloned from the fission yeast Schizosaccharomyces pombe. The nucleotide sequence determined was found to contain 2110 base pairs including an open reading frame of 242 amino acids that would encode a protein of a molecular mass of 26,620 Da. The cloned GSTIII gene could be expressed in S. pombe, S. cerevisiae and Escherichia coli cells which gave 1.4-, 2.1-, and 3.0-fold higher GST activity in an assay using 1-chloro-2,4-dinitrobenzene as a substrate, respectively. The cloned GSTIII gene caused higher survivals of S. pombe cells on solid media with cadmium chloride or mercuric chloride. The GSTIII protein has 16% and 18% homologies with the GSTI and GSTII proteins, respectively. To independently monitor the regulation of the GSTIII gene, its 1168 bp upstream region and N-terminal 33 amino acid-coding region was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357. The synthesis of beta-galactosidase from the fusion plasmid pGY357 was greatly enhanced by cadmium chloride (50 microM), cupric chloride (10 microM), aluminum chloride (5 mM, 10 mM), mercuric chloride (1 microM), and zinc chloride (10 mM). However, the synthesis of beta-galactosidase from the fusion plasmid pGY357 was not affected by superoxide-generating menadione, and o-dinitrobenzene, whereas they could significantly induce the expression of the GSTI and GSTII genes of S. pombe. The overproduced Pap1 inhibited the induction of beta-galactosidase synthesis from the fusion plasmid pGY357 by cadmium chloride, which is opposite to the previously known role of Pap1 in the response to oxidative stress. Our results collectively indicate that the three GST genes of S. pombe are subjected to different regulatory mechanisms. The major role of the GSTIII protein in S. pombe may be the detoxification of various metals.
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PMID:Characterization, expression and regulation of a third gene encoding glutathione S-transferase from the fission yeast. 1215 Nov 11

The genomic DNA encoding a second glutathione S-transferase (GSTII) was previously isolated from the fission yeast Schizosaccharomyces pombe. Its expression was shown to be induced by menadione, mercuric chloride, o-dinitrobenzene, and NO-generating S-nitroso-N-acetylpenicillamine using the GSTII-lacZ fusion harboring the 910 bp upstream region from the translational initiation point. In this study, the additional fusion plasmids pGST50-590 and pGST50-6R-590 were constructed to carry the 590 bp upstream region in the vectors YEp357 and YEp367R, respectively. The synthesis of beta-galactosidase from the fusion plasmid pGST50-590 was about 3-fold higher than that from the fusion plasmid pGST50-F, indicating the presence of negatively activating sequence in the -910 to approximately -590 region. It was also enhanced by the same agents, which induced the synthesis of beta-galactosidase from the fusion plasmid pGST50-F. The synthesis of beta-galactosidase from both fusion plasmids pGST50-F and pGST50-590 was enhanced by the overexpressed Pap1 protein. The synthesis of beta-galactosidase from the two YEp367R derivatives pGST50-6R-F and pGST50-6R-590 was greatly decreased in the Pap1-negative strain TP108-3C. These results propose the Pap1-dependent regulation of the GSTII gene from the fission yeast.
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PMID:Pap1-dependent regulation of the GSTII gene from the fission yeast. 1252 8

In the yeast Saccharomyces cerevisiae, the mineral zinc is essential for growth and metabolism. Depletion of zinc from the growth medium of wild type cells results in changes in phospholipid metabolism, including an increase in phosphatidylinositol content (Iwanyshyn, W. M., Han, G.-S., and Carman, G. M. (2004) J. Biol. Chem. 279, 21976-21983). We examined the effects of zinc depletion on the regulation of the PIS1-encoded phosphatidylinositol synthase, the enzyme that catalyzes the formation of phosphatidylinositol from CDP-diacylglycerol and inositol. Phosphatidylinositol synthase activity increased when zinc was depleted from the growth medium. Analysis of a zrt1Delta zrt2Delta mutant defective in plasma membrane zinc transport indicated that the cytoplasmic levels of zinc were responsible for the regulation of phosphatidylinositol synthase. PIS1 mRNA, its encoded protein Pis1p, and the beta-galactosidase activity driven by the P(PIS1)-lacZ reporter gene were elevated in zinc-depleted cells. This indicated that the increase in phosphatidylinositol synthase activity was the result of a transcriptional mechanism. The zinc-mediated induction of the P(PIS1)-lacZ reporter gene, Pis1p, and phosphatidylinositol synthase activity was lost in zap1Delta mutant cells. These data indicated that the regulation of PIS1 gene expression by zinc depletion was mediated by the zinc-regulated transcription factor Zap1p. Direct interaction between glutathione S-transferase (GST)-Zap1p(687-880) and a putative upstream activating sequence (UAS) zinc-responsive element in the PIS1 promoter was demonstrated by electrophoretic mobility shift assays. Mutations in the UAS zinc-responsive element in the PIS1 promoter abolished the GST-Zap1p(687-880)-DNA interaction in vitro and abolished the zinc-mediated regulation of the PIS1 gene in vivo. This work advances understanding of phospholipid synthesis regulation by zinc and the transcription control of the PIS1 gene.
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PMID:Regulation of the PIS1-encoded phosphatidylinositol synthase in Saccharomyces cerevisiae by zinc. 1598 62

Mouse Pem, a homeobox gene, encodes a protein consisting of 210 amino acid residues. To study the function of mouse Pem protein, we used the yeast two-hybrid system to screen the library of 7-day mouse embryo with full-length mouse Pem cDNA. Fifty-two colonies were obtained after 1.57 x 10(8) colonies were screened by nutrition limitation and beta-galactosidase assay. Seven individual insert fragments were obtained from the library, and three of them were identified, one of which was confirmed to be the cell division cycle 37 (Cdc37) homolog gene by sequencing. The interaction between mouse Pem and Cdc37 homolog was then confirmed by glutathione S-transferase pull-down assay, and the possible interaction model was suggested.
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PMID:Interaction of mouse Pem protein and cell division cycle 37 homolog. 1627 Jan 59


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