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)

Choleragen exerts its effect on cells through activation of adenylate cyclase. Choleragen initially interacts with cells through binding of the B subunit of the toxin to the ganglioside GM1 on the cell surface. Subsequent events are less clear. Patching or capping of toxin on the cell surface may be an obligatory step in choleragen action. Studies in cell-free systems have demonstrated that activation of adenylate cyclase by choleragen requires NAD. In addition to NAD, requirements have been observed for ATP, GTP, and calcium-dependent regulatory protein. GTP also is required for the expression of choleragen-activated adenylate cyclase. In preparations from turkey erythrocytes, choleragen appears to inhibit an isoproterenol-stimulated GTPase. It has been postulated that by decreasing the activity of a specific GTPase, choleragen would stabilize a GTP-adenylate cyclase complex and maintain the cyclase in an activated state. Although the holotoxin is most effective in intact cells, with the A subunit having 1/20th of its activity and the B subunit (choleragenoid) being inactive, in cell-free systems the A subunit, specifically the A1 fragment, is required for adenylate cyclase activation. The B protomer is inactive. Choleragen, the A subunit, or A1 fragment under suitable conditions hydrolyzes NAD to ADP-ribose and nicotinamide (NAD glycohydrolase activity) and catalyzes the transfer of the ADP-ribose moiety of NAD to the guandino group of arginine (ADP-ribosyltransferase activity). The NAD glycohydrolase activity is similar to that exhibited by other NAD-dependent bacterial toxins (diphtheria toxin, Pseudomonas exotoxin A), which act by catalyzing the ADP-ribosylation of a specific acceptor protein. If the ADP-ribosylation of arginine is a model for the reaction catalyzed by choleragen in vivo, then arginine is presumably an analog of the amino acid which is ADP-ribosylated in the acceptor protein. It is postulated that choleragen exerts its effects on cells through the NAD-dependent ADP-ribosylation of an arginine or similar amino acid in either the cyclase itself or a regulatory protein of the cyclase system.
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PMID:Mechanism of action of choleragen. 21 41

Synthetic and natural amphiphiles, octyl glucoside, Nonidet P40, sodium dodecyl sulfate (SDS), gangliosides GM1 and GD1a, interact with cholera toxin (CLT) and with its active region (promoter A). The formation of CLT-amphiphile complex leads to inhibition of ADP-ribosyltransferase activity, a characteristic of promoter A elicited after thiol-reagents treatment. In all cases the interaction produces the maximum inhibitory effect above the critical micellar concentration of amphiphiles, although monomers of SDS show inhibition activity as well. The gangliosides appear to be capable of altering bilayer organization of membrane, similar to synthetic detergents. When CLT-ganglioside complexes were incubated with cell culture medium containing 10% fetal calf serum (FCS) and ADP-ribosyltransferase activity was completely restored both in cholera toxin and in promoter A. Some protein of FCS, which is avid of gangliosides, seems to be responsible for reversibility of inhibition. The results indicate that the active site of promoter A may be located in a hydrophobic pocket of the toxin structure. Furthermore, CLT was bound to reconstituted Sendai virus envelopes (RSVEs), containing a small amount of GM1. The RSVEs are made of membranous vesicles, capable of binding and fusing with host cell membrane. The incubation for 1 1hr of RSVE bearing CLT with Friend's erythroleukemic cells produced the stimulation of adenylate cyclase. This stimulation appears to be due to the translocation of the active subunit of CLT in the inner half of plasma membrane.
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PMID:Biological activity of preformed cholera toxin-ganglioside GM1 complex. 609 37

The catalytic A subunit of cholera toxin (CT-A) is capable of ADP-ribosylating the guanine nucleotide-binding protein, which regulates cell adenylyl cyclase, leading to the life-threatening diarrhea of cholera. Amino acids involved in the enzymatic activity of CT-A have previously been identified. By means of site-directed mutagenesis, an analog of the CT-A subunit gene was created with codon substitutions for both Arg-7 and Glu-112, each of which has been shown to produce subunits lacking ADP-ribosyltransferase activity. The mutated gene fragment was exchanged for the wild-type copy in the previously cloned ctxAB operon from El Tor biotype, Ogawa serotype Vibrio cholerae strain 3083, which produces CT-2. Further, the zonula occludens toxin gene, zot, was inactivated by an insertional mutation to create the new plasmid construct pCT-2*. Additionally, a DNA fragment encoding the B subunit of CT-1 (CT produced by classical biotype, Inaba serotype V. cholerae strain 569B) was exchanged for the homologous part in pCT-2*, resulting in the creation of pCT-1*. These plasmid constructs were introduced into the CT-negative V. cholerae mutant strain JBK70 (E1 Tor biotype, Inaba serotype); CT-A-B+ derivatives CVD101 and CVD103 of classical biotype Ogawa and Inaba serotype strains 395 and 569B, respectively; El Tor biotype Inaba and Ogawa serotype strains C6706 and C7258, respectively, recently isolated in Peru; and O139 (synonym Bengal) strain SG25-1 from the current epidemic in India. Recombinant toxins (CT-1* and CT-2*), partially purified from culture supernatants of transformed JBK70, were shown to be inactive on mouse Y1 adrenal tumor cells and in an in vitro ADP-ribosyltransferase assay. CT-1* and CT-2* reacted with polyclonal and monoclonal antibodies against both A and B subunits of CT. The toxin analogs reacted with antibodies against CT-A and CT-B on cellulose acetate strips and in a GM1 enzyme-linked immunosorbent assay; they reacted appropriately with B-subunit epitype-specific monoclonal antibodies in checkerboard immunoblots, and they formed precipitin bands with GM1-ganglioside in Ouchterlony tests. However, the reactions of the modified proteins with anti-A-subunit monoclonal antibodies were weaker than the reactions with wild-type holotoxins. V, cholerae strains carrying ctxA*, with either ctxB-1 or ctxB-2, and inactivated zot genes were created by homologous recombination. The recombinant strains and the purified toxin analogs were inactive in the infant rabbit animal model.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Construction and characterization of recombinant Vibrio cholerae strains producing inactive cholera toxin analogs. 803 72

