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:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Arginine-specific ADP-ribosyltransferase (ART) activity has been implicated in white cell chemotaxis. In this study, we examined the capacity of a panel of structurally unrelated inhibitors and pseudosubstrates of ART to inhibit chemotaxis of A7r5 rat vascular smooth muscle cells in response to PDGF-BB. 2. The IC50 values for nicotinamide (12 mM) and novobiocin (165 microM) were similar to those observed for inhibition of chemotaxis by human polymorphonuclear neutrophil leucocytes (PMN), whereas vitamins K3 (IC50=22 microM) and K1 (IC50=95 microM) were less potent than previously described in PMNs. The pseudo-substrates for the enzyme (DEA-BAG, agmatine and arginine-methylester) also inhibited A7r5 chemotaxis, and in addition inhibited cell adhesion at similar concentrations. Vitamin K3 was unique among the inhibitors of ART, in that it also inhibited cell adhesion. 3. A rat ART1 transcript was amplified by rtPCR from rat skeletal muscle, and was noted to share 94% homology with the mouse ART1 cDNA sequence. No such transcript could be detected in A7r5 cells by Northern blot analysis or rtPCR. 4. Evidence for ART activity on the surface of A7r5 cells was investigated using 32P-NAD+ as substrate, and labelled membrane proteins were observed with MWt values of 116, 100, 90 and 70 kDa. Exposure of the labelled proteins to phosphodiesterase yielded 32P-AMP, and hydrolysis with NaOH yielded 32P-NAD+. These results indicated that the labelled proteins were adducts with NAD+, and not the products of ART activity. The absence of ART catalytic activity in A7r5 cells was confirmed in protocols designed to show ADP-ribosylation of agmatine. 5. We conclude that the chemotactic activity of A7r5 cells is independent of ART activity, and the mechanism whereby the novel panel of inhibitors reduced cell migration remains undefined.
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PMID:Inhibition of chemotaxis in A7r5 rat smooth muscle cells by a novel panel of inhibitors. 977 55

Mono(ADP-ribosyl)transferases regulate the function of target proteins by attaching ADP-ribose to specific amino acid residues in the proteins. We have characterized the gene for mouse arginine-specific ADP-ribosyltransferase, Art1. Southern blot analyses indicate that Art1 is a single-copy gene. Northern blot and reverse transcription-PCR analyses demonstrate prominent expression of Art1 in cardiac and skeletal muscle, and lower levels in spleen, lung, liver and fetal tissues. While human ART1 is not represented in the public expressed sequence tag (EST) database, the database contains 14 mouse Art1 ESTs. The Art1 gene encompasses four exons spanning 20 kb of genomic DNA. The deduced amino acid sequence of Art1 exhibits the characteristic features of a glycosylphosphatidylinositol-anchored membrane protein. It shows 75-77% sequence identity with its orthologues from the human and rabbit, and 33-34% identity with its paralogues from the mouse, Art2-1 and Art2-2. Separate exons encode the N- and C-terminal signal peptides, and a single long exon encodes the entire predicted native polypeptide chain. We expressed Art1 in 293T cells as a recombinant fusion protein with the Fc portion of human IgG1. This soluble protein exhibits enzyme activities characteristic of arginine-specific ADP-ribosyltransferases. The availability of the Art1 gene provides the basis for applying transgene and knockout technologies to further probe the function of this gene product.
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PMID:Molecular characterization and expression of the gene for mouse NAD+:arginine ecto-mono(ADP-ribosyl)transferase, Art1. 984 66

