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)

Bacterial toxin ADP-ribosyltransferases, e.g. diphtheria toxin (DT) and pertussis toxin, have in common consensus sequences involved in catalytic activity, which are localized to three regions. Region I is notable for a histidine or arginine; region II, approximately 50-75 amino acids downstream, is rich in aromatic/hydrophobic amino acids; and region III, further downstream, has a glutamate and other acidic amino acids. A similar motif was observed in the sequence of the glycosylphosphatidylinositol-linked muscle ADP-ribosyltransferase. Site-directed mutagenesis was performed to verify the role of this motif. Proteins were expressed in rat adenocarcinoma cells, released from the cell with phosphatidylinositol-specific phospholipase C, and quantified with polyclonal antibodies. Transferase His114 in region I aligned with His21 of DT; as with DT, the H114N mutant was active. Aromatic/hydrophobic amino acids (region II) were found approximately 30-50 amino acids downstream of this histidine. Although transferase has a Glu278-Tyr-Ile sequence characteristic of region III in DT, Glu278 was not critical for activity. In an alternative region III containing Glu238-Glu239-Glu240, Glu238 and Glu240 but not Glu239 were critical. Glu240 aligned with critical glutamates in DT, Pseudomonas exotoxin, and C3 transferase. Thus, the mammalian ADP-ribosyltransferases have motifs similar to toxin ADP-ribosyltransferases, suggesting that these sequences are important in ADP-ribose transfer reactions.
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PMID:Conservation of a common motif in enzymes catalyzing ADP-ribose transfer. Identification of domains in mammalian transferases. 782 77

RT6.2 is a 26-kDa alloantigen expressed only on post-thymic T cells and attached to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. It has been reported that expression of RT6.2 in animal models may correlate with lymphopenia and genetically-induced insulin-dependent diabetes mellitus. Its physiological function is unclear. Since RT6.2 has significant amino acid identity with a GPI-anchored rabbit muscle NAD:arginine ADP-ribosyltransferase, RT6.2 was expressed in rat mammary adenocarcinoma cells and the ability of the expressed protein to catalyze ADP-ribose transfer reactions was examined. Cells transformed with the RT6.2 gene expressed NAD glycohydrolase activity that was released from intact cells by phosphatidylinositol-specific phospholipase C, consistent with its presence on the cell surface. A similar activity was not detected with vector-transformed cells. RT6.2 did not ADP-ribosylate simple guanidino compounds. The molecular weight of the phosphatidylinositol-specific phospholipase C-released NAD glycohydrolase, determined by SDS-polyacrylamide gel electrophoresis, was 22,000-24,000, in good agreement with that of native RT6.2. These results strongly suggest that the rat T cell alloantigen RT6.2 is a GPI-anchored NAD glycohydrolase.
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PMID:Expression of NAD glycohydrolase activity by rat mammary adenocarcinoma cells transformed with rat T cell alloantigen RT6.2. 814 25

Mono ADP-ribosyltransferases catalyze the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) to proteins. It was reported by Wang et al (J Immunol 153:4048, 1994) that incubation of mouse cytotoxic T lymphocytes (CTL) with NAD resulted in the ADP-ribosylation of membrane proteins and inhibition of cell proliferation and cytotoxicity. Treatment of CTL with phosphatidylinositol-specific phospholipase C (PI-PLC) before incubation with NAD prevented the inhibitory effects of NAD on the cells, consistent with the removal of a glycosylphosphatidylinositol (GPI)-anchored ADP-ribosyltransferase on the lymphocyte surface. We have identified and cloned a GPI-linked ADP-ribosyltransferase from Yac-1 mouse T-cell lymphoma cells. The deduced amino acid sequence of the Yac-1 transferase was 70% and 41% identical to those of the rabbit skeletal muscle and chicken heterophil, respectively. It contained three noncontiguous sequences similar to those found in several of the bacterial toxin and vertebrate ADP-ribosyltransferases. Based on crystallography of the bacterial toxins, these regions are believed to form, in part, the catalytic site consistent with a common mechanism for the ADP-ribose transfer reaction. In rat mammary adenocarcinoma (NMU) cells transformed with the Yac-1 transferase cDNA, transferase activity was present on the cell surface and was released into the medium by treatment of cells with PI-PLC. Thus, we have cloned a novel gene that has properties identical to the transferase detected in CTL, and may be involved in the NAD-dependent regulation of proliferation and cytotoxicity.
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PMID:Molecular characterization of a glycosylphosphatidylinositol-linked ADP-ribosyltransferase from lymphocytes. 870 49

