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 cowpox virus (CPV) CrmA and the equivalent rabbitpox virus (RPV) SPI-2 proteins have anti-inflammatory and antiapoptosis activity by virtue of their ability to inhibit caspases, including the interleukin-1beta-converting enzyme (ICE; caspase-1). Infection of LLC-PK1 pig kidney cells with a CPV CrmA mutant, but not with wild-type (wt) CPV, results in the induction of many of the morphological features of apoptosis (C. A. Ray and D. J. Pickup, Virology 217:384-391, 1996). In our study, LLC-PK1 cells infected with CPV delta crmA, but not those infected with wt CPV, showed induction of poly(ADP-ribose) polymerase (PARP)- and lamin A-cleaving activities and processing of the CPP32 (caspase-3) precursor to a mature 18-kDa form. Surprisingly, infection of LLC-PK1 cells with either wt RPV (despite the presence of the SPI-2 protein) or RPV delta SPI-2 resulted in cleavage activity against PARP and lamin A and the appearance of the mature subunit of CPP32/caspase-3. The biotinylated specific peptide inhibitor Ac-Tyr-Val-Lys(biotinyl)-Asp-2,6-dimethylbenzoyloxymethylketone [AcYV(bio)KD-aomk] labeled active caspase subunits of 18, 19, and 21 kDa in extracts from LLC-PK1 cells infected with CPV delta crmA, wt RPV, or RPV delta SPI-2 but not wt CPV. Mixed infection of LLC-PK1 cells with wt RPV and wt CPV gave no PARP-cleaving activity, and all PARP cleavage mediated by SPI-2 and CrmA mutants of RPV and CPV, respectively, could be eliminated by coinfection with wt CPV. These results suggest that the RPV SPI-2 and CPV CrmA proteins are not functionally equivalent and that CrmA, but not SPI-2 protein, can completely prevent apoptosis in LLC-PK1 cells under these conditions.
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PMID:Activation of caspases in pig kidney cells infected with wild-type and CrmA/SPI-2 mutants of cowpox and rabbitpox viruses. 955 31

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

The actin ADP-ribosylating Clostridium botulinum C2 toxin is a binary toxin composed of the binding component C2II and the enzyme component C2I. C2I ADP-ribosylates G-actin at arginine 177, resulting in the depolymerization of the actin cytoskeleton. Here, we studied the structure-function relationship of C2I by site-directed mutagenesis. Exchange of Glu389 to glutamine caused the complete loss of ADP-ribosyltransferase and NAD-glycohydrolase activities of C2I. In contrast, exchange of Glu387 to glutamine blocked ADP-ribosyltransferase but not NAD-glycohydrolase activity. Whereas photoaffinity labeling of the double mutant E387Q/E389Q C2I with [carbonyl-14C]NAD was blocked, labeling of the single C2I mutants was reduced (E389Q) or not changed (E387Q). Exchange of the STS motif (amino acid residues 348-350) of C2I caused a decrease in transferase activity by more than 99 (S348A) and 90% (T349V), or did not affect activity (S350A). Exchange of Arg299 and Arg300 to lysine reduced transferase activity to <0.1 and approximately 35% of wild-type activity. The data indicate that the amino acid residues Glu389, Glu387, Ser348, and Arg299, which are conserved in various prokaryotic and eukaryotic arginine-modifying ADP-ribosyltransferases, are essential for ADP-ribosyltransferase activity of the enzyme component of C. botulinum C2 toxin.
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PMID:Characterization of the catalytic site of the ADP-ribosyltransferase Clostridium botulinum C2 toxin by site-directed mutagenesis. 979 57

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that recognizes and binds to the nicks and ends of DNA, and catalyses successive ADP-ribosylation reactions. To clarify the function of PARP at the molecular level, we searched proteins which interact with PARP. In the auto-modification domain of PARP in Drosophila, there is a putative leucine-zipper motif which can interact with other protein molecules. To find interacting proteins we examined the auto-modification domain of Drosophila PARP, using the Far-Western screening method. From six independent cDNA clones isolated, we characterized two clones, PBP-3 and PBP-12. The predicted amino acid sequences from 109 to 269 of PBP-3 and from 184 to 312 of PBP-12 had more than 62% identities to mammalian L23a (rpl23a) and L22 (rpl22), the ribosomal proteins of the large subunit. This indicated that PBP-3 and PBP-12 are Drosophila homologues of L23a and L22, respectively. These Drosophila ribosomal protein L22 and L23a have additional Ala-, Lys- and Pro-rich sequences at the amino terminus, which have a resemblance to the carboxy-terminal portion of histone H1. Thus, Drosophila L22 and L23a might have two functions, namely the role of DNA-binding similar to histone H1 and the role of organizing the ribosome.
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PMID:Poly(ADP-ribose) polymerase interacts with novel Drosophila ribosomal proteins, L22 and l23a, with unique histone-like amino-terminal extensions. 993 8