mRNA from human polymorphonuclear neutrophil leucocytes (PMNs) was probed with cDNA encoding human skeletal muscle arginine-specific ADP-ribosyltransferase (ART1). A single 2.6-kb transcript was identified, which was similar in size to that observed in human skeletal muscle RNA. An 872-bp cDNA fragment, corresponding to the amino acid sequence of the processed human skeletal muscle enzyme, was generated by reverse transcription-PCR amplification of RNA from human PMNs, and was found to be identical to the ART1 cDNA derived from human skeletal muscle. ART1 was expressed as a fusion protein with glutathione S-transferase (GST) in insect cells, and antibodies were raised against the fusion protein in a rabbit. Following removal of GST immunoreactivity by immunoprecipitation, these antibodies were used to measure the abundance of immunoreactive ART1 on the surface of PMNs. Exposure of PMNs to formyl-Met-Leu-Phe (FMLP) was followed by a rapid increase in the abundance of cell surface ART1 (T1/2 = 1.9 min), and the concentration of FMLP for half-maximum response was 28.6 nM. Similar responses were observed after exposure of the cells to platelet-activating factor or interleukin-8, and we conclude that some of the effects of these chemotaxins are mediated by translocation of an intracellular pool of ART1 to its site of catalytic activity on the outer aspect of the plasma membrane.
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PMID:Chemotaxin-dependent translocation of immunoreactive ADP-ribosyltransferase-1 to the surface of human neutrophil polymorphs. 1009 75

NAD:arginine mono-ADP-ribosyltransferases catalyze the transfer of ADP-ribose from NAD to the guanidino group of arginine on a target protein. Deduced amino acid sequences of one family (ART1) of mammalian ADP-ribosyltransferases, cloned from muscle and lymphocytes, show hydrophobic amino and carboxyl termini consistent with glycosylphosphatidylinositol (GPI)-anchored proteins. The proteins, overexpressed in mammalian cells transfected with the transferase cDNAs, are released from the cell surface with phosphatidylinositol-specific phospholipase C (PI-PLC), and display immunological and biochemical characteristics consistent with a cell surface, GPI-anchored protein. In contrast, the deduced amino acid sequence of a second family (ART5) of transferases, cloned from murine lymphoma cells and expressed in high abundance in testis, displays a hydrophobic amino terminus, consistent with a signal sequence, but lacks a hydrophobic signal sequence at its carboxyl terminus, suggesting that the protein is destined for export. Consistent with the surface localization of the GPI-linked transferases, multiple surface substrates have been identified in myotubes and activated lymphocytes, and, notably, include integrin alpha subunits. Similar to the bacterial toxin ADP-ribosyltransferases, the mammalian transferases contain the characteristic domains involved in NAD binding and ADP-ribose transfer, including a highly acidic region near the carboxy terminus, which, when disrupted by in vitro mutagenesis, results in a loss of enzymatic activity. The carboxyl half of the protein, synthesized as a fusion protein in E. coli, possessed NADase, but not ADP-ribosyltransferase activity. These findings are consistent with the existence at the carboxyl terminus of ART1 of a catalytically active domain, capable of hydrolyzing NAD, but not of transferring ADP-ribose to a guanidino acceptor.
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PMID:Characterization of NAD:arginine ADP-ribosyltransferases. 1033 46

A number of well-known bacterial toxins ADP-ribosylate and thereby inactivate target proteins in their animal hosts. Recently, several vertebrate ecto-enzymes (ART1-ART7) with activities similar to bacterial toxins have also been cloned. We show here that PSIBLAST, a position-specific-iterative database search program, faithfully connects all known vertebrate ecto-mono(ADP-ribosyl)transferases (mADPRTs) with most of the known bacterial mADPRTs. Intriguingly, no matches were found in the available public genome sequences of archaeabacteria, the yeast Saccharomyces cerevisiae or the nematode Caenorhabditis elegans. Significant new matches detected by PSIBLAST from the public sequence data bases included only one open reading frame (ORF) of previously unknown function: the spvB gene contained in the virulence plasmids of Salmonella enterica. Structure predictions of SpvB indicated that it is composed of a C-terminal ADP-ribosyltransferase domain fused via a poly proline stretch to a N-domain resembling the N-domain of the secretory toxin TcaC from nematode-infecting enterobacteria. We produced the predicted catalytic domain of SpvB as a recombinant fusion protein and demonstrate that it, indeed, acts as an ADP-ribosyltransferase. Our findings underscore the power of the PSIBLAST program for the discovery of new family members in genome databases. Moreover, they open a new avenue of investigation regarding salmonella pathogenesis.
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PMID:The spvB gene-product of the Salmonella enterica virulence plasmid is a mono(ADP-ribosyl)transferase. 1097 29