Rat RT6 proteins, and perhaps mouse Rt6, identify a set of immunoregulatory T lymphocytes. Rat RT6.1 (RT6.1) and rat RT6.2 (RT6. 2) are NAD glycohydrolases, which catalyze auto-ADP-ribosylation, but not ADP-ribosylation of exogenous proteins. Mouse Rt6.1 (mRt6.1) also catalyzes auto-ADP-ribosylation. The activity of mouse cytotoxic T lymphocytes is reportedly inhibited by ADP-ribosylation of surface proteins, raising the possibility that mRt6 may participate in this process. The reactions catalyzed by mRt6, would, however, need to be more diverse than those of the rat homologues and include the ADP-ribosylation of acceptors other than itself. To test this hypothesis, mRt6.1 and rat RT6.2 were synthesized in Sf9 insect cells and rat mammary adenocarcinoma (NMU) cells. mRt6.1, but not rat RT6.2, catalyzed the ADP-ribosylation of guanidino-containing compounds (e.g. agmatine). Unlike RT6.2, mRt6.1 was a weak NAD glycohydrolase. In the presence of agmatine, however, the ratio of [adenine-14C]ADP-ribosylagmatine formation from [adenine-14C]NAD to [carbonyl-14C]nicotinamide formation from [carbonyl-14C]NAD was approximately 1.0, demonstrating that mRt6.1 is primarily a transferase. ADP-ribosylarginine hydrolase, which preferentially hydrolyzes the alpha-anomer of ADP-ribosylarginine, released [U-14C]arginine from ADP-ribosyl[U-14C]arginine synthesized by mRT6.1, consistent with the conclusion that mRt6.1 catalyzes a stereospecific Sn2-like reaction. Thus, mRt6.1 is an NAD:arginine ADP-ribosyltransferase capable of catalyzing a multiple turnover, stereospecific Sn2-like reaction.
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PMID:Characterization of mouse Rt6.1 NAD:arginine ADP-ribosyltransferase. 902 Jan 54

Transfection of NMU (rat mammary adenocarcinoma) cells with NAD:arginine ADP-ribosyltransferase cDNAs from Yac-1 murine lymphoma cells or rabbit muscle increased NAD glycohydrolase and ADP-ribosyltransferase activities. The ADP-ribosyltransferase activity was released from transformed NMU cells by phosphatidylinositol-specific phospholipase C (PI-PLC) and hence glycosylphosphatidylinositol (GPI)-anchored, whereas the NAD glycohydrolase (NADase) activity remained cell-associated. By gel permeation chromatography, the size of the PI-PLC-released transferase was approximately 40 kDa and that of the detergent-solubilized NADase was approximately 100 kDa. Using polyclonal antibodies against rabbit muscle transferase on Western blots, approximately 18- and approximately 30-kDa band were visualized among proteins from the NADase fractions and 38-40-kDa bands with protein from the transferase fractions. Incubation of blots with [32P]NAD led to the incorporation of radioactivity into the immunoreactive transferase bands of 38 kDa and the immunoreactive NADase band of approximately 18 kDa. These data suggest that proteolysis of ADP-ribosyltransferase synthesized in transformed NMU cells might result in the formation of aggregates of an 18-kDa NAD glycohydrolase. A fusion protein with glutathione S-transferase linked to the amino terminus of Yac-1 transferase, from which the amino-terminal 121 amino acids had been deleted (GST-Yac-1-delta121), exhibited NADase, but not transferase, activity. The size of the recombinant fusion protein was similar to that of the proteolytic fragment seen in NMU cells transformed with transferase cDNA. These results are compatible with the conclusion that the NAD glycohydrolase activity was generated in NMU cells by proteolysis of ADP-ribosyltransferase, with release of a carboxyl-terminal fragment that possesses glycohydrolase but not transferase activity, i.e. the carboxyl-terminal portion of the transferase can exist as a catalytically active NADase.
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PMID:An 18-kDa domain of a glycosylphosphatidylinositol-linked NAD:arginine ADP-ribosyltransferase possesses NAD glycohydrolase activity. 908 12

