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)

ADP-ribosylation of regulatory proteins is an important pathological mechanism by which various bacterial toxins affect eukaryotic cell functions. While diphtheria toxin catalyses the ADP-ribosylation of elongation factor 2, which results in inhibition of protein synthesis, cholera toxin and pertussis toxin ADP-ribosylate Ns and Ni, respectively, the GTP-binding regulatory components of the adenylate cyclase system, thereby modulating the bidirectional hormonal regulation of the adenylate cyclase. Botulinum C2 toxin is another toxin which has been reported to possess ADP-ribosyltransferase activity. This extremely toxic agent is produced by certain strains of Clostridium botulinum and induces hypotension, an increase in intestinal secretion, vascular permeability and haemorrhaging in the lungs. In contrast to botulinum neurotoxins, the botulinum C2 toxin apparently lacks any neurotoxic effects. Here we report that botulinum C2 toxin ADP-ribosylates a protein of relative molecular mass 43,000 (43K) in intact cells and in cell-free preparations. We present evidence that the 43K protein substrate is actin, which is apparently mono-ADP-ribosylated by the toxin. Botulinum C2 toxin also ADP-ribosylated purified liver G-actin, whereas liver F-actin was only poorly ADP-ribosylated and skeletal muscle actin was not ADP-ribosylated in either its G form or its F form. ADP-ribosylation of liver G-actin by botulinum C2 toxin resulted in a drastic reduction in viscosity of actin polymerized in vitro.
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PMID:Botulinum C2 toxin ADP-ribosylates actin. 373 64

An ADP-ribosyltransferase was found in elongation factor 2 (EF-2) preparations from polyoma virus-transformed baby hamster kidney (pyBHK) cells. Like fragment A of diphtheria toxin and Pseudomonas toxin A, this eukaryotic cellular enzyme transfers [14C]adenosine from NAD+ to EF-2. However, the cellular transferase is immunologically distinct from fragment A. The transferase also can be distinguished from fragment A and Pseudomonas toxin A by the inhibition of the activity of the former by cytoplasmic extracts and by histamine. Snake venom phosphodiesterase digestion of the [14C]adenosine-labeled EF-2 product of the cellular transferase reaction yielded [14C]AMP, indicating that the cellular enzyme is a mono(ADP-ribosyl)transferase. The forward ADP-ribosylation reaction catalyzed by the cellular enzyme is reversed by fragment A, yielding [14C]NAD+. The results strongly suggest that the cellular transferase is a mono(ADP-ribosyl)transferase, which ADP-ribosylates the same diphthamide residue of EF-2 as does fragment A and Pseudomonas toxin A.
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PMID:Cellular ADP-ribosyltransferase with the same mechanism of action as diphtheria toxin and Pseudomonas toxin A. 632 38

A cellular ADP-ribosyltransferase activity has been found in a variety of animals and tissues. The enzyme transfers ADP-ribose from NAD to elongation factor 2, inactivating the factor and thus inhibiting in vitro protein synthesis. Although, the mechanism of action of the cellular enzyme appears similar to diphtheria toxin and Pseudomonas exotoxin A, it differs from the toxins in that only a fraction of the EF-2 pool is modified. The endogenously ADP-ribosylated EF-2 has been detected by a variety of methods including two-dimensional electrophoresis and immunoprecipitation with elongation factor 2 antibody. The nature of the cellular ADP-ribosyltransferase and its physiological significance are unknown.
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PMID:Cellular ADP-ribosylation of elongation factor 2. 789 55

Two rabbit polyclonal antisera have been produced by immunization with two fragments corresponding to sequences 392 to 404 and 392 to 613 of Pseudomonas aeruginosa exotoxin A. Both antisera inhibit the ADP-ribosyltransferase activity of exotoxin A but do not inhibit its NAD-glycohydrolase activity. In addition, only the second antiserum was capable of neutralizing exotoxin A cytotoxicity in cell culture and in vivo. Consequently, the common sequence 392 to 404 of the two fragments is not a neutralizing epitope and such an epitope should reside within residues 405 to 613 of exotoxin A. The sequence 392 to 404 was shown to be hidden in the native molecule, and the results suggest that this sequence is most likely in close proximity to residues involved in eukaryotic elongation factor 2 binding.
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PMID:Comparative immunochemistry of two fragments from domains Ib and III of Pseudomonas aeruginosa exotoxin A. 822 17

