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 ADP-ribosyltransferase activity of pertussis toxin resides within the S-1 subunit of the toxin. Deletion mapping of a recombinant S-1 subunit produced in Escherichia coli showed that amino acids 2 through 180 are required for ADP-ribosylation of Gi protein. Mutants of the S-1 subunit which lacked either amino acids 2 through 22 or amino acids 153 through 180 failed to express enzyme activity, implicating a functional or structural role for these residues in catalysis. The catalytic carboxy-terminal S-1 deletion, C-180, was found to be more soluble than the recombinant S-1 subunit, making it a useful construct for future structure-function studies on enzyme catalysis. Four independent single-amino-acid substitutions which decreased ADP-ribosyltransferase activity were constructed in the recombinant S-1 subunit. Substitution of Asp-11 by Ser, Arg-13 by Leu, or Trp-26 by Ile decreased enzyme activity to below detectable levels (less than 1.0% of that of the recombinant S-1 subunit). The Glu-139-to-Ser substitution reduced ADP-ribosyltransferase activity to 15% of that of the recombinant S-1 subunit. Both the oxidized and reduced forms of the recombinant S-1 subunit and recombinant S-1 subunits containing single-amino-acid substitutions were degraded through identical immunoreactive tryptic peptides, suggesting that the conformations of the mutants are similar to that of the recombinant S-1 subunit. Identification of noncatalytic forms of the S-1 subunit of pertussis toxin which have conserved protein structure is an initial step in the generation of a recombinant noncatalytic form of pertussis toxin which may be tested as a candidate for an acellular vaccine against Bordetella pertussis.
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PMID:ADP-ribosyltransferase mutations in the catalytic S-1 subunit of pertussis toxin. 313 65

Cholera toxin (CTX) is composed of two subunits, subunit A, which possesses ADP-ribosyltransferase activity, and subunit B, which is responsible for receptor binding. It has previously been shown that agents that increase cyclic AMP (cAMP) levels in cells induce differentiation of PC12 cells into neurite-like cells. In this report, we show that as little as 100 pg of CTX per ml induces such changes. CTX was found to ADP-ribosylate at least four membrane proteins of PC12 cells in vitro and in vivo and to increase intracellular cAMP levels. We have developed an inducible ctx gene expression system in Vibrio cholerae by using the tac promoter. The culture medium of the CTX-producing bacteria was able to induce the morphological changes and the ADP-ribosylation of the PC12 cell membrane proteins. We have constructed two CTX-cross-reactive mutant proteins (CTX-CRM) by site-directed mutagenesis. The choice of glutamic acid 29 as the target amino acid was based on sequence similarities with other bacterial toxins. CTX-CRM-E29 delta, in which the Glu-29 of the A subunit was deleted, showed strongly reduced ADP-ribosyltransferase activity and did not induce significant morphological changes of PC12 cells. In contrast, CTX-CRM-E29D, in which the Glu-29 was replaced by an aspartic acid, was as active as the wild-type protein. We conclude that the ADP-ribosylation activity of CTX is important for the toxin-induced differentiation of PC12 cells. Pertussis toxin, which had no visible effect on PC12 cell morphology, was also able to ADP-ribosylate a membrane-bound protein(s) in vitro and in vivo. Pertussis toxin alone did not significantly increase cAMP levels in PC12 cells, but it acted synergistically with CTX.
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PMID:Importance of ADP-ribosylation in the morphological changes of PC12 cells induced by cholera toxin. 792 73

