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)

Poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) is a zinc finger DNA-binding protein involved in DNA repair processes in eukaryotes. By deletion and extensive site-directed mutagenesis, its DNA-binding domain fused to the N-terminus of beta-galactosidase was shown to contain a nuclear localization signal (NLS) of the form KRK-X(11)-KKKSKK (residues 207-226). In vitro, both the DNA-binding capacity and the polymerizing activity of PARP are independent of the nuclear location function. Each basic cluster is essential but not sufficient on its own for this function, while both motifs together are. Crucial basic amino acids (K207, R208 and K222) in each of these two motifs are required for nuclear homing. The results presented here support the concept that the human PARP NLS is an autonomous functional element and belongs to the class of bipartite NLSs. We show that the linear distance between the two basic clusters is not crucial. Insertional mutation analysis leading to a partial reversion of the cytoplasmic phenotype displayed by the mutant K222I highlights the crucial positioning of this lysine. The structure-function relationship of the second cluster of basic residues is discussed.
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PMID:The human poly(ADP-ribose) polymerase nuclear localization signal is a bipartite element functionally separate from DNA binding and catalytic activity. 150 17

The interleukin 4 (IL 4) receptor is expressed on various cells of the immune system, including T and B lymphocytes, macrophages and mast cells. We have constructed a recombinant protein, DAB389-mIL 4, that is composed of the enzymatically active and membrane translocation domains of diphtheria toxin fused to murine IL 4. We demonstrate that this fusion toxin selectively inhibits protein synsthesis in eukaryotic cells which express the murine IL 4 receptor. The cytotoxic potency of this fusion toxin is shown to be directly proportional to the reported number of IL 4 receptors on the surface of target cells. Since the action of DAB389-mIL 4 can be blocked with either excess mIL 4 or antibody to mIL 4, we conclude that its entry into target cells is mediated through the mIL 4 receptor. A mutant form of DAB389-mIL 4, DA(197)B389-mIL 4, in which the fragment A-associated ADP-ribosyltransferase is inactive, is not cytotoxic to murine IL 4 receptor-bearing cells. Finally, we demonstrate that DAB389-mIL 4 administered subcutaneously to DBA/2 mice results in suppression of delayed-type hypersensitivity (DTH); whereas, the non-toxic DA(197)B389-mIL 4 fails to dampen the DTH response.
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PMID:Interleukin 4 receptor targeted cytotoxicity: genetic construction and in vivo immunosuppressive activity of a diphtheria toxin-related murine interleukin 4 fusion protein. 167 15

To be capable of selective killing of tumor cells, the non-selective Pseudomonas aeruginosa exotoxin A must have its cell-binding domain inactivated or removed and then be chemically linked to, or genetically fused with, a specific targeting agent. In the present study, epsilon-NH2 groups of lysine residues of the cell-binding domain of exotoxin A were extensively propionylated with N-succinimidyl-3-propionate (NSP). The NSP-treated exotoxin retained its cytocidal ADP-ribosyltransferase activity, but it could no longer bind to, and inhibit the proliferation of, Friend murine erythroleukemia cells. Cytotoxicity (i.e., the ability to inhibit proliferation) for the Friend erythroid cells was restored completely to the NSP-inactivated exotoxin by conjugating it to ADIF, an autocrine factor secreted by chicken erythroleukemia cells which selectively inhibits the differentiation of erythroid cells such as Friend erythroleukemia cells without inhibiting their proliferation.
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PMID:The cytotoxicity of Pseudomonas exotoxin A, inactivated by modification of the cell-binding domain I, is restored when conjugated to an erythroid cell-specific targeting agent. 210 50

The ras-related rho A protein expressed in E. coli, was ADP-ribosylated by botulinum ADP-ribosyltransferase C3. C3 also modified the valine-14 mutant rho protein but not the products of H-ras, R-ras, ral, ypt, and rap 1 genes. A ras-rho chimaera consisting of 60 amino acids from the amino terminus of ras fused to 133 amino acids from the carboxy terminus of rho was not modified by C3. Antibodies raised against the porcine brain cytosolic substrate of C3 cross reacted with the rho, valine-14 rho and ras-rho proteins, but not with the gene products of H-ras, R-ras, ral or rap 1. Polyclonal anti-H-ras antibodies cross reacted with H-ras but not with ral, rho, or the C3 substrate purified from porcine brain.
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PMID:The rho gene product expressed in E. coli is a substrate of botulinum ADP-ribosyltransferase C3. 249 92