Phospholipase D (PLD) has been implicated as a crucial signaling enzyme in secretory pathways. Two 20-kDa guanine nucleotide-binding proteins, Rho and ADP-ribosylation factor (ARF), are involved in the regulation of secretion and can activate PLD in vitro. We investigated in intact (human adenocarcinoma A549 cells) the role of RhoA and ARF in activation of PLD by phorbol 12-myristate 13-acetate, bradykinin, and/or sphingosine 1-phosphate. To express recombinant Clostridium botulinum C3 exoenzyme (using double subgenomic recombinant Sindbis virus C3), an ADP-ribosyltransferase that inactivates Rho, or dominant-negative Rho containing asparagine at position 19 (using double subgenomic recombinant Sindbis virus Rho19N), cells were infected with Sindbis virus, a novel vector that allows rapid, high level expression of heterologous proteins. Expression of C3 toxin or Rho19N increased basal and decreased phorbol 12-myristate 13-acetate-stimulated PLD activity. Bradykinin or sphingosine 1-phosphate increased PLD activity with additive effects that were abolished in cells expressing C3 exoenzyme or Rho19N. In cells expressing C3, modification of Rho appeared to be incomplete, suggesting the existence of pools that differed in their accessibility to the enzyme. Similar results were obtained with cells scrape-loaded in the presence of C3; however, results with virus infection were more reproducible. To assess the role of ARF, cells were incubated with brefeldin A (BFA), a fungal metabolite that disrupts Golgi structure and inhibits enzymes that catalyze ARF activation by accelerating guanine nucleotide exchange. BFA disrupted Golgi structure, but did not affect basal or agonist-stimulated PLD activity, i.e. it did not alter a rate-limiting step in PLD activation. It also had no effect on Rho-stimulated PLD activity, indicating that RhoA action did not involve a BFA-sensitive pathway. A novel PLD activation mechanism, not sensitive to BFA and involving RhoA, was identified in human airway epithelial cells by use of a viral infection technique that preserves cell responsiveness.
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PMID:Effect of Rho and ADP-ribosylation factor GTPases on phospholipase D activity in intact human adenocarcinoma A549 cells. 1037 71

E2F -1 is a transcription factor that regulates cell cycle progression into S-phase. Deregulation of E2F-1 activity has been associated with cellular commitment to apoptosis. Also critical in the regulation of S-phase are the actions of the cyclin dependent kinases, Cdk2 and cdc2. Inhibition of these cyclin dependent kinases has been similarly associated with disrupting orderly S-phase progression and causing subsequent apoptosis in certain cancer cells. In this study, we examine the ability of adenovirus-mediated E2F-1 overexpression to induce apoptosis in human gastric carcinoma cells. Furthermore, we investigate the effect of the cyclin dependent kinase inhibitors, olomoucine and roscovitine, on E2F-1-mediated apoptosis in human gastric carcinoma cells. AGS and SNU-1 gastric adenocarcinoma cells were infected with adenoviral vectors expressing E2F-1 (Ad5CMVE2F-1) or control viruses expressing beta-galactosidase (Ad5CMVLacZ) or lacking a transgene (Ad5). Gastric adenocarcinoma cells were then independently treated with roscovitine or olomoucine. Finally, gastric adenocarcinoma cells were infected with the various adenoviral vectors in combination with roscovitine or olomoucine. E2F-1 overexpression resulted in an 85% reduction in cell viability at 72 h compared to controls. Combining E2F-1 overexpression with roscovitine resulted in >99% reduction in cell viability by 72 h. Overexpression of E2F-1 resulted in premature S-phase entry and G2/M arrest at 24 h, followed by apoptosis by 72 h. Combining E2F-1 overexpression with roscovitine resulted in an earlier G2/M arrest, followed by a more complete, widespread apoptotic response by 24 h. Caspase 3/CPP32 activation and PARP cleavage in response to E2F-1 overexpression, alone and in combination with roscovitine, implicate the caspase cascade in E2F-1-mediated apoptosis of gastric cancer cells. Bax levels also increased in response to E2F-1 gene transfer, alone and in combination with roscovitine. E2F-1 overexpression induces widespread apoptosis in human gastric carcinoma cells. Combining E2F-1 overexpression with cyclin-dependent kinase inhibitors results in an enhanced apoptotic response, causing nearly complete gastric tumor cell death within 72 h. E2F-1 gene therapy in combination with cyclin dependent kinase inhibitors is a potentially active chemogene therapy strategy for the treatment of human gastric cancer.
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PMID:Adenovirus-mediated E2F-1 gene transfer induces an apoptotic response in human gastric carcinoma cells that is enhanced by cyclin dependent kinase inhibitors. 1085 Dec 67

