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)

Previous studies of cholera toxin and Escherichia coli heat-labile enterotoxin have suggested that proteolytic cleavage plays an important role in the expression of ADP-ribosyltransferase activity and toxicity. Specifically, several studies have implicated a trypsin-like cleavage at arginine 192, which lies within an exposed region subtended by a disulfide bond in the intact A subunit, in toxicity. To investigate the role of this modification in the enzymatic and cytotonic properties of heat-labile enterotoxin, the response of purified, recombinant A subunit to tryptic activation and the effect of substituting arginine 192 with glycine on the activities of the holotoxin were examined. The recombinant A subunit of heat-labile enterotoxin exhibited significant levels of ADP-ribosyltransferase activity that were only nominally increased (approximately twofold) by prior limited trypsinolysis. The enzymatic activity also did not appear to be affected by auto-ADP-ribosylation that occurs during the high-level synthesis of the recombinant A subunit in E. coli. A mutant form of the holotoxin containing the arginine 192-to-glycine substitution exhibited levels of cytotonic activity for CHO cells that were similar to that of the untreated, wild-type holotoxin but exhibited a marked delay in the ability to increase intracellular levels of cyclic AMP in Caco-2 cells. The results indicate that trypsin-like cleavage of the A subunit of E. coli heat-labile enterotoxin at arginine 192 is not requisite to the expression of enzymatic activity by the A subunit and further reveal that this modification, although it enhances the biological and enzymatic activities of the toxin, is not absolutely required for the enterotoxin to elicit cytotonic effects.
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PMID:Role of trypsin-like cleavage at arginine 192 in the enzymatic and cytotonic activities of Escherichia coli heat-labile enterotoxin. 792 84

The two active-site tryptophans of diphtheria toxin, Trp-50 and Trp-153, were individually or jointly replaced with phenylalanine or alanine by directed mutagenesis of a synthetic gene for the toxin's catalytic A fragment. Substitution of Trp-50 with alanine (W50A) decreased the ADP-ribosyltransferase activity by nearly 10(5)-fold and reduced NAD-glycohydrolase activity beyond the limits of our detection. Effects of the W153A mutation on these activities were less dramatic, < 40-fold decrease in ADP-ribosylation and < 10-fold decrease in NAD glycohydrolysis. The W50F and W153F substitutions caused only minimal reductions (< 2-fold) in enzyme activities and NAD affinity. Decreases in affinity for NAD in the initial, ground state complex, as measured by intrinsic protein fluorescence, correlated well with the reductions in enzyme activity. None of the mutations caused greater than a 10-fold decrease in NAD affinity for the ternary Michaelis complex in the ADP-ribosylation reaction; and none caused significant increase in susceptibility to proteolytic digestion by trypsin. The results indicate that Trp-50 is a major determinant of NAD affinity. Also, they identify this residue as a candidate for modification in the development of inactive forms of the toxin for use in vaccine development.
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PMID:Active-site mutations of diphtheria toxin. Tryptophan 50 is a major determinant of NAD affinity. 808 36

An arginine-specific mono-ADP-ribosyltransferase is expressed on the surface of differentiated mouse skeletal muscle cells and is anchored in the membrane via a glycosylphosphatidylinositol tail. Following incubation of intact cells with [adenylate-32P]NAD and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 97-kDa [32P]ADP-ribosylated protein was observed under reducing conditions and a 140-kDa complex under nonreducing conditions. The ADP-ribosylated protein was purified on a laminin affinity column. Based on its N-terminal sequence (FNLDVM-GAIRKEGEPGSLFGF) and a partial internal sequence (GLMRSEELSFVAGAP), the modified protein was identified as integrin alpha 7. Following partial trypsin digestion, a 39-kDa/79-kDa radiolabeled fragment was produced (reduced/nonreduced SDS-PAGE), narrowing the ADP-ribosylation site to a 39-kDa segment in the extracellular domain of integrin alpha 7. Labeling under optimal conditions was at least 0.4 mol of ADP-ribose/mol of integrin alpha 7. Selective expression of both ADP-ribosyltransferase and integrin alpha 7 in cardiac and skeletal muscle, a similar developmental appearance, and the apparently specific ADP-ribosylation, are consistent with a regulatory association between these proteins. ADP-ribosylation may modulate integrin receptor signaling and could play a significant role in the regulation of muscle cell function by the extracellular matrix.
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PMID:Integrin alpha 7 as substrate for a glycosylphosphatidylinositol-anchored ADP-ribosyltransferase on the surface of skeletal muscle cells. 824 57