Diphtheria toxin (DT) and Pseudomonas aeruginosa exotoxin A have the same molecular mechanism of toxicity; both toxins ADP-ribosylate a modified histidine residue in elongation factor 2. To help identify amino acids involved in this reaction, sequences in DT that share homology with P. aeruginosa exotoxin A were synthesized and examined for a role in the ADP-ribosyltransferase reaction. By using this approach, residues 32 to 54 of DT were found to define an epitope associated with antibody-mediated inhibition of DT enzyme activity. This lends further support to the notion that residues in this region of DT are involved in the enzymatic reaction.
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PMID:Use of synthetic peptides and site-specific antibodies to localize a diphtheria toxin sequence associated with ADP-ribosyltransferase activity. 842 59

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

Burn patients suffer a break in the physical barrier (skin), which, when combined with their generalized state of immunodeficiency, creates an open window for opportunistic infections, mainly with Pseudomonas aeruginosa. Infection of the burn wound has always been a major factor in retardation of wound healing, and sepsis remains the leading cause of death in burn patients. Because studies have shown that topical treatment with antiexotoxin A (ETA) antibodies significantly increases survival in rats infected with toxin-producing strains of P. aeruginosa, we examined 11 synthetic peptides encompassing 12 to 45 amino acid (aa) residues, representing what were predicted by computer analysis to be the most hydrophilic and antigenic regions of ETA. These synthetic peptides were injected into rabbits for antibody production. Different groups of rabbits were immunized with a combination of peptides, with each combination representing one of the three distinct domains of ETA. Animals immunized with various peptide combinations produced peptide-specific antibodies that exhibited cross-reactivity to ETA. Two major epitopes were identified on the ETA molecule by experiments with peptide-specific antibodies in enzyme-linked immunosorbent assay and immunoprecipitation. One of these epitopes was located in the translocation domain (II) (aa 297 to 310), while the other was mapped to the last 13 aa residues at the carboxy-terminal end of the enzymatic domain (III) (aa 626 to 638). Of these two regions, the epitope in the enzymatic domain induced a much higher level of neutralizing antibodies that abrogated the cytotoxic activity of ETA in vitro. Antibodies to this epitope blocked the ADP-ribosyltransferase activity of ETA and appeared to interfere with binding of the substrate elongation factor 2 to the enzymatic active site of the ETA molecule. We conclude that polyclonal, as well as monoclonal, antibodies to short peptides, representing small regions of ETA, may have therapeutic potential in passive immunization or topical treatment of burn patients infected with toxin-producing strains of P. aeruginosa.
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PMID:Generation of neutralizing antipeptide antibodies to the enzymatic domain of Pseudomonas aeruginosa exotoxin A. 957 4

CRM45 is a mutant form of diphtheria toxin (DTx) that lacks a 17-kDa carboxyl-terminal segment of the receptor-binding B subunit (DTB). The missing segment is a discrete structural domain of DTB that normally rests against the NAD binding pocket of the enzymically-active A subunit (DTA). Proteolytic cleavage and disulfide bridge reduction in the DTA-DTB linker region of DTx are required for optimal ADP-ribosylation of elongation factor 2 (EF-2). Here, we show that cleaved and uncleaved preparations of X-ray crystal grade CRM45 both exhibit an ADP-ribosyltransferase activity similar to that of cleaved DTx. Crystal-grade preparations of CRM45 also display a potent deoxyribonuclease activity. However, as observed with DTx, cleavage and reduction of CRM45 are not required for expression of this nuclease activity. After SDS-PAGE in a gel that contains DNA embedded in the matrix, renaturable Ca++/Mg(++)-dependent nuclease-active bands co-migrate with intact CRM45 (45 kDa) as well as with the DTA subunit (24 kDa) of CRM45. Because the 45-kDa nuclease-active band is unique to the CRM45 form of DTx, it offers direct proof that this activity is intrinsic to the DTA domain of DTx and its homologues.
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PMID:Characterization of the deoxyribonuclease and ADP-ribosyltransferase activities of CRM45, a truncated homologue of diphtheria toxin. 978 63

