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)

The enzymatic ADP-ribosyltransferase activity associated with the S1 subunit of pertussis toxin is considered to be responsible for its biological effects. Although pertussis toxin has no significant homology to other ADP-ribosylating toxins such as diphtheria toxin and Pseudomonas aeruginosa exotoxin A, the results presented in this paper show that, as for diphtheria toxin and exotoxin A, tryptophan and glutamic acid residues are essential for the enzymatic activities of pertussis toxin. Moreover, a structural motif can be identified around the critical glutamic acid residue. Chemical modification or site-directed deletion or replacement of Trp-26 abolishes ADP-ribosyltransferase and the associated NAD glycohydrolase activities. Both enzymatic activities are also abolished when Glu-129 is deleted or replaced by aspartic acid. Mutations at the Glu-106 position do not significantly reduce the enzymatic activities of the S1 subunit. The mutations do not affect the ability of the different S1 forms to be recognized by a variety of monoclonal antibodies, including neutralizing antibodies. Pertussis toxin containing a deletion or replacement of Trp-26, Glu-129, or both in the S1 subunit should thus be devoid of toxic activities without losing its reactivity with protective antibodies and, therefore, could be safely included in new generation vaccines against whooping cough.
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PMID:Identification of amino acid residues essential for the enzymatic activities of pertussis toxin. 247 88

The structural gene of the S-1 subunit of pertussis toxin (rS-1) and the catalytic C180 peptide of the S-1 subunit (C180 peptide) were independently subcloned downstream of the tac promoter in Escherichia coli. Both constructions included DNA encoding for the predicted leader sequence of the S-1 subunit which was inserted between the tac promoter and the structural gene. E. coli containing the plasmids encoding for rS-1 and C180 peptide produced a peptide that reacted with anti-pertussis toxin antibody and had a molecular weight corresponding to that of the cloned gene; some degradation of rS-1 was observed. Extracts of E. coli containing plasmids encoding for rS-1 and the C180 peptide possessed ADP-ribosyltransferase activity. Subcellular fractionation showed that both rS-1 and the C180 peptide were present in the periplasm, indicating that E. coli recognized the pertussis toxin peptide leader sequence. The protein sequence of the amino terminus of the C180 peptide was identical to that of authentic S-1 subunit produced by Bordetella pertussis, which showed that E. coli leader peptidase correctly processed the pertussis toxin peptide leader sequence. Two single amino acid substitutions at residue 26 (C180I-26) and residue 139 (C180S-139) which were previously shown to reduce ADP-ribosyltransferase activity were introduced into the C180 peptide. C180I-26 possessed approximately 1% of the NAD-glycohydrolase activity of the C180 peptide, suggesting that tryptophan 26 functions in the interaction of NAD with the C180 peptide. In contrast, C180S-139 possessed essentially the same level of NAD-glycohydrolase activity as the C180 peptide, suggesting that glutamic acid 139 does not function in the interaction of NAD but plays a role in a later step in the ADP-ribosyltransferase reaction.
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PMID:Expression and secretion of the S-1 subunit and C180 peptide of pertussis toxin in Escherichia coli. 254 19

A recently constructed chromosome 13-enriched library was used to isolate the entire human ADP-ribosyltransferase (NAD+) pseudogene 1 gene (ADPRTP1) on 13q34. Recently, ADPRTP1 was shown to detect a restriction fragment length polymorphism that was associated with a predisposition to cancer. The complete sequence of the ADPRTP1 (3808 bases) was determined and found to resemble an intronless cDNA, including 137 bases of the 5' untranslated region and a short poly(A) tail. A previously uncharacterized, endogenous pol-like element (1.53 kb) in which the DNA sequences were interrupted by the polymorphic ADPRTP1 was identified. The solitary pol-related retroviral element may represent a multimember family of viral-like DNA sequences dispersed throughout the human genome. Altogether a 9.25-kb genomic region that also encompassed two Alu elements and the long-terminal repeat-like element of the "O"-LTR family was characterized.
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PMID:The polymorphic ADP-ribosyltransferase (NAD+) pseudogene 1 in humans interrupts an endogenous pol-like element on 13q34. 790 81