The carboxyl-terminal catalytic domain of the human poly(ADP-ribose) polymerase (PARP) exhibits sequence homology with the NAD(P)(+)-dependent leucine and glutamate dehydrogenases. To clarify the role played by some conserved residues between PARP and NAD(P)(+)-dependent dehydrogenases, point mutations were introduced into the whole enzyme context. Non-conservative mutations of Lys-893 (K893I) and Asp-993 (D993A) completely inactivate human PARP, whereas conservative and nonconservative mutations of Asp-914 (D914E and D914A, respectively) and Lys-953 (K953R and K953I, respectively) partially alter PARP activity. The consequences of conservative substitution of Lys-893 and Asp-993 on the kinetic properties of human poly(ADP-ribose) polymerase enzyme and the polymer it synthesizes suggest that these 2 amino acids are directly involved in the covalent attachment of the first ADP-ribosyl residue from NAD+ onto the acceptor amino acid. In addition, the recent resolution of the three-dimensional structure of the NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum (Baker, P.J., Britton, K.L., Engel, P.C., Farrants, G.W., Lilley, K.S., Rice, D.W., and Stillman, T.J. (1992) Proteins 12, 75-86) strongly supports our alignment with leucine and glutamate dehydrogenases and provides an interesting structural framework for the analysis of our results of site-directed mutagenesis.
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PMID:Identification of potential active-site residues in the human poly(ADP-ribose) polymerase. 847 97

Clostridium limosum ADP-ribosyltransferase modifies low molecular mass GTP-binding proteins of the Rho subtype family. Here we cloned and sequenced the gene of the transferase and expressed it in Escherichia coli. The gene encodes a protein of 250 amino acids (M(r) = 27,840), with a putative signal peptide of 45 amino acids, that shows about 60-65% identity with C3 transferases from Clostridium botulinum. The mature C. limosum transferase was expressed as a maltose-binding fusion protein in E. coli and purified to apparent homogeneity. To study the functional role of Glu174 of C. limosum transferase, which was recently photoaffinity-labeled with [carbonyl-14C]NAD [Jung, M., et al. (1993) J. Biol. Chem. 268, 23215-23218], two mutants E174D and E174Q were constructed by a polymerase chain reaction-based system. The E174D and E174Q mutants showed a dramatic decrease in kcat, but no major changes in Km,NAD. Furthermore, replacement of Glu174 by aspartic acid and glutamine largely reduced and completely blocked UV-induced incorporation of [carbonyl-14C]NAD into the transferase. The data indicate that Glu174 is an active site residue of C. limosum transferase.
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PMID:Active site mutation of the C3-like ADP-ribosyltransferase from Clostridium limosum--analysis of glutamic acid 174. 855 86

We have identified and characterized a novel cysteine protease named CMH-1 that is a new member of the interleukin 1 beta converting enzyme (ICE) family of proteases with substrate specificity for Asp-X. CMH-1 has the highest similarity to CPP32 (52% amino acid identity) and MCH2 (31% identical). CMH-1 shares conserved amino acid residues that form the core structure of ICE as well as those residues involved in catalysis and in the P1 aspartate binding. Overexpression of CMH-1 in COS cells resulted in the processing of CMH-1 and the induction of apoptosis of transfected cells. Coexpression of CMH-1 with poly(ADP-ribose) polymerase (PARP) also resulted in a specific cleavage of PARP. Purified recombinant CMH-1 cleaved PARP but not interleukin 1 beta precursor in vitro.
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PMID:Identification and characterization of CPP32/Mch2 homolog 1, a novel cysteine protease similar to CPP32. 856 22

The new and growing family of interleukin-1beta-converting enzyme (ICE) cysteine proteases are now recognised to be major effectors of cellular death by apoptosis. Like other members of this family, the CPP32/Yama proform is activated by processing to its active heterodimeric enzyme or apopain when it likely contributes to the process of apoptosis by cleaving poly(ADP-ribose) polymerase (PARP) and thereby inhibiting much of its DNA repair activity. Apoptosis plays a fundamental role in the regulation of the immune system where it is involved in the selection of both T and B lymphocytes bearing antigen receptor (AgR) for non-self. Cells of the Ramos Epstein-Barr virus (EBV)-genome-negative Burkitt lymphoma (BL) B cell line (Ramos-BL) can be triggered into growth arrest and apoptosis by treating with the calcium ionophore ionomycin or by crosslinking their surface AgR with antibodies directed against immunoglobulin (Ig)M (anti-IgM). Ionomycin- and AgR-triggered growth arrest and apoptosis are arrested by signals transduced through the surface CD40 of Ramos-BL B cells. Both ionomycin and anti-IgM trigger activation of CPP32 and cleavage of PARP prior to the onset of apoptosis; this process is abrogated by treatment with anti-CD40 and is independent of Bcl-2 expression. A tripeptide inhibitor of ICE family cysteine proteases, Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) inhibits ionomycin- and AgR-triggered CPP32 activation, PARP cleavage and apoptosis, but not growth arrest, in Ramos-BL B cells. Thus, in this report we demonstrate that in a physiological system, activation of endogenous members of the ICE family, including CPP32, and cleavage of the death substrate PARP act as major effectors of apoptotic death.
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PMID:Ligation of CD40 rescues Ramos-Burkitt lymphoma B cells from calcium ionophore- and antigen receptor-triggered apoptosis by inhibiting activation of the cysteine protease CPP32/Yama and cleavage of its substrate PARP. 864 64