The coding region of diphtheria toxin gene from Corynebacterium diphtheriae PW8 was cloned in Escherichia coli on pUC19 plasmid. 5' end of the gene was deleted. The deleted gene codes for a toxin like protein truncated for 36 N-terminal aminoacids. The modified gene was fused with 5' terminal part of the lacZ gene on pUC19 plasmid having kept the translational frame intact. The hybrid gene transcription depends on lacP promoter, Mr of the resulting toxoid is 54,000. The toxoid is devoid of ADP-ribosyltransferase activity peculiar for diphtheria toxin and thus is not toxic. The constructed version of the toxin gene can be used for production of diphtheria anatoxin which does not require to be inactivated by formalin.
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PMID:[Cloning of the structural gene for diphtheria toxin from Corynebacterium diphtheriae PW8 and expression of its deletion derivative in E. coli]. 312 95

ABM508 is a recombinant fusion protein consisting of the N-terminal 485 amino acids of diphtheria toxin joined to alpha-melanocyte-stimulating hormone. When expressed in Escherichia coli under the control of the tox promoter and signal sequence, ABM508 is severely degraded. When overexpressed from a thermoinducible lambda pR promoter fusion, ABM508 is largely insoluble. We compared the expression of ABM508 (501 amino acids) to a full-length mutant form of the toxin (CRM197; 535 amino acids) and found that CRM197 showed minimal proteolysis. Thus, the removal of the C-terminal 50 amino acids of the toxin destabilizes the protein, making it a target for proteases. Proteolysis of ABM508 could be reduced by removal of the tox signal sequence (thereby directing the protein to the cytoplasm) and growth in lon and htpR mutant strains of E. coli. We also showed that the solubility of tox gene products expressed in E. coli was directly related to the growth temperature of the culture. Thus, a fragment A fusion protein (223 amino acids), ABM508, and CRM197 were found in soluble extracts when expressed at 30 degrees C but could not be released by the same procedures after growth at 42 degrees C. On the basis of these observations, we fused the coding sequences for mature ABM508 to the trc promoter (inducible at 30 degrees C by isopropyl-beta-D-thiogalactoside) and expressed this construct in a lon htpR strain of E. coli. This plasmid made 10 mg of soluble tox protein per liter of culture (7.7% of the total cell protein) or 14 times more than our previous maximal level. Extracts from lon htpR cells harboring this plasmid had high levels of ADP-ribosyltransferase activity, and although proteolysis still occurred, the major tox product corresponded to full-length ABM508.
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PMID:High-level expression of a proteolytically sensitive diphtheria toxin fragment in Escherichia coli. 331 66

An activator of rat brain phospholipase D (PLD) that is distinct from the already identified PLD activator, ADP-ribosylation factor (ARF), was partially purified from bovine brain cytosol by a series of chromatographic steps. The partially purified preparation contained a 22-kDa substrate for Clostridium botulinum C3 exoenzyme ADP-ribosyltransferase, which strongly reacted with anti-rhoA p21 antibody, but not with anti-rac1 p21 or anti-cdc42Hs p21 antibody. Treatment of the partially purified PLD-activating factor with both C3 exoenzyme and NAD significantly inhibited the PLD-stimulating activity. These results suggest that rhoA p21 is, at least in part, responsible for the PLD-stimulating activity in the preparation. Recombinant isoprenylated rhoA p21 expressed in and purified from Sf9 cells activated rat brain PLD in a concentration- and GTP gamma S (guanosine 5'-O-(3-thiotriphosphate))-dependent manner. In contrast, recombinant non-isoprenylated rhoA p21 (fused to glutathione S-transferase) expressed in Escherichia coli failed to activate the PLD. This difference cannot be explained by a lower affinity of non-isoprenylated rhoA p21 for GTP gamma S, as the rates of [35S]GTP gamma S binding were very similar for both recombinant preparations and the GTP gamma S-bound form of non-isoprenylated rhoA p21 did not induce PLD activation. Interestingly, recombinant isoprenylated rhoA p21 and ARF synergistically activated rat brain PLD; a similar pattern was seen with the partially purified PLD-activating factor. The synergistic activation was inhibited by C3 exoenzyme-catalyzed ADP-ribosylation of recombinant isoprenylated rhoA p21 in a NAD-dependent manner. Inhibition correlated with the extent of ADP-ribosylation. These findings suggest that rhoA p21 regulates rat brain PLD in concert with ARF, and that isoprenylation of rhoA p21 is essential for PLD regulation in vitro.
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PMID:Synergistic activation of rat brain phospholipase D by ADP-ribosylation factor and rhoA p21, and its inhibition by Clostridium botulinum C3 exoenzyme. 759 44