4-Hydroxynonenal (HNE), a diffusible product of lipid peroxidation, has been suggested to be a key mediator of oxidative stress-induced cell death. In this study, we partially characterized the mechanism of HNE-mediated cytotoxicity. Incubation of human T lymphoma Jurkat cells with 20-50 microM HNE led to cell death accompanied by DNA fragmentation. Western blot analysis showed that HNE-treatment induced time- and dose-dependent activation of caspase-8, caspase-9 and caspase-3. HNE-induced caspase-3 processing was confirmed by a flow cytometric demonstration of increased catalytic activity on the substrate peptide. HNE treatment also led to remarkable cleavage of poly(ADP-ribose) polymerase (PARP), which was prevented by pretreatment of cells with DEVD-FMK as a caspase-3 inhibitor. The HNE-mediated activation of caspases, cleavage of PARP and DNA fragmentation were blocked by antioxidants cysteine, N-acety-L-cysteine and dithiothreitol, but not by two other HNE-reactive amino acids lysine and histidine, or by cystine, the oxidized form of cysteine. HNE rapidly decreased levels of intracellular reduced glutathione (GSH) and its oxidized form GSSG, and these were also attenuated by the reductants. Coincubation of Jurkat cells with a blocking anti-Fas antibody prevented Fas-induced but not HNE-induced activation of caspase-3. HNE also activated caspase-3 in K562 cells that do not express functional Fas. Our results thereby demonstrate that HNE triggers oxidative stress-linked apoptotic cell death through activation of the caspase cascade. The results also suggest a possible mechanism involving a direct scavenge of intracellular GSH by HNE.
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PMID:4-hydroxynonenal induces a cellular redox status-related activation of the caspase cascade for apoptotic cell death. 1065 56

The iota(a) component (i(a)) of Clostridium perfringens ADP ribosylates nonmuscle beta/gamma actin and skeletal muscle alpha-actin. Replacement of Arg-295 in i(a) with alanine led to a complete loss of NAD(+)-glycohydrolase (NADase) and ADP-ribosyltransferase (ARTase); that of the residue with lysine caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them. Substitution of alanine for Glu-378 and Glu-380 caused a complete loss of NADase and ARTase. However, exchange of Glu-378 to aspartic acid or glutamine resulted in little effect on NADase activity but a drastic reduction in ARTase activity (<0.1% of the wild-type activity). Exchange of Glu-380 to aspartic acid caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them; that of the residue to glutamine caused a complete loss of ARTase activity. Replacement of Ser-338 with alanine resulted in 0.7 to 2.3% wild-type activities, and that of Ser-340 and Thr-339 caused a reduction in these activities of 5 to 30% wild-type activities. The kinetic analysis showed that Arg-295 and Ser-338 also play an important role in the binding of NAD(+) to i(a), that Arg-295, Glu-380, and Ser-338 play a crucial role in the catalytic rate of NADase activity, and that these three amino acid residues and Glu-378 are essential for ARTase activity. The effect of amino acid replacement in i(a) on ARTase activity was similar to that on lethal and cytotoxic activities, suggesting that lethal and cytotoxic activities in i(a) are dependent on ARTase activity.
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PMID:Characterization of the enzymatic component of Clostridium perfringens iota-toxin. 1073 50

In a number of nitrogen-fixing bacteria, nitrogenase is posttranslationally regulated by reversible ADP-ribosylation of dinitrogenase reductase. The structure of the dinitrogenase reductase from Azotobacter vinelandii is known. In this study, mutant forms of dinitrogenase reductase from A. vinelandii that are affected in various protein activities were tested for their ability to be ADP-ribosylated or to form a complex with dinitrogenase reductase ADP-ribosyltransferase (DRAT) from Rhodospirillum rubrum. R140Q dinitrogenase reductase could not be ADP-ribosylated by DRAT, although it still formed a cross-linkable complex with DRAT. Thus, the Arg 140 residue of dinitrogenase reductase plays a critical role in the ADP-ribosylation reaction. Conformational changes in dinitrogenase reductase induced by an F135Y substitution or by removal of the Fe(4)S(4) cluster resulted in dinitrogenase reductase not being a substrate for ADP-ribosylation. Through cross-linking studies it was also shown that these changes decreased the ability of dinitrogenase reductase to form a cross-linkable complex with DRAT. Substitution of D129E or deletion of Leu 127, which result in altered nucleotide binding regions of these dinitrogenase reductases, did not significantly change the interaction between dinitrogenase reductase and DRAT. Previous results showed that changing Lys 143 to Gln decreased the binding between dinitrogenase reductase and dinitrogenase (L. C. Seefeldt, Protein Sci. 3:2073-2081, 1994); however, this change did not have a substantial effect on the interaction between dinitrogenase reductase and DRAT.
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PMID:ADP-Ribosylation of variants of Azotobacter vinelandii dinitrogenase reductase by Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyltransferase. 1076 64

