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)

We have identified a novel gene, USP15, encoding a human ubiquitin-specific protease (USP). The USP15 protein consists of 952 amino acids with a predicted molecular mass of 109.2 kDa and contains the highly conserved Cys and His boxes present in all members of the UBP family of deubiquitinating enzymes. USP15 shares 60.5% sequence identity and 76% sequence similarity with the human homolog (UNP/Unph/USP4) of the mouse Unp proto-oncogene. Recombinant USP15 demonstrated ubiquitin-specific protease activity against engineered linear fusions of ubiquitin to beta-galactosidase and glutathione S-transferase. USP15 can also cleave the ubiquitin-proline bond, a property previously unique to Unp/UNP. Chromosomal mapping by fluorescence in situ hybridization and radiation hybrid analyses localized the USP15 gene to chromosome band 12q14, a different location than that of UNP (3p21.3). Analysis of expressed sequence tag databases reveals evidence of alternate polyadenylation sites in the USP15 gene and also indicates that the gene may possess an exon/intron structure similar to that of the Unp gene, suggesting they have descended from a common ancestor. A systematic nomenclature for the human USPs is proposed.
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PMID:Identification, functional characterization, and chromosomal localization of USP15, a novel human ubiquitin-specific protease related to the UNP oncoprotein, and a systematic nomenclature for human ubiquitin-specific proteases. 1044 27

The pollen of Parietaria, a weed of the Urticaceae family, is a major cause of respiratory allergy in Europe, where the most common species are P. judaica and P. officinalis. Previously, we reported that a beta-galactosidase fusion protein (6a-BG) expressing a 26-bp cDNA fragment (6a cDNA) contained a dominant IgE-binding epitope (6a epitope) of the major allergens Par o 1 and Par j 1. The present study aimed to define the amino-acid sequence containing the 6a epitope. We analyzed the reactivity of anti-Par o 1 antibodies affinity purified from allergic patient sera with: 1) a panel of synthetic peptides deduced from the 6a nucleotide sequence using different reading frames 2) glutathione S-transferase (GST) fusion proteins containing selected peptides. The peptide NSARARADSCRI (p102) specifically bound anti-Par o 1 antibodies affinity purified from allergic patient sera or from rabbit anti-Par o 1 antiserum (ELISA). The related peptide NSARAGTSSCRI (p101) reacted to human but not to rabbit, anti-Par o 1 antibodies. GST fusion proteins containing p101 (GST 3.5) or p102 (GST 3.2) extensively inhibited the binding between Par o 1 and IgE or IgG antibodies from an allergic patient serum pool according to a dose-response curve. Percent inhibition of IgE antibodies binding obtained by absorbing a solution (50 microl) of affinity-purified antibodies with 5 microg of GST 3.2 or with 1.2 mg of GST 3.5 was 69% and 66%, respectively. In conclusion, the results of the present study indicate that the amino-acid sequences NSARARADSCRI (p102) and NSARAGTSSCRI (p101) contain the dominant epitope of Par o 1 and Par j 1 for human IgE and IgG antibodies indicated as 6a epitope. Moreover, the study shows that the epitope is conserved in recombinant molecules containing these peptides, irrespective of the fused polypeptide (beta-galactosidase or GST). The knowledge of the amino-acid sequence of this dominant epitope is important in therapeutic approaches to the development of allergen-derived haptens.
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PMID:Characterization of a dodecapeptide containing a dominant epitope of Par j 1 and Par o 1, the major allergens of P. judaica and P. officinalis pollen. 1053 83

The major sporozoite surface antigen of Theileria annulata (SPAG-1) is a candidate for inclusion in a subunit vaccine. In this paper we summarize the results of 4 vaccination experiments using recombinant SPAG-1 expressed in different systems and presented in different adjuvants. The antigen has been presented as either a C terminal 108 amino acid peptide (called SR1) expressed as both beta-galactosidase and hepatitis B core antigen fusions or as a full-length form expressed as a GST fusion with an N terminal His6 tag. We used different adjuvants, namely Freund's, saponin, ISCOMs and a proprietary adjuvant supplied by SmithKline Beecham, which we call SKBA. The data point to the conclusion that SPAG-1 can elicit partial protection and is therefore suitable for inclusion in an eventual multicomponent subunit vaccine.
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PMID:Evaluation of recombinant sporozoite antigen SPAG-1 as a vaccine candidate against Theileria annulata by the use of different delivery systems. 1054 Mar 14