Cholera toxin (CT) consists of a pentameric B subunit that binds to specific cell surface receptors identified as ganglioside GM1 and an A subunit that activates adenylylcyclase. The A subunit consists of A1 and A2 peptides linked by a disulfide bond; A2 acts to connect A to B, whereas A1 is an ADP-ribosyltransferase that modifies the alpha subunit of the stimulatory G protein (Gs). How the toxin is oriented when it binds to the cell surface and the related issue of the mechanism by which A1 gains access to Gs alpha are not known. In the present study, we used subunit-specific antibodies and their corresponding Fab fragments to assess their affects on holotoxin binding to target cells and their immunoreactivity to cell-bound toxin. Our results suggest that CT binds with A1 facing away from the membrane. Our hypothesis is further supported by the ability to assemble active CT on the cell surface of cultured human intestinal and neurotumor cells by the sequential addition of purified B and A subunits. We also observed that when cells containing bound CT were incubated at 37 degrees C, both subunits rapidly became inaccessible to their respective antibodies. We propose that the holotoxin binds with its A subunit facing away from the membrane and must enter the cell in order for A1 to be released, gain access to Gs alpha, and activate adenylylcyclase.
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PMID:Orientation of cholera toxin bound to target cells. 834 92

Since it has been reported that a single amino acid mutation of Gly-->Arg in the CAGYC region of the beta chain of human thyroid stimulating hormone (hTSH) was responsible for congenital isolated TSH deficiency, and that the same amino acid substitution in this site of hTSH and human chorionic gonadotropin (hCG) introduced by site-directed mutagenesis resulted in loss of activity, the authors studied the role of glutamic acid at position 11 (Glu-11) from the N-terminus of the B subunit of cholera toxin (CT), which corresponds to the glycine in the CAGYC region of the beta chain of hTSH and hCG. A mutant CT constructed by site-directed mutagenesis in which Glu-11 was replaced by Arg (CT-E11R) did not induce either morphological changes or accumulation of cytosolic cyclic AMP in Chinese hamster ovary cells, although it formed the holotoxin AB5, retained the ability to bind to GM1-ganglioside and showed ADP-ribosyltransferase activity. Weak assembly of the B subunits in mutant CT-E11R demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-heating conditions might explain the loss of biological activity.
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PMID:Loss of biological activity due to Glu-->Arg mutation at residue 11 of the B subunit of cholera toxin. 940 7

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

A promising novel concept in mucosal adjuvant research is demonstrated here. The adjuvant and toxic effects of the cholera toxin (CT) have been successfully separated in a gene fusion protein, CTA1-DD. This protein consists of the ADP-ribosylating A1 subunit of CT linked to a synthetic analogue of protein A. The CTA1-DD protein was found to exert comparable adjuvant activity to that of CT after systemic as well as mucosal immunizations with soluble protein antigens, such as KLH or ovalbumin (OVA). However, contrary to CT it was completely non-toxic. The CTA1-DD approach to the construction of a potential vaccine adjuvant is unique and highly promising. Conceptually, the CTA1-DD fusion protein demonstrates that: (i) contrary to CT the CTA1-DD is a highly targeted adjuvant, directed to B cells and possibly other antigen-presenting cells; (ii) it is possible to introduce ADP-ribosyltransferase activity into cells via an alternative pathway to the GM1 receptor pathway used by CTB; (iii) the adjuvant effect of CTA1-DD, and possibly also of CT, depend on the enzymatic activity; and (iv) one possible mechanism, shared by CT, that may explain the adjuvant effect of CTA1-DD is its ability to induce expression of the costimulatory molecule CD86 on B cells.
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PMID:A novel concept in mucosal adjuvanticity: the CTA1-DD adjuvant is a B cell-targeted fusion protein that incorporates the enzymatically active cholera toxin A1 subunit. 968 72