1. Arginine-specific ADP-ribosyltransferase (ART1) is expressed on the surface of a number of cell types, and catalyses the transfer of ADP-ribose from NAD(+) to target proteins. We investigated whether extracellular proteins such as growth factors may serve as substrates for this enzyme, with subsequent alteration in their biological activity. Experiments were performed with rat skeletal muscle membranes and V79 Chinese hamster lung fibroblasts with doxycycline-inducible expression of human ART. 2. From a panel of growth factors, platelet-derived growth factor-BB (PDGF-BB) was found to be the best substrate for ART1, whereas the structural homologue PDGF-AA was not a substrate. Under conditions of maximum labelling 5 mol ADP-ribose was incorporated per mol of PDGF-BB. 3. Purified (ADP-ribosyl)-PDGF-BB did not stimulate a mitogenic or chemotactic response in human pulmonary smooth muscle cells, and showed a reduced capacity to bind to PDGF receptors in competition binding experiments, when compared to unmodified PDGF-BB. 4. PDGF-dependent [(3)H-methyl]-thymidine incorporation was measured in the ART1-transfected fibroblast cell line at physiological concentrations of PDGF-BB, and without addition of extracellular NAD(+). Fibroblasts expressing human ART1 at the cell surface showed reduced mitogenic responses to PDGF-BB, but not to PDGF-AA. This loss of mitogenic response in cells expressing ART1 activity was reversed by the addition of agmatine (an ART1 substrate). 5. In conclusion, we propose that PDGF-BB-dependent signalling may be regulated by post-translational modification of the growth factor by ART1 at the cell surface. This has been demonstrated in membranes of rat skeletal muscle, and the reaction confirmed in ART1-transfected fibroblasts.
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PMID:Inactivation of platelet-derived growth factor-BB following modification by ADP-ribosyltransferase. 1149 6

Mono(ADP-ribosyl)transferases regulate the function of target proteins by attaching ADP-ribose to specific amino acid residues in their target proteins. The purpose of this study was to determine the structure, chromosomal localization, and expression profile of the gene for mouse ecto-ADP-ribosyltransferase ART5. Southern blot analyses indicate that Art5 is a single copy gene which maps to mouse chromosome 7 at offset 49.6 cM in close proximity to the Art1, Art2a and Art2b genes. Northern blot and RT-PCR analyses demonstrate prominent expression of Art5 in testis, and lower levels in cardiac and skeletal muscle. Sequence analyses reveal that the Art5 gene encompasses six exons spanning 8 kb of genomic DNA. The 5' end of the Art5 gene overlaps with that of the Art1 gene. A single long exon encodes the predicted ART5 catalytic domain. Separate exons encode the N-terminal leader peptide and a hydrophilic C-terminal extension. Sequencing of RT-PCR products and ESTs identified six splice variants. The deduced amino acid sequence of ART5 shows 87% sequence identity to its orthologue from the human, and 37 and 32% identity to its murine paralogues ART1 and ART2. Unlike ART1 and ART2, ART5 lacks a glycosylphosphatidylinositol-anchor signal sequence and is predicted to be a secretory enzyme. This prediction was confirmed by transfecting an Art5 cDNA expression construct into Sf9 insect cells. The secreted epitope-tagged ART5 protein resembled rat ART2 in exhibiting potent NAD-glycohydrolase activity. This study provides important experimental tools to further elucidate the function of ART5.
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PMID:Structure, chromosomal localization, and expression of the gene for mouse ecto-mono(ADP-ribosyl)transferase ART5. 1158 54