The role of the tryptophan residues in the substrate-binding and catalytic mechanism of an enzymatically active C-terminal fragment of Pseudomonas aeruginosa exotoxin A was studied by individually or jointly replacing these residues with phenylalanine. Substitution of W-466 decreased the ADP-ribosyltransferase and NAD(+)-glycohydrolase activities by 20- and 3-fold, respectively. In contrast, substitution of W-417 or W-558 with phenylalanine both resulted in a 3-fold decrease in ADP-ribosyltransferase activity with, however, only a decrease by 40% and 70% in NAD(+)-glycohydrolase activity, respectively. Simultaneous replacement of W-466 and W-558 resulted in a 200-fold decrease in ADP-ribosyltransferase and an 6-fold decrease in NAD(+)-glycohydrolase activities, suggesting that W-466 may play a minor role in the transfer of ADP-ribose to the eEF-2 protein. Chemical modification of the tryptophan residues in the wild-type toxin fragment by N-bromosuccinimide revealed the presence of a single residue important for enzymatic activity, W-466, with a minor contribution from W-558. Additionally, tryptophan residues, W-305 and W-417, were refractory to oxidation by N-bromosuccinimide, which likely indicated the buried nature of these residues within the protein structure. Titration of the wild-type toxin fragment with NAD+ resulted in the quenching of the intrinsic tryptophan fluorescence to 58% of the initial value. Titration of the various single and a double tryptophan replacement mutant protein(s) indicated that W-558 and W-466 are responsible for the substrate-induced fluorescence quenching, with the former being responsible for the largest fraction of the observed quenching in the wild-type toxin. Consequently, a molecular mechanism is proposed for the substrate-induced fluorescence quenching of both W-466 and W-558. Furthermore, molecular modeling of the recent crystal structures for both exotoxin A (domain III fragment) and diphtheria toxin, combined with a variety of previous results, has led to the proposal for a catalytic mechanism for the ADP-ribosyltransferase reaction. This mechanism features a SN1 attack (instead of the previously purported SN2 mechanism) by the diphthamide residue (nucleophile) of eukaryotic elongation factor 2 on the C-1 of the nicotinamide ribose of NAD+, which results in an inversion of configuration likely due to steric constraints within the NAD(+)-toxin-elongation factor 2 complex.
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PMID:Investigation into the catalytic role for the tryptophan residues within domain III of Pseudomonas aeruginosa exotoxin A. 895 60

Intracutaneous injection of cholera toxin (CT) into rabbits increases vascular permeability and induces epidermal proliferation. To understand the mechanisms of these effects on the skin, we evaluated the involvement of the ADP-ribosyltransferase activity of the A subunit of CT and receptor-binding interactions between GM1-ganglioside and the B subunit of CT. We constructed two mutant CTs, E112K and W88K, by site-directed mutagenesis. Mutant CT-E112K, in which glutamic acid at position 112 (E112) of the A subunit of CT was replaced by lysine, has been shown to have lost its biological activity on Chinese hamster ovary (CHO) cells because of its abolished ADP-ribosyltransferase activity. Mutant CT-W88K, in which tryptophan at position 88 (W88) of the B subunit of CT was replaced by lysine, has been shown to have lost its binding ability to GM1-ganglioside. Intracutaneous injection of these mutant CTs evoked less vascular permeability and less epidermal proliferation than recombinant wild-type CT. These results suggest that: (1) the ADP-ribosyltransferase activity carried by E112 of the A subunit of CT; and (2) the binding ability to GM1-ganglioside via W88 of the B subunit of CT are essential for these effects of CT on the skin.
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PMID:Analysis of mechanisms of epidermal proliferation induced by intracutaneous injection of cholera toxin by the use of site-specifically mutated cholera toxins. 965 15