An androgen receptor (AR) interacting protein was isolated from a HeLa cell cDNA library by two-hybrid screening in yeast using the AR DNA+ligand binding domains as bait. The protein has sequence identity with human protein inhibitor of activated signal transducer and activator of transcription (PIAS1) and human Gu RNA helicase II binding protein (GBP). Binding of PIAS1 to human AR DNA+ligand binding domains was androgen dependent in the yeast liquid beta-galactosidase assay. Activation of binding by dihydrotestosterone was greater than testosterone > estradiol > progesterone. PIAS1 binding to full-length human AR in a reversed yeast two hybrid system was also androgen dependent. [35S] PIAS1 bound a glutathione S-transferase-AR-DNA binding domain (amino acids 544-634) fusion protein in affinity matrix assays. In transient cotransfection assays using CV1 cells with full-length human AR and a mouse mammary tumor virus luciferase reporter vector, there was an androgen-dependent 3- to 5-fold greater increase in luciferase activity with PIAS1 over that obtained with an equal amount of control antisense cDNA or mutant PIAS1. Constitutive transcriptional activity of the AR N-terminal+DNA binding domain was increased 6-fold by PIAS1. PIAS1 also enhanced glucocorticoid receptor transactivation in response to dexamethasone but inhibited progesterone-induced progesterone receptor transactivation in the same assay system. mRNA for PIAS1 was highly expressed in testis of human, monkey, rat, and mouse. In rat testis the onset of PIAS1 mRNA expression coincided with the initiation of spermatogenesis between 25-30 days of age. Immunostaining of human and mouse testis with PIAS1-specific antiserum demonstrated coexpression of PIAS1 with AR in Sertoli cells and Leydig cells. In addition, PIAS1 was expressed in spermatogenic cells. The results suggest that PIAS1 functions in testis as a nuclear receptor transcriptional coregulator and may have a role in AR initiation and maintenance of spermatogenesis.
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PMID:Protein inhibitor of activated STAT-1 (signal transducer and activator of transcription-1) is a nuclear receptor coregulator expressed in human testis. 1062 44

Phosducin (Phd) and Phd-like proteins (PhLPs) selectively bind guanine nucleotide protein (G protein) betagamma subunits (Gbetagamma), while Phd-like orphan proteins (PhLOPs) lack the major functional domain for the binding of Gbetagamma. A retina- and pineal gland-specific transcription factor, cone-rod homeobox (CRX), was identified by a yeast two-hybrid screen using PhLOP1 as the bait. Direct protein-protein interactions between Phd or PhLOP1 and CRX were demonstrated using a beta-galactosidase quantitative assay in the yeast two-hybrid system and were confirmed by an in vitro binding assay and a glutathione S-transferase (GST) pull-down assay. To determine if the interaction with Phd or PhLOP1 affected CRX transactivation, a 120-bp interphotoreceptor retinoid binding protein (IRBP) promoter-luciferase reporter construct containing a CRX consensus element (GATTAA) was cotransfected into either COS-7 or retinoblastoma Weri-Rb-1 cells with expression constructs for CRX and either Phd or PhLOP1. Phd and PhLOP1 inhibited the transcriptional activation activity of CRX by 50% during transient cotransfection in COS-7 cells and by 70% in Weri-Rb-1 cells and COS-7 cells stably transfected with CRX. Phd inhibited CRX transactivation in a dose-dependent manner. Whereas Phd is a cytoplasmic phosphoprotein, coexpression of Phd with CRX results in Phd being localized both in the cytoplasm and nucleus. By contrast, PhLOP1 is found in the nucleus even without CRX coexpression. To address the physiological relevance of these potential protein interacting partners, we identified immunoreactive proteins for Phd and CRX in retinal cytosolic and nuclear fractions. Immunohistochemical analysis of bovine retinas reveals colocalization of Phd isoforms with CRX predominantly in the inner segment of cone cells, with additional costaining in the outer nuclear layer and the synaptic region. Our findings demonstrate that both Phd and PhLOP1 interact directly with CRX and that each diminishes the transactivation activity of CRX on the IRBP promoter. A domain that interacts with CRX is found in the carboxyl terminus of the Phd isoforms. Phd antibody-immunoreactive peptides are seen in light-adapted mouse retinal cytosolic and nuclear extracts. Neither Phd nor PhLOP1 affected CRX binding to its consensus DNA element in electrophoretic mobility shift assays. A model that illustrates separate functional roles for interactions between Phd and either SUG1 or CRX is proposed. The model suggests further a mechanism by which Phd isoforms could inhibit CRX transcriptional activation.
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PMID:Modulation of CRX transactivation activity by phosducin isoforms. 1086 77

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

The VacA toxin is the major virulence factor of Helicobacter pylori. The studies on VacA intracellular expression suggest that it interacts with cytosolic proteins and that this interaction contributes significantly to vacuolization. The aim of this study was to identify the host protein(s) that interacts with the VacA protein. We used the fragments of VacA protein fused with GAL4-BD as the baits in the yeast two-hybrid approach. The yeast transformed with plasmids encoding bait proteins were screened with human gastric mucosa cDNA library, encoded C-terminal fusion proteins with GAL4-AD. Three independent His-beta-Gal-positive clones were identified in VacA-b1 screen; they matched two different lengths of cDNA encoding RACK1 protein. The specific activity of beta-galactosidase found in the yeast expressing both VacA-b1 and RACK1 fusion proteins was 12-19 times higher compared to all negative controls used. VacA is capable of binding the RACK1 in vitro as was confirmed by the pull-down assay with GST fusion VacA protein and [(35)S]Met-labeled RACK1 protein fragments.
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PMID:RACK1 protein interacts with Helicobacter pylori VacA cytotoxin: the yeast two-hybrid approach. 1170 84

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


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