The edema factor (EF) and lethal factor (LF) components of anthrax toxin require anthrax protective antigen (PA) for binding and entry into mammalian cells. After internalization by receptor-mediated endocytosis, PA facilitates the translocation of EF and LF across the membrane of an acidic intracellular compartment. To characterize the translocation process, we generated chimeric proteins composed of the PA recognition domain of LF (LFN; residues 1-255) fused to either the amino-terminus or the carboxy-terminus of the catalytic chain of diphtheria toxin (DTA). The purified fusion proteins retained ADP-ribosyltransferase activity and reacted with antisera against LF and diphtheria toxin. Both fusion proteins strongly inhibited protein synthesis in CHO-K1 cells in the presence of PA, but not in its absence, and they showed similar levels of activity. This activity could be inhibited by adding LF or the LFN fragment (which blocked the interaction of the fusion proteins with PA), by adding inhibitors of endosome acidification known to block entry of EF and LF into cells, or by introducing mutations that attenuated the ADP-ribosylation activity of the DTA moiety. The results demonstrate that LFN fused to either the amino-terminus or the carboxy-terminus of a heterologous protein retains its ability to complement PA in mediating translocation of the protein to the cytoplasm. Besides its importance in understanding translocation, this finding provides the basis for constructing a translocation vector that mediates entry of a variety of heterologous proteins, which may require a free amino- or carboxy-terminus for biological activity, into the cytoplasm of mammalian cells.
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PMID:Protective antigen-binding domain of anthrax lethal factor mediates translocation of a heterologous protein fused to its amino- or carboxy-terminus. 778 38

We describe an efficient, general approach for cloning, expression and purification of heterologous proteins in Escherichia coli host strains. The affinity expression cassette polymerase chain reaction (AEC-PCR) allows the insertion of virtually any coding sequence in suitable expression vectors. For ease of purification of the (over)produced protein the gene expression cassettes are engineered by specifically designed oligonucleotide primers in the polymerase chain reaction (PCR) to contain either 3' or 5' additional nucleotides coding for a short amino acid sequence constituting an 'affinity block' fused to either end of the protein. This allows a one-step purification by affinity chromatography. In combination with PCR-mediated site-specific mutagenesis this approach is a highly efficient way for the expression and isolation of proteins and for the analysis and dissection of functional domains. The application of AEC-PCR is demonstrated by the cloning, production and purification of the native, active and the mutagenized, inactive ADP-ribosyltransferase (S1 subunit) of pertussis toxin. In this example, a string of six histidines has been engineered to either the N-terminal or the C-terminal end of the protein to serve as 'affinity block' for the isolation of the recombinant protein by immobilized metal ion affinity chromatography (IMAC). Thus, the S1 subunit can now be produced in sufficient quantities to facilitate further studies on its immunological and molecular properties.
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PMID:Efficient production of active and mutated ADP-ribosyltransferase (S1) of pertussis toxin using affinity expression cassette polymerase chain reaction. 800 55

We made use of ADP-ribosylarginine hydrolase to detect arginine-ADP- ribosylated proteins. The hydrolase was expressed in Escherichia coli as a protein fused with glutathione S-transferase (GST). The fusion protein GST-ADP-ribosylarginine hydrolase catalyzed the hydrolysis of alpha-ADP-ribosylarginine to produce ADP-ribose and arginine. Casein ADP-ribosylated with [32P]NAD and chicken heterophil arginine-specific ADP-ribosyltransferase served as a substrate for the recombinant ADP-ribosylarginine hydrolase and the released ADP-ribose was determined. Protein ADP-ribosylated by cholera toxin could serve as substrate of the hydrolase but protein ADP-ribosylated by pertussis toxin, diphtheria toxin, or C(3) enzyme of Clostridium botulinum could not. The hydrolase did not release the radioactivity incorporated into isolated rat liver nuclei incubated with [(32)P]NAD or in bovine brain cytosol incubated with [(32)P]ADP-ribose. In homogenate of mouse heart which contained arginine-specific ADP-ribosyltransferase, labeling of a 55-kDa protein by incubation with [(32)P]NAD was removed by ADP-ribosylarginine hydrolase treatment; hence, the specific hydrolysis of ADP-ribose-arginine bond by GST-ADP-ribosylarginine hydrolase can be used to detect the arginine-ADP-ribosylated proteins in crude preparations. Arginine--ADP-ribosylated proteins in crude preparations. Arginine-ADP-ribosylated proteins in mouse spleen lymphocytes were identified using this method.
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PMID:Detection of arginine-ADP-ribosylated protein using recombinant ADP-ribosylarginine hydrolase. 867 89


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