A rat T-cell antigen RT6.1 catalyzes NAD glycohydrolysis but not ADP-ribose transfer, even though the antigen has significant amino acid identity with eucaryotic arginine-specific ADP-ribosyltransferases. Since a highly conserved Glu in the catalytic region of these transferases is substituted with Gln at position 207 in RT6.1, we replaced the Gln with Glu, Asp, or Ala, by site-directed mutagenesis. The Glu-207 mutant produced ADP-ribosylarginine during incubation with NAD and L-arginine. The Asp-207 mutant but not the Ala-207 mutant produced ADP-ribosylarginine, but at a lower rate. In contrast, these mutations affected NAD glycohydrolase activity of RT6.1 to a much lesser extent. Kinetic studies of transferase reaction revealed that kcat of the Glu-207 mutant increased compared to findings with the Asp-207 mutant. Moreover, the mouse homologue of rat RT6 lost arginine-specific ADP-ribosyltransferase activity when Glu-207 was replaced with Gln. Thus, Glu-207 in rodent T-cell RT6 antigens is essential for transfer reaction of ADP-ribose to arginine.
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PMID:Glutamic acid 207 in rodent T-cell RT6 antigens is essential for arginine-specific ADP-ribosylation. 893 82

ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins and members of the Ras superfamily, originally identified and purified by their ability to enhance the ADP-ribosyltransferase activity of cholera toxin and more recently recognized as critical participants in vesicular trafficking pathways and phospholipase D activation. ARD1 is a 64-kDa protein with an 18-kDa carboxyl-terminal ARF domain (p3) and a 46-kDa amino-terminal extension (p5) that is widely expressed in mammalian tissues. Using recombinant proteins, we showed that p5, the amino-terminal domain of ARD1, stimulates the GTPase activity of p3, the ARF domain, and appears to be the GTPase-activating protein (GAP) component of this bifunctional protein, whereas in other members of the Ras superfamily a separate GAP molecule interacts with the effector region of the GTP-binding protein. p5 stimulated the GTPase activity of p3 but not of ARF1, which differs from p3 in several amino acids in the effector domain. After substitution of 7 amino acids from p3 in the appropriate position in ARF1, the chimeric protein ARF1(39-45p3) bound to p5, which increased its GTPase activity. Specifically, after Gly40 and Thr45 in the putative effector domain of ARF1 were replaced with the equivalent Asp and Pro, respectively, from p3, functional interaction of the chimeric ARF1 with p5 was increased. Thus, Asp25 and Pro30 of the ARF domain (p3) of ARD1 are involved in its functional and physical interaction with the GTPase-activating (p5) domain of ARD1. After deletion of the amino-terminal 15 amino acids from ARF1(39-45p3), its interaction with p5 was essentially equivalent to that of p3, suggesting that the amino terminus of ARF1(39-45p3) may interfere with binding to p5. These results are consistent with the conclusion that the GAP domain of ARD1 interacts with the effector region of the ARF domain and thereby stimulates GTP hydrolysis.
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PMID:Interaction of the GTP-binding and GTPase-activating domains of ARD1 involves the effector region of the ADP-ribosylation factor domain. 902 91