ADP-ribosyltransferase activity was shown to be present on the surface of human monocytes. Incubating the cells in the presence of BSA leads to an increase in enzyme activity. The acceptor amino acid mainly responsible for the ADP-ribose bond was identified as a cysteine residue. An increase in ADP-ribosyltransferase activity was observed when cells were treated for 16 h with bacterial lipopolysaccharide (LPS). Possible candidates for catalysing the reaction are mono-ADP-ribosyltransferases (ARTs). When measuring expression of the mRNA of ART1, 3, 4 and 5, only ART3 mRNA was detected in unstimulated monocytes. Upon stimulation for 16 h with LPS, lipoteichoic acid or peptidoglycan, ART4 mRNA was found to be expressed. No ART4 signal appeared after a 4 h exposure of the cells to LPS. Cell-surface proteins were labelled when incubating monocytes with [(32)P]NAD(+). Their molecular masses were 29, 33, 43, 45, 60 and 82 kDa. In response to LPS an additional protein of 31 kDa was found to be labelled. The bound label was resistant to treatment with NH(2)OH but sensitive to HgCl(2), characteristic of a cysteine-linked ADP-ribosylation.
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PMID:Mono-ADP-ribosyltransferases in human monocytes: regulation by lipopolysaccharide. 1187

Mammalian ecto ADP-ribosyltransferases (ARTs) constitute a family of structurally related proteins expressed on the cell surface or secreted in the extracellular compartment. Using NAD+ as substrate, they transfer ADP-ribose groups onto target proteins. In contrast to intracellular poly(ADP-ribosyl)transferases (PARPs), these enzymes transfer a single ADPR and are thus mono-ARTs. Five paralogs (ART1-5) have been cloned but only four of them are expressed in human due to a defective ART2 gene, and six in the mouse as the result of ART2 gene duplication. The recent determination of the crystal structure of rat ART2 reveals homologies with bacterial ART toxins and provides a molecular basis for understanding the specificity of ARTs for their targets. A combination of different technological approaches reveals that ecto-ARTs are expressed in different tissues with privileged sites such as heart and skeletal muscles for ART1, T lymphocytes for ART2 or testis for ART5. It also indicates that ART expression is highly regulated. ADP-ribosylation of target proteins on cell surfaces or circulating in body fluids leads to reversible post-translational modifications which can inhibit the targets, as known for bacterial ARTs, or activate them, as in the crosstalk between mouse ART2 and the cytolytic P2X7 receptor on T lymphocytes. ART activity in the extracellular compartment provides sophisticated regulatory mechanisms for cell communication. This designates ecto-ARTs as new candidates for drug targeting.
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PMID:Ecto-ADP-ribosyltransferases (ARTs): emerging actors in cell communication and signaling. 1507 70

ADP-ribosyltransferases (ARTs) transfer ADP-ribose from NAD to arginine, asparagine, or cysteine residues in target proteins. This post-translational protein modification is the mechanism by which cholera-toxin and other bacterial toxins cause pathology in human host cells. Molecular cloning has identified five toxin-related GPI-anchored cell surface ARTs in the mouse (ART1, ART2.1, ART2.2, ART3, and ART4) and three in the human (ART1, ART3, and ART4). ART2-which has sparked interest because of its ability to activate the cytolytic P2X7 purinergic receptor by ADP-ribosylation-is encoded by two functional gene copies in the mouse genome while the human genome carries two inactivated ART2 pseudogenes. We generated stable transfectants for FLAG-tagged versions of each of the functional human and mouse ARTs. Using genetic immunization we raised monoclonal antibodies that recognize the native human ARTs on the surface of living cells. Some of these mAbs recognize an epitope shared with the mouse ART orthologue but not with more distant ART paralogues. Screening of primary cells and established cell lines by FACS revealed expression of ART1 by monocytes, neutrophils and myeloid leukemia cell lines but not by cell lines derived from solid tumors. ART1 and ART4 have been assigned the designations: CD296, and CD297, respectively.
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PMID:Use of genetic immunization to raise antibodies recognizing toxin-related cell surface ADP-ribosyltransferases in native conformation. 1627 11


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