Pertussis toxin (PT)-catalyzed ADP-ribosylation of transducin (Gt) is stimulated by ATP. In the absence of ATP, PT exhibited an approximately 20-fold lower linear velocity than the recombinant S1 subunit (rS1) in catalyzing the ADP-ribosylation of Gt. In the presence of 0.1 mM ATP, the linear velocities of rS1 and PT were essentially identical. ATP increased the kcat of PT-catalyzed ADP-ribosylation of Gt without altering the Kmapp for either Gt or NAD. Further, in the presence of ATP, PT exhibited similar kinetic constants under conditions of variable Gt and variable NAD as rS1 in catalyzing the ADP-ribosylation of Gt. The S1 subunit of PT was cleaved by chymotrypsin to a single immunoreactive peptide in the absence of ATP, while three immunoreactive peptides were generated in the presence of ATP. The S1 subunit of PT was not cleaved by trypsin in the absence of ATP, at the concentrations of trypsin used, while two immunoreactive peptides were produced in the presence of ATP. The immunoreactive peptides produced either by chymotrypsin or trypsin cleavage of the S1 subunit of PT in the presence of ATP were indistinguishable from those produced by cleavage of rS1 with the same protease. Chymotryptic and tryptic cleavage of rS1 was not altered by ATP. When PT was incubated with ATP prior to Bio-Gel P-100 gel filtration, approximately 8% of the S1 subunit dissociated from the B oligomer, as determined by ADP-ribosyltransferase assays of the column eluant. This increased to 20% when ATP was included in the column buffer. The presence of dithiothreitol and NAD in addition to ATP did not affect the amount of dissociated S1 subunit. Our data further indicated that activation of PT by ATP was a reversible process. Together, these data showed that ATP quantitatively converted the S1 subunit of PT to a form which was kinetically and conformationally identical with rS1, while only a fraction of the S1 subunit was dissociated from the B oligomer. These results indicate that both S1 subunit which is bound to the B oligomer as well as dissociated S1 subunit are capable of catalyzing the ADP-ribosylation of Gt.
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PMID:Molecular characterization of the in vitro activation of pertussis toxin by ATP. 850 98

Exoenzyme S of Pseudomonas aeruginosa (ExoS) is a member of the family of bacterial ADP-ribosylating exotoxins (bAREs). Site-directed mutagenesis of glutamic acids within the catalytic domain of ExoS (termed delta N222) allowed the identification of the preferential inactivation of ADP-ribosyltransferase activity by alanine substitution of E381. The specific activity of E381A mutant was 0.02% of wild-type delta N222. Delta N222(E381A) retained the requirement of factor activating exoenzyme S (FAS) activation for the expression of ADP-ribosyltransferase activity. In contrast, E387A, E399A, and E414A mutants possessed ADP-ribosyltransferase activity similar to that of wild-type delta N222. Kinetic evaluation of E381A and two other mutants, E381D and E381S, showed that their primary defect was a lower kcat in the ADP-ribosylation of soybean trypsin inhibitor (SBTI). The Km for NAD and SBTI and activation by FAS varied 2- and 10-fold relative to delta N222. In addition, the E381 mutants possessed identical protease patterns during thrombin and trypsin digestion as delta N222, which indicated that E381 mutants had retained their overall conformation. Together, these data identify E381 as contributing to the catalytic activity of exoenzyme S.
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PMID:Identification of glutamic acid 381 as a candidate active site residue of Pseudomonas aeruginosa exoenzyme S. 861 82

Photoaffinity labelling of the human poly(ADP-ribose) polymerase (PARP) catalytic domain (40 kDa) with the NAD+ photoaffinity analogue 2-azido-[alpha-32P]NAD+ has been used to identify NAD+-binding residues. In the presence of UV, photo-insertion of the analogue was observed with a stoichiometry of 0.73 mol of 2-azido-[alpha-32P]NAD+ per mol of catalytic domain. Competition experiments indicated that 3-aminobenzamide strongly protected the insertion site. Residues binding the adenine ring of NAD+ were identified by trypsin digestion and boronate affinity chromatography in combination with reverse-phase HPLC. Two major NAD+-binding residues, Trp1014 of peptide Thr1011-Trp1014 and Lys893 of peptide Ile979-Lys893, were identified. The site-directed mutagenesis of these two residues revealed that Lys893, but not Trp1014, is critical for activity. The close positioning of Lys893 near the adenine ring of NAD+ has been confirmed by the recently solved crystallographic structure of the chicken PARP catalytic domain [Ruf, Menissier-de Murcia, de Murcia and Schulz (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 7481-7485].
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PMID:Photoaffinity labelling of human poly(ADP-ribose) polymerase catalytic domain. 906 65

We examined the role in toxicity of histidine-44 of the A subunit of Escherichia coli enterotoxin, which is located in the active site cavity close to glutamic acid-112. Although amino acid substitution of histidine-44 usually renders a mutant toxin unstable to trypsin, one mutant, alanine-44 (His44Ala) was found to be stable. His44Ala did not show any agmatine:ADP-ribosyltransferase activity in the presence or absence of recombinant ADP-ribosylation factor. It showed no diarrheal or rabbit skin permeability activity and was a competitor in enterotoxin-ADP-ribosyltransferase assays containing recombinant ADP-ribosylation factor. These results suggest that like glutamic acid-112, histidine-44 plays an essential role in toxicity. A tentative model, which explains NAD+ catalysis and the transfer of the ADP-ribosyl moiety to a target amino acid, is proposed for histidine-44 and glutamic acid-112.
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PMID:Histidine-44 of the A subunit of Escherichia coli enterotoxin is involved in its enzymatic and biological activities. 923 14