We here report an alternatively spliced form of PARP lacking exon 5 of the Drosophila PARP gene encoding the auto-modification domain. The alternative form of PARP (PARP II) consists 804 amino acids with a molecular weight of 92.3 kDa. The deduced amino acid sequence of PARP II was completely matched to that of PARP I encoded by a full-length Drosophila PARP cDNA, except it lacks the region corresponding to the auto-modification domain. To examine the function of PARP II, stable transformants of Rat-1 cells in which PARP II was ectopically expressed by MMTV-LTR were isolated and characterized. After induction with dexamethasone, PARP II transformants showed slower growth and showed morphological changes with loss of spindled shape compared to cells transformed with the vector or PARP I. The PARP II-transformed cells incorporated propidium iodide after induction; however, Annexin V and TUNEL analysis indicated these changes were not due to apoptosis.
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PMID:An alternative form of poly(ADP-ribose) polymerase in Drosophila melanogaster and its ectopic expression in rat-1 cells. 979 Sep 3

Phorbol 12-myristate 13-acetate (PMA)-induced HIV-1 production in U1 cells was markedly suppressed by inhibitors of poly (ADP-ribose) polymerase (PARP). Northern blot analysis revealed that the PARP-inhibitors suppressed the virus production at a level of transcription. In order to examine the effect of PARP on transcriptional regulation of HIV-1 genes, we transfected a reporter plasmid containing HIV-1-LTR-promoted luciferase gene to L-1210 cell clones, which expressed varying decreased level of PARP. In wild type L-1210 cells, the expression of LTR-promoted luciferase gene was stimulated approximately 4-fold in response to PMA, whereas the PMA-dependent response was almost abolished in mutant cells, which expressed only 8% of PARP of the wild type cells. The effect of decrease in PARP content on the function of HIV-1-LTR was confirmed also in human wild type cells, Jurkat and J111, which were co-transfected with the reporter plasmid and a plasmid expressing a PARP-antisense RNA: Down-regulation of PARP in the cells by the expression of the antisense RNA significantly suppressed the PMA-dependent, LTR-function of the reporter plasmid in both Jurkat and J111 cells. NF-kappaB, which is known to mediate the PMA-induced activation of HIV-1 in U1 cells, was found to be activated approximately 5-fold in PMA-treated U1 cells. PARP-inhibitor, unexpectedly, did not suppress but rather stimulated (approximately 2-fold) the NF-kappaB activation. Combining the results with the finding that the LTR-function was minimum in a PARP-defective mutant cells in spite of a very high level of the activated NF-kappaB in the cells, we suggest that PARP, in addition to activated NF-kappaB, is essential for the function of HIV-1 LTR.
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PMID:Poly (ADP-ribose) polymerase is involved in PMA-induced activation of HIV-1 in U1 cells by modulating the LTR function. 1044 6

The transcription factor NF-kappaB plays a critical role in immune and inflammatory responses. Here we show that poly (ADP ribose) polymerase (PARP) is required for specific NF-kappaB transcriptional activation in vivo. The activation of the HIV-LTR promoter and an NF-kappaB-dependent artificial promoter was drastically reduced in PARP (-/-) cells, independently of the signaling pathway through which NF-kappaB was induced. Furthermore NF-kappaB-dependent gene activation was restored in vivo by the expression of PARP in PARP (-/-) cells. Finally, we show that both NF-kappaB and PARP formed a stable immunoprecipitable nuclear complex. This interaction did not need DNA binding. Our results suggest that PARP is an important cofactor in the activation cascade of NF-kappaB-dependent target genes.
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PMID:A role of poly (ADP-ribose) polymerase in NF-kappaB transcriptional activation. 1049 47