Two mutants of cholera toxin (CTS106 containing a Pro106-->Ser substitution and CTK63 containing a Ser63-->Lys substitution) with greatly reduced or no toxicity respectively, were expressed in the naturally attenuated IEM101 Vibrio cholerae strain (El Tor, Ogawa) which does not express cholera toxin (CT). Expression was driven by the natural promoter of CT, or by a promoter known to induce strong in vivo expression such as nirB. In the rabbit ileal loop assay, where 10(4) wild type bacteria were sufficient to induce fluid accumulation, 10(9) IEM101 expressing CTS106 bacteria were needed to induce some fluid accumulation, while IEM101 expressing CTK63 was inactive, even when 10(10) cells were used. When used to immunize mice intranasally, all bacteria induced vibriocidal antibodies; however, anti-CT antibodies were not induced by bacteria expressing low levels of CTK63 under the control of the ct promoter. Anti-CT antibodies were successfully induced by bacteria expressing high levels of CTK63 under the control of the nirB promoter, or by bacteria expressing low levels of CTS106. These data show that antibodies against cholera toxin can be induced in vivo by high level expression of a non toxic mutant, or by using a mutant with residual ADP-ribosyltransferase activity. In conclusion, we have shown that IEM101, a naturally attenuated Vibrio strain known to be safe and immunogenic in humans, can be engineered to express immunogenic levels of CTK63, and may represent a good candidate for vaccination against cholera.
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PMID:IEM101, a naturally attenuated Vibrio cholerae strain as carrier for genetically detoxified derivatives of cholera toxin. 1092 89

Suicide gene therapy utilizing the herpes simplex thymidine kinase (HSVtk) / ganciclovir (GCV) system has been performed to kill cancer cells. However, the low transduction efficiency of HSVtk gene into cancer cells critically limits its efficacy in cancer treatment in clinical situations. To improve delivery of the HSVtk gene into cancer cells, we transduced U-87MG and U-373MG glioma cells with adenovirus (Adv) vectors with a fiber mutant, F / K20, which has a stretch of 20 lysine residues added at the C-terminus of the fiber, for the HSVtk gene (Adv-TK-F / K20), and compared the cytopathic effect of Adv-TK-F / K20 with that of the Adv for HSVtk with wild-type fiber (Adv-TK). The cytopathic effect of Adv-TK-F / K20 in U-87MG and U-373MG cells was approximately 140 and 40 times, respectively, stronger than that of Adv-TK. At the same multiplicity of infection (MOI) in each cell line, Adv-TK-F / K20 induced a higher degree of apoptosis (U-87MG, 35%; U-373MG, 77%) than Adv-TK (U-87MG, 0.11%; U-373MG, 27%) in U-87MG (MOI 0.03) and U-373MG cells (MOI 0.1). Cleavage of poly(ADP-ribose)polymerase (PARP) was more marked in the cells that were infected with Adv-TK-F / K20 than in cells that were infected with Adv-TK. These results indicate that gene therapy utilizing Adv-TK-F / K20 may be a promising therapeutic modality for the treatment of gliomas.
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PMID:Transduction of a fiber-mutant adenovirus for the HSVtk gene highly augments the cytopathic effect towards gliomas. 1105 Apr 74

Conflicting reports have suggested that the silent information regulator 2 (SIR2) protein family employs NAD(+) to ADP-ribosylate histones [Tanny, J. C., Dowd, G. J., Huang, J., Hilz, H. & Moazed, D. (1999) Cell 99, 735-745; Frye, R. A. (1999) Biochem. Biophys. Res. Commun. 260, 273-279], deacetylate histones [Landry, J., Sutton, A., Tafrov, S. T., Heller, R. C., Stebbins, J., Pillus, L. & Sternglanz, R. (2000) Proc. Natl. Acad. Sci. USA 97, 5807-5811; Smith, J. S., Brachmann, C. B., Celic, I., Kenna, M. A., Muhammad, S., Starai, V. J., Avalos, J. L., Escalante-Semerena, J. C., Grubmeyer, C., Wolberger, C. & Boeke, J. D. (2000) Proc. Natl. Acad. Sci. USA 97, 6658-6663], or both [Imai, S., Armstrong, C. M., Kaeberlein, M. & Guarente, L. (2000) Nature (London) 403, 795-800]. Uncovering the true enzymatic function of SIR2 is critical to the basic understanding of its cellular function. Therefore, we set out to authenticate the reaction products and to determine the intrinsic catalytic mechanism. We provide direct evidence that the efficient histone/protein deacetylase reaction is tightly coupled to the formation of a previously unidentified acetyl-ADP-ribose product (1-O-acetyl-ADP ribose). One molecule of NAD(+) and one molecule of acetyl-lysine are readily catalyzed to one molecule of deacetylated lysine, nicotinamide, and 1-O-acetyl-ADP-ribose. A unique reaction mechanism involving the attack of enzyme-bound acetate or the direct attack of acetyl-lysine on an oxocarbenium ADP-ribose intermediate is proposed. We suggest that the reported histone/protein ADP-ribosyltransferase activity is a low-efficiency side reaction that can be explained through the partial uncoupling of the intrinsic deacetylation and acetate transfer to ADP-ribose.
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PMID:Silent information regulator 2 family of NAD- dependent histone/protein deacetylases generates a unique product, 1-O-acetyl-ADP-ribose. 1111 64


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