Oxidative injury is believed to be a major factor in the pathogenesis of a variety of neurodegenerative diseases. Additionally, the mode of cell death in oxidant-stressed cells can vary. The present study was conducted to evaluate the use of a primary neuronal cell-based bioassay in which different modes of oxidant-induced cell death could be studied and in which putative neuroprotective agents could be screened. Addition of 50 microM H(2)O(2) to primary cortical neuronal cultures for 1 h under normal ATP conditions resulted in approximately 40% cell death, almost exclusively of an apoptotic nature. In this condition, cell death was effectively blocked by GM1 ganglioside, the semi-synthetic ganglioside derivative LIGA20, the dopamine receptor agonist pramipexole (PPX) and the caspase inhibitor Z-VAD-FMK but not by the poly (ADP-ribose) polymerase (PARP) inhibitor 3-aminobenzamide (3-AB). Pretreatment of cells with 0.01 microM oligomycin for 45 min prior to addition of 50 microM H(2)O(2) caused significant ATP depletion and approximately the same amount of cell death as H(2)O(2) alone. However, under these conditions, cell death was primarily non-apoptotic in nature and GM1, LIGA20 and Z-VAD-FMK had no protective effects. In contrast, AB and PPX effectively blocked cell death. These results suggest that cellular ATP plays a critical role in determining the mode of cell death in primary neurons and that these types of in vitro models may provide a useful system for screening putative neuroprotective agents.
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PMID:Modulation of ATP levels alters the mode of hydrogen peroxide-induced cell death in primary cortical cultures: effects of putative neuroprotective agents. 1471 52

We provide compelling evidence that delivery of Ag in the absence of ADP-ribosylation can promote tolerance, whereas ADP-ribosyltransferase activity induces IgA immunity and prevents tolerance. By linking Ag to the ADP-ribosylating enzyme, cholera toxin subunit A1 (CTA1), we could show that the combination of targeting to antigen-presenting cells (APC) and enzymatic activity is a highly effective means of controlling the induction of tolerance or immunity. Firstly, we demonstrated that cholera toxin (CT), although potentially binding to all nucleated cells, in fact, bound preferentially to dendritic cells (DC) in vivo. Following injection of CT-conjugated Ag, we found that DC in the marginal zone (MZ) of the spleen accumulated Ag, a process that was GM1-ganglioside receptor dependent. Contrary to CTB, which also delivered Ag to the MZ DC, CT matured and activated co-stimulatory functions in the targeted DC and greatly augmented immune responses to Ag. Secondly, when Ag was incorporated into the CTA1-DD fusion protein, which equals the CT in adjuvant function but lacks GM1-ganglioside-binding ability, we greatly augmented specific responses to Ag. The DD-bound Ag was distinctly targeted to B cells and probably also to follicular dendritic cells (FDC) in vivo. Thus, in both constructs Ag was targeted to APC and associated with an ADP-ribosylating enzyme, which resulted in greatly enhanced immunogenicity. When the enzymatic activity was absent, as in CT B-subunit (CTB) or in the inactive CTA1R7K-DD mutant, Ag largely failed to stimulate an active immune response. Rather, this type of Ag exposure resulted in Ag-specific tolerance, especially when mucosal delivery of Ag was attempted. Therefore, targeting to APC in the absence or presence of the CTA1-enzyme appears to be an effective means to control tolerance and active protective IgA immunity.
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PMID:From toxin to adjuvant: basic mechanisms for the control of mucosal IgA immunity and tolerance. 1575 58

Plant polyphenols, RG-tannin, and applephenon had been reported to inhibit cholera toxin (CT) ADP-ribosyltransferase activity and CT-induced fluid accumulation in mouse ileal loops. A high molecular weight fraction of hop bract extract (HBT) also inhibited CT ADP-ribosyltransferase activity. We report here the effect of those polyphenols on the binding and entry of CT into Vero cells. Binding of CT to Vero cells or to ganglioside GM1, a CT receptor, was inhibited in a concentration-dependent manner by HBT and applephenon but not RG-tannin. These observations were confirmed by fluorescence microscopy using Cy3-labeled CT. Following toxin binding to cells, applephenon, HBT, and RG-tannin suppressed its internalization. HBT or applephenon precipitated CT, CTA, and CTB from solution, creating aggregates larger than 250 kDa. In contrast, RG-tannin precipitated CT poorly; it formed complexes with CT, CTA, or CTB, which were demonstrated with sucrose density gradient centrifugation and molecular weight exclusion filters. In agreement, CTA blocked the inhibition of CT internalization by RG-tannin. These data suggest that some plant polyphenols, similar to applephenon and HBT, bind CT, forming large aggregates in solution or, perhaps, on the cell surface and thereby suppress CT binding and internalization. In contrast, RG-tannin binding to CT did not interfere with its binding to Vero cells or GM1, but it did inhibit internalization.
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PMID:Differential activities of plant polyphenols on the binding and internalization of cholera toxin in vero cells. 1581 10


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