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 biochemical and biologic properties of a purified disulfide conjugate of diphtheria toxin fragment A and human placental lactogen (toxin A-hPL) have been studied by (a) assaying the ADP-ribosyltransferase activity of the intact conjugate, (b) assaying the binding of the intact conjugate to mammary gland plasma membrane lactogenic receptors, and (c) assaying the effect of the conjugate on the rate of protein synthesis in rabbit mammary gland explants maintained in organ culture. The toxin A-hPL conjugate retains one-third of the NAD+:EF-2 ADP-ribosyltransferase activity of toxin A, and 26% of the hPL-binding activity to lactogenic receptors. Binding activity was demonstrated by radioreceptor assay and by assaying toxin A activity bound to membranes which was competitively displaced by excess hPL. Since the toxin A-hPL conjugate retained activities of its separate subunits, it could be regarded as a structural analogue of nicked diphtheria toxin with replacement of the original membrane-binding chain by another binding chain that is specific for lactogenic receptor. However, the conjugate failed to inhibit protein synthesis in organ-cultured mammary gland explants, although these were sensitive to native diphtheria toxin and could bind hPL. It is concluded from these results that the toxin A-hPL conjugate does not act as a functional analogue of diphtheria toxin with altered receptor specificity, and that the hPL receptor cannot mediate the entry of toxin A or toxin A-hPL from membrane-bound conjugate into the cytosol site of action of toxin A.
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PMID:Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. II. Biochemical and biologic properties of diphtheria toxin fragment A-S-S-human placental lactogen. 19 Feb 37

Antibodies highly selective for two functionally distinct regions of diphtheria toxin (DTx) were prepared using synthetic peptide conjugates as immunogens. Three peptides were selected for synthesis: sequence DTx141-157 on fragment A, which contains the putative protein elongation factor (EF-2) ADP-ribosyltransferase site; DTx224-237 on fragment B, selected on the basis of forming a predicted surface loop; and DTx513-526 on fragment B, forming a part of the region containing the putative receptor binding domain. All of the anti-peptide antibodies recognized the corresponding peptide, and also reacted with the toxin, specifically with the fragment containing the sequence against which they were raised, confirming the utility of this approach in generating fragment-specific antibodies. The anti-peptide antibody with the highest binding titre both to the peptide and to the native toxin was the one prepared against the sequence with the highest surface and loop likelihood indices of the three peptides selected. The similarity of the reactivity profiles with peptide and native and denatured toxin is consistent with the prediction that the region selected occurs in a surface loop and that the structure of the peptide is similar to the conformation of this region in the native protein. The epitopes for two of the anti-peptide antibodies were mapped. The results indicated that even though the antisera were raised to peptides containing 14 amino acids (aa) they were directed predominantly against a narrow region within the peptide, consisting of only 5-6 aa residues.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Targeting of specific domains of diphtheria toxin by site-directed antibodies. 128 34

Glutamic acid-148, an active-site residue of diphtheria toxin identified by photoaffinity labeling with NAD, was replaced with aspartic acid, glutamine, or serine by directed mutagenesis of the F2 fragment of the toxin gene. Wild-type and mutant F2 proteins were synthesized in Escherichia coli, and the corresponding enzymic fragment A moieties (DTA) were derived, purified, and characterized. The Glu----Asp (E148D), Glu----Gln (E148Q), and Glu----Ser (E148S) mutations caused reductions in NAD:EF-2 ADP-ribosyltransferase activity of ca. 100-, 250-, and 300-fold, respectively, while causing only minimal changes in substrate affinity. The effects of the mutations on NAD-glycohydrolase activity were considerably different; only a 10-fold reduction in activity was observed for E148S, and the E148D and E148Q mutants actually exhibited a small but reproducible increase in NAD-glycohydrolytic activity. Photolabeling by nicotinamide-radiolabeled NAD was diminished ca. 8-fold in the E148D mutant and was undetectable in the other mutants. The results confirm that Glu-148 plays a crucial role in the ADP-ribosylation of EF-2 and imply an important function for the side-chain carboxyl group in catalysis. The carboxyl group is also important for photochemical labeling by NAD but not for NAD-glycohydrolase activity. The pH dependence of the catalytic parameters for the ADP-ribosyltransferase reaction revealed a group in DTA-wt that titrates with an apparent pKa of 6.2-6.3 and is in the protonated state in the rate-determining step.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine. 198 Feb 8