Prodigiosin is a red pigment produced by various bacteria including Serratia marcescens. Colorectal cancer is one of the most frequent malignancies and one of the most frequent causes of cancer death in the Western world. Its treatment is far from satisfactory and the challenge to oncologists is to find novel chemical entities with less toxicity and greater effectiveness than those used in current chemotherapy. Here we characterize the apoptotic action of prodigiosin in colon cancer cells. DLD-1 and SW-620 human colon adenocarcinoma cells, NRK and Swiss-3T3 nonmalignant cells were assayed by the MTT assay, fragmentation pattern of DNA, Hoechst 33342 staining and study of PARP cleavage by Western blot, in order to characterize the prodigiosin-induced apoptosis. Prodigiosin was purified and its structure was confirmed. Metastatic SW-620 cells were more sensitive to prodigiosin (IC50: 275 nM) than DLD-1. We did not observe a significant decrease in the viability of NRK cells. We confirmed that prodigiosin induces apoptosis in both cancer cell lines by the characteristic DNA laddering pattern and condensed nuclei or apoptotic bodies identified by fluorescence microscopy. These results indicate that prodigiosin induces apoptosis in colon cancer cells.
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PMID:Prodigiosin-induced apoptosis in human colon cancer cells. 1138 4

The present study was performed to gain insight into the role of p53 on the cytotoxicity of tubulin-binding agents (TBA) on cancer cells. Drug sensitivity, cell cycle distribution and drug-induced apoptosis were compared in 2 lines derived from the mammary adenocarcinoma MCF-7: the MN-1 cell line containing wild-type p53 (wt-p53) and the MDD2 line, containing a dominant negative variant of the p53 protein (mut-p53). The MDD2 cell line was significantly more resistant to the cytotoxic effects of vinblastine and paclitaxel than the MN1 cell line. MN1 cells, but not MDD2 cells, displayed wt-p53 protein accumulation as well as p21/WAF1 and cyclin G1 induction after exposure to TBA. Both cell lines arrested at G(2)/M after drug treatment. However exposure of MN1 cells to TBA resulted in a stronger variation in mitochondrial membrane potential, associated with cleavage of PARP, and more apoptosis, as measured by annexin V expression. After exposure to vinblastine, Raf 1 kinase activity was reduced in MDD2 cells but not in MN1 cells. Addition of flavopiridol to vinblastine- and paclitaxel-treated cells reversed the MDD2-resistant phenotype by inducing G(1)cell cycle arrest and inhibiting endoreduplication. We conclude that the p53 status of cancer cells influences their sensitivity to TBA cytotoxicity. This effect is likely to involve differences in the apoptotic cascade.
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PMID:Inactivation of wild-type p53 by a dominant negative mutant renders MCF-7 cells resistant to tubulin-binding agent cytotoxicity. 1155 44

The human monoclonal antibody SC-1 was isolated from a patient with a diffuse-type adenocarcinoma of the stomach using somatic cell hybridization. The immunoglobulin (Ig)M antibody reacts specifically with diffuse- (70%) and intestinal-type (25%) gastric adenocarcinoma and induces apoptosis in vitro and in vivo. When used in clinical trials with stomach carcinoma patients, significant apoptotic and regressive effects in primary tumors have been observed with the antibody SC-1. The SC-1 receptor is a new 82 kd membrane-bound isoform of glycosylphosphatidylinositol (GPI)-linked CD55 (decay-accelerating factor, DAF). CD55 is known to protect cells from lysis through autologous complement and is coexpressed with the ubiquitously distributed 70 kd isoform. The SC-1-specific CD55 isoform is up-regulated shortly after antibody binding, followed by an internalization of the antibody/receptor-complex, whereas the membranous expression of wild-type CD55 remains unchanged. The apoptotic process is marked by cleavage of cytokeratin 18, indicating the involvement of caspase-6 in the apoptotic process. In contrast to other apoptotic pathways, a cleavage of poly(ADP-ribose)polymerase (PARP) is not observed. The expression of the cell-cycle regulator c-myc becomes up-regulated, whereas expression of topoisomerase IIalpha is down-regulated. Induction of apoptosis leads to an increase in the internal Ca(2+) concentration, which is not necessary for the apoptotic process but for the transport of newly synthesized SC-1-specific CD55 isoform to the membrane.
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PMID:Regulation of the new coexpressed CD55 (decay-accelerating factor) receptor on stomach carcinoma cells involved in antibody SC-1-induced apoptosis. 1170 63


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