Cyclic GMP phosphodiesterase, a key enzyme in phototransduction, is composed of P alpha beta and two P gamma subunits. Interaction of P gamma with P alpha beta or with the alpha subunit (T alpha) of transducin is crucial for the regulation of cGMP phosphodiesterase in retinal photoreceptors. Here we have investigated phosphorylation of P gamma by cAMP-dependent protein kinase and its functional effect on the P gamma interaction with P alpha beta or T alpha in vitro. P gamma, but not P gamma complexed with T alpha (both GTP and GDP forms), is phosphorylated. Measurement of 32P radioactivity in phosphorylated P gamma, analysis of phosphorylated P gamma by laser mass spectrometry, identification of phosphoamino acid, and phosphorylation of mutant forms of P gamma indicate that only threonine 35 in P gamma is phosphorylated. Phosphorylation of P gamma mutants also reveals that the C and N terminals of P gamma which are required for the regulation of P alpha beta functions are not involved in the P gamma phosphorylation but that arginine 33, which is ADP-ribosylated by an endogenous ADP-ribosyltransferase, is required for the phosphorylation. Phosphorylated P gamma has a higher inhibitory activity for trypsin-activated cGMP phosphodiesterase than nonphosphorylated P gamma, indicating that the P gamma-P alpha beta interaction is affected by P gamma phosphorylation. Nonphosphorylated P gamma inhibits both the GTPase activity of T alpha and the binding of a hydrolysis-resistant GTP analogue to T alpha, while P gamma phosphorylation reduces these inhibitory activities. These observations suggest that a P gamma domain containing threonine 35 is involved in the P gamma-T alpha interaction, and P gamma phosphorylation regulates the P gamma-T alpha interaction. Our observation suggests that P gamma phosphorylation by cAMP-dependent protein kinase may function for the regulation of phototransduction in vertebrate rod photoreceptors.
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PMID:Phosphorylation of the gamma subunit of the retinal photoreceptor cGMP phosphodiesterase by the cAMP-dependent protein kinase and its effect on the gamma subunit interaction with other proteins. 955 60

beta-Lapachone (beta-lap) effectively killed MCF-7 and T47D cell lines via apoptosis in a cell-cycle-independent manner. However, the mechanism by which this compound activated downstream proteolytic execution processes were studied. At low concentrations, beta-lap activated the caspase-mediated pathway, similar to the topoisomerase I poison, topotecan; apoptotic reactions caused by both agents at these doses were inhibited by zVAD-fmk. However at higher doses of beta-lap, a novel non-caspase-mediated "atypical" cleavage of PARP (i.e., an approximately 60-kDa cleavage fragment) was observed. Atypical PARP cleavage directly correlated with apoptosis in MCF-7 cells and was inhibited by the global cysteine protease inhibitors iodoacetamide and N-ethylmaleimide. This cleavage was insensitive to inhibitors of caspases, granzyme B, cathepsins B and L, trypsin, and chymotrypsin-like proteases. The protease responsible appears to be calcium-dependent and the concomitant cleavage of PARP and p53 was consistent with a beta-lap-mediated activation of calpain. beta-Lap exposure also stimulated the cleavage of lamin B, a putative caspase 6 substrate. Reexpression of procaspase-3 into caspase-3-null MCF-7 cells did not affect this atypical PARP proteolytic pathway. These findings demonstrate that beta-lap kills cells through the cell-cycle-independent activation of a noncaspase proteolytic pathway.
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PMID:Activation of a cysteine protease in MCF-7 and T47D breast cancer cells during beta-lapachone-mediated apoptosis. 1069 31

We asked whether the antiangiogenic action of 16K human PRL (hPRL), in addition to blocking mitogen-induced vascular endothelial cell proliferation, involved activation of programmed cell death. Treatment with recombinant 16K hPRL increased DNA fragmentation in cultured bovine brain capillary endothelial (BBE) and human umbilical vein endothelial (HUVE) cells in a time- and dose-dependent fashion, independent of the serum concentration. The activation of apoptosis by 16K hPRL was specific for endothelial cells, and the activity of the peptide could be inhibited by heat denaturation, trypsin digestion, and immunoneutralization, but not by treatment with the endotoxin blocker, polymyxin-B. 16K hPRL-induced apoptosis was correlated with the rapid activation of caspases 1 and 3 and was blocked by pharmacological inhibition of caspase activity. Caspase activation was followed by inactivation of two caspase substrates, poly(ADP-ribose) polymerase (PARP) and the inhibitor of caspase-activated deoxyribonuclease (DNase) (ICAD). Furthermore, 16K hPRL increased the conversion of Bcl-X to its proapoptotic form, suggesting that the Bcl-2 protein family may also be involved in 16K hPRL-induced apoptosis. These findings support the hypothesis that the antiangiogenic action of 16K hPRL includes the activation of programmed cell death of vascular endothelial cells.
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PMID:The antiangiogenic factor 16K PRL induces programmed cell death in endothelial cells by caspase activation. 1104 70


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