The aim of the present study was to improve the viability of marginal livers from non-heart beating donors upon cold preservation using two different techniques for the provision of tissue aerobiosis. Livers from male Wistar rats (250-300 g bw) were harvested after 60 min of cardiac arrest, flushed via the portal vein with 20 mL of heparinized Ringer's solution and 60 mL of histidine-tryptophan-ketoglutarate (HTK) preservation solution. Control livers were then stored submerged in HTK for 24 h at 4 degrees C while other organs were subjected to aerobic conditions by either insufflation of gaseous oxygen via the venous vascular system of the cold stored organ (VSOP) or pulsatile machine perfusion (MP) with oxygenated HTK at 5 mL/min at 4 degrees C. Superoxide dismutase (SOD) (7500 IU) was added to the last 10 mL of HTK in order to prevent adverse effects of high oxygen tensions at hypothermia. Viability of the livers was assessed upon isolated perfusion in vitro with oxygenated Krebs-Henseleit buffer at constant flow. VSOP or MP, both significantly improved vascular conductivity upon reperfusion as evaluated by portal venous pressure, reduced hepatic enzyme release and led to a rise in hepatic bile production upon reperfusion. Induction of apoptosis was also looked for in tissue homogenates by Western analysis for cleavage of poly(ADP-ribose)polymerase (PARP). Expression of cleaved PARP fragment could be found in reperfused control livers but also, though to a lesser extend, after VSOP or MP. In conclusion, provision of oxygen during cold preservation significantly contributes to improve organ viability upon reperfusion and must be regarded as a useful adjunct for marginal or pre-damaged livers. HTK has been shown for the first time to be also suitable for long-term MP preservation of the liver, but, as inferred from these data, simple insufflation of gaseous O2 may be considered a feasible alternative.
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PMID:Liver preservation with HTK: salutary effect of hypothermic aerobiosis by either gaseous oxygen or machine perfusion. 1201 Jan 45

Recently, the endoplasmic reticulum (ER) has been disclosed as subcellular target reactive to ischaemia/reperfusion and possibly influenced by hypothermic machine preservation. Here, the respective role of perfusate, perfusion itself, and the effect of continuous oxygenation to trigger ER-stress in the graft should be investigated. Livers were retrieved 30 min after cardiac arrest of male Wistar rats and preserved by cold storage (CS) in histidine-tryptophan-ketoglutarate (HTK) for 18 h at 4 degrees C. Other organs were subjected to aerobic conditions either by oxygenated machine perfusion with HTK (MP-HTK) or Belzer solution (MP-Belzer) at 4 degrees C or by venous insufflation of gaseous oxygen during cold storage (VSOP). Viability of livers was evaluated upon reperfusion in vitro according to previously validated techniques for 120 min at 37 degrees C. Oxygenation during preservation (MP-HTK, MP-Belzer or VSOP) concordantly improved functional recovery (bile flow, ammonia clearance), reduced parenchymal enzyme leakage and histological signs of necrosis and significantly attenuated mitochondrial induction of apoptosis (cleavage of caspase 9) compared to CS. However, MP with either medium produced about 500% elevated protein expression of CHOP/GADD153, suggesting pro-apoptotic ER-stress responses, paralleled by a significant elevation of caspase-12 enzyme activity compared to CS or VSOP. Although MP also promoted a slight (20%) induction of the cytoprotective ER-protein Bax inhibitor protein (BI-1), prevailing of proapoptotic reactions was seen by increased cleavage of caspase-3 and poly (ADP-Ribase)-polymerase (PARP) in both MP-groups. Endoplasmic stress activation is conjectured a specific side effect of long-term machine preservation irrespective of the medium, actually promoting cellular apoptosis via activation of caspase-12. The simple insufflation of gaseous O2 may be considered a feasible alternative, apparently indifferent to the endoplasmic reticulum.
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PMID:Role of perfusion medium, oxygen and rheology for endoplasmic reticulum stress-induced cell death after hypothermic machine preservation of the liver. 1800 84


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