Immunotoxins composed of antibodies linked to plant or bacterial toxins are being evaluated in the treatment of cancer. It is known that the toxin moieties of immunotoxins, including Pseudomonasexotoxin A (PE), diphtheria toxin, and ricin, are capable of inducing apoptosis. Since the efficiency of induction of apoptosis and the apoptosis pathway may have direct effects on the therapeutic usefulness of immunotoxins, we have studied how B3(Fv)-PE38, a genetically engineered immunotoxin in which the Fv fragment of an antibody is fused to a mutated form of PE, induces apoptosis of the MCF-7 breast cancer cell line. We show for the first time that a PE-containing immunotoxin activates ICE/ced-3 proteases, now termed caspases, and causes characteristic cleavage of the "death substrate" poly(ADP)-ribose polymerase (PARP) to an 89 kDa fragment with a time course of cleavage comparable to that induced by TNFalpha. Also the fluorescent substrate, DEVD-AFC, is cleaved 2-4-fold more rapidly by lysates from B3(Fv)-PE38 treated MCF-7 cells than untreated control cells, suggesting that a CPP32-like caspase is involved in B3(Fv)-PE38-mediated apoptosis. B3(Fv)-PE38-induced PARP cleavage is inhibited by several protease inhibitors known to inhibit caspases (zVAD-fmk, zDEVD-fmk, zIETD-fmk) as well as by overexpression of Bcl-2 providing additional evidence for caspase involvement. zVAD-fmk, a broad spectrum inhibitor of most mammalian caspases, prevents the early morphological changes and loss of cell membrane integrity produced by B3(Fv)-PE38, but not its ability to inhibit protein synthesis, arrest cell growth, and subsequently kill cells. Despite inhibition of apoptosis, the immunotoxin is still capable of selective cell killing, which indicates that B3(Fv)-PE38 kills cells by two mechanisms: one requires caspase activation, and the other is due to the arrest of protein synthesis caused by inactivation of elongation factor 2. The fact that an immunotoxin can specifically kill tumor cells without the need of inducing apoptosis makes such agents especially valuable for the treatment of cancers that are protected against apoptosis, e.g., by overexpression of Bcl-2.
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PMID:Role of caspases in immunotoxin-induced apoptosis of cancer cells. 983 86

Here we examined the intrinsic nuclease activity of diphtheria toxin (DTx) to determine the mechanism by which it catalyzes DNA degradation. Results show that DTx degrades double-stranded DNA (dsDNA) by non-processive, endonucleolytic attack, without apparent specificity for nucleotide sequence. Moreover, divalent cation composition determines whether supercoiled dsDNA is cleaved by the introduction of single-strand nicks or double-strand breaks. Circular single-stranded DNA (ssDNA) is also a substrate for endonucleolytic attack. Pre-incubation of DTx with a 2000-fold excess of NAD, the natural substrate for the toxin's ADP-ribosyltransferase (ADPrT) activity, inhibited the transfer of radiolabeled ADP-ribose to elongation factor 2 but had no effect on the degradation of radiolabeled DNA. Based on this result and the fact that compounds known to inhibit the ADPrT activity of DTx had no effect on its nuclease activity and pre-incubation of DTx with DNA had no effect on ADPrT activity, we conclude that the ADPrT and nuclease active sites of DTx are functionally and spatially distinct. Moreover, studies with an ADPrT-inactivated form of DTx indicate that nuclease activity alone can lead to target cell lysis.
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PMID:Mechanistic aspects of the deoxyribonuclease activity of diphtheria toxin. 1568 Feb 46


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