Interleukin-1beta-converting enzyme (ICE) is a novel cysteine protease responsible for the cleavage of pre-interleukin-1beta (pre-IL-1beta) to the mature cytokine and a member of a family of related proteases (the caspases) that includes the Caenorhabditis elegans cell death gene product, CED-3. In addition to their sequence homology, these cysteine proteases display an unusual substrate specificity for peptidyl sequences with a P1 aspartate residue. We have examined the kinetics of processing pre-IL-1beta to the mature form by ICE and three of its homologs, TX, CPP-32, and CMH-1. Of the ICE homologs, only TX processes pre-IL-1beta, albeit with a catalytic efficiency 250-fold less than ICE itself. We also investigated the ability of these four proteases to process poly(ADP-ribose) polymerase, a DNA repair enzyme that is cleaved within minutes of the onset of apoptosis. Every caspase examined cleaves PARP, with catalytic efficiencies ranging from 2.3 x 10(6) M-1 s-1 for CPP32 to 1.0 x 10(3) M-1 s-1 for TX. In addition, we report kinetic constants for several reversible inhibitors and irreversible inactivators, which have been used to implicate one or more caspases in the apoptotic proteolysis cascade. Ac-Asp-Glu-Val-Asp aldehyde (DEVD-CHO) is a potent inhibitor of CPP-32 with a Ki value of 0.5 nM, but is also potent as inhibitor of CMH-1 (Ki = 35 nM) and ICE (Ki = 15 nM). The x-ray crystal structure of DEVD-CHO complexed to ICE presented here reveals electrostatic interactions not present in the Ac-YVAD-CHO co-complex structure (Wilson, K. P., Black, J.-A. F., Thomson, J. A., Kim, E. E., Griffith, J. P., Navia, M. A., Murcko, M. A., Chambers, S. P., Aldape, R. A., Raybuck, S. A., and Livingston, D. J. (1994) Nature 370, 270-275), accounting for the surprising potency of this inhibitor against ICE.
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PMID:Substrate and inhibitor specificity of interleukin-1 beta-converting enzyme and related caspases. 905 18

Identification of the processing/activation of multiple interleukin-1beta converting enzyme (ICE)-like proteases and their target substrates in the intact cell is critical to our understanding of the apoptotic process. In this study we demonstrate processing/activation of at least four ICE-like proteases during the execution phase of apoptosis in human monocytic tumor THP.1 cells. Apoptosis was accompanied by processing of Ich-1, CPP32, and Mch3alpha to their catalytically active subunits, and lysates from these cells displayed a proteolytic activity with kinetics, characteristic of CPP32/Mch3alpha but not of ICE. Fluorescence-activated cell sorting was used to obtain pure populations of normal and apoptotic cells. In apoptotic cells, extensive cleavage of Ich-1, CPP32, and Mch3alpha. was observed together with proteolysis of the ICE-like protease substrates, poly (ADP-ribose) polymerase (PARP), the 70-kD protein component of U1 small nuclear ribonucleoprotein (U1-70K), and lamins A/B. In contrast, no cleavage of CPP32, Mch3alpha or the substrates was observed in normal cells. In cells exposed to an apoptotic stimulus, some processing of Ich-1 was detected in morphologically normal cells, suggesting that cleavage of Ich-1 may occur early in the apoptotic process. The ICE-like protease inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK), inhibited apoptosis and cleavage of Ich-1, CPP32, Mch3alpha, Mch2alpha, PARP, U1-70K, and lamins. These results suggest that Z-VAD.FMK inhibits apoptosis by inhibiting a key effector protease upstream of Ich-1, CPP32, Mch3alpha, and Mch2alpha. Together these observations demonstrate that processing/activation of Ich-1, CPP32, Mch3alpha, and Mch2alpha accompanies the execution phase of apoptosis in THP.1 cells. This is the first demonstration of the activation of at least four ICE-like proteases in apoptotic cells, providing further evidence for a requirement for the activation of multiple ICE-like proteases during apoptosis.
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PMID:Processing/activation of at least four interleukin-1beta converting enzyme-like proteases occurs during the execution phase of apoptosis in human monocytic tumor cells. 912 56


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