The amino-acid sequences of two diphtheria toxin-related, non-toxic proteins, CRM45 and CRM197 , were deduced from the complete sequence of their genes: tox 45 and tox 197. CRM45 lacks the last 149 C-terminal amino-acid residues, but is otherwise identical to diphtheria toxin: a single C----T transition introduces an "ochre" (TAA) termination signal in tox 45, after the codon for threonine-386. A single G----A transition was also found in tox 197, leading to the substitution of glycine-52, present in the wild-type toxin, with glutamic acid in CRM197 . This aminoacid change is responsible for the loss of the NAD:EF2 ADP-ribosyltransferase activity in CRM197 , due most probably to an alteration of the NAD+ binding site.
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PMID:The amino-acid sequence of two non-toxic mutants of diphtheria toxin: CRM45 and CRM197. 642 53

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

Biotin- or digoxigenin-conjugated NAD has been used successfully to label EF-2 by diphtheria toxin, an alpha subunit of G protein by pertussis toxin, and poly(ADP-ribose) synthase through auto-poly(ADP-ribosyl)ation (J. Zhang, unpublished result, 1996). It is likely that many other ADP-ribosyl-transferases are capable of using modified NAD as substrates. Compared to radioactive labeling, biotinylation has several advantages. Commercially available precursors make synthesis of biotinylated NAD simple and economic. No extensive purification of the product is required. Because biotinylated NAD can be separated from NAD readily, there is no dilution, in contrast to [32P]NAD, in which only a small proportion of the NAD molecules are radioactive. Once purified, biotinylated NAD can be stored for a long time without decay (unlike radioactive NAD, which does decay). Most importantly, the system described here may afford an efficient means for purifying and identifying ADP-ribosylated proteins. Biotinylated NAD can be used for in situ labeling to study the cellular localization and tissue distribution of the ADP-ribosylated proteins.
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PMID:Use of biotinylated NAD to label and purify ADP-ribosylated proteins. 921 21

The intersection of small molecular weight drugs and antibody-based therapeutics is rarely studied in large scale. Both types of agents are currently part of the cancer armamentarium. However, very little is known about how to combine them in optimal ways. Immunotoxins are antibody-toxin gene fusion proteins engineered to target cancer cells via antibody binding to surface antigens. For fusion proteins derived from Pseudomonas exotoxin (PE), potency relies on the enzymatic domain of the toxin which catalyzes the ADP-ribosylation of EF2 causing inhibition of protein synthesis leading to cell death. Candidate immunotoxins have demonstrated clear value in clinical trials but generally have not been curative as single agents. Therefore we undertook three screens to discover effective combinations that could act synergistically. From the MIPE-3 library of compounds we identified various enhancers of immunotoxin action and at least one major class of inhibitor. Follow-up experiments confirmed the screening data and suggested that immunotoxins when administered with everolimus or nilotinib exhibit favorable combinatory activity and would be candidates for preclinical development. Mechanistic studies revealed that everolimus-immunotoxin combinations acted synergistically on elements of the protein synthetic machinery, including S61 kinase and 4E-BP1 of the mTORC1 pathway. Conversely, PARP inhibitors antagonized immunotoxins and also blocked the toxicity due to native ADP-ribosylating toxins. Thus, our goal of investigating a chemical library was justified based on the identification of several approved compounds that could be developed preclinically as 'enhancers' and at least one class of mitigator to be avoided.
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PMID:Chemical Screens Identify Drugs that Enhance or Mitigate Cellular Responses to Antibody-Toxin Fusion Proteins. 2755 70