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:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The basis of the high potency of salmon calcitonin (sCT) in radioligand binding competition and cAMP accumulation studies with cloned calcitonin (CT) receptors from rats, pigs, and humans was examined using two sets of CT analogues, i.e., chimeric sCT/human CT (hCT) analogues and analogues of sCT with differing capacities to form an amphipathic alpha-helix. In competition for 125I-sCT binding the following relative specificities were observed for the chimeric peptides: rat C1a CT receptor, sCT > or = (1-16)hCT/(17-32)sCT (ACT-15) > (1-16)sCT/(17-32)hCT (ACT-27); rat C1b CT receptor, sCT >> ACT-15 > ACT-27; hCT receptor, sCT = ACT-15 > ACT-27; porcine CT receptor, sCT > ACT-27 > ACT-15. In contrast, in ligand-induced cAMP accumulation studies the relative efficacies were as follows: rat C1a CT receptor, sCT = ACT-15 > ACT-27; rat C1b CT receptor, sCT = ACT-15 > ACT-27; hCT receptor, sCT = ACT-15 > or = to ACT-27; porcine CT receptor, sCT = ACT-15 = ACT-27. The data demonstrate that residues present in the carboxyl-terminal half of sCT are more important for binding competition with the rat C1a, rat C1b, and human CT receptors, whereas residues in the amino-terminal half of sCT are more important for binding competition with the porcine CT receptor. Carboxyl-terminal sCT residues are also important for full potency in adenylate cyclase activation with the rat C1a and rat C1b CT receptors but are less important for activation via the hCT receptor. The disparity in the relative potencies of the peptides in studies of binding competition and cAMP accumulation is suggestive of significant differences in the relative affinities of the peptides for active and inactive conformations of the CT receptor. The use of sCT analogues with varying capacities to form alpha-helices also revealed divergence in the responses of different receptors. This was most apparent for the stimulation of cAMP production by the rat receptor isoforms C1a and C1b. In cells expressing the C1a receptor, the helical analogues sCT and des-Ser2-sCT were equipotent with [Gly8]-des-Leu19-sCT and des-1-amino-[Ala1,7,Gly8]-des-Leu19 sCT, analogues that have reduced or absent helical structure, respectively. In contrast, the nonhelical analogues were 100-1000-fold less potent than sCT and des-Ser2-sCT at the C1b receptor. In general, reduction in the ability of sCT analogues to form helix structures had a greater impact on the potency of the analogues in competition for 125I-sCT binding than in cAMP accumulation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Divergent structural requirements exist for calcitonin receptor binding specificity and adenylate cyclase activation. 772 41

Bordetella pertussis adenylate cyclase (AC) toxin-hemolysin (ACT-Hly) can penetrate a variety of eukaryotic cells. Recombinant AC toxoids have therefore been recently used for delivery of CD8(+) T-cell epitopes into antigen-presenting cells in vivo and for induction of protective antiviral, as well as therapeutic antitumor cytotoxic T-cell responses. We have explored the carrier potential of the ACT molecule by insertional mutagenesis scanning for new permissive sites, at which integration of two- to nine-residue-long peptides does not interfere with membrane interaction and translocation of ACT. A model CD8(+) T-cell epitope of ovalbumin was incorporated at 10 of these permissive sites along the toxin molecule, and the capacity of ACT constructs to penetrate into cell cytosol and deliver the epitope into the major histocompatibility complex (MHC) class I antigen processing and presentation pathway was examined. While all six constructs bearing the epitope within the Hly portion of ACT failed to deliver the epitope to the MHC class I molecules, all four toxoids with inserts within different permissive sites in the AC domain efficiently delivered the epitope into this cytosolic pathway, giving rise to stimulation of a specific CD8(+) T-cell hybridoma. The results suggest that, in contrast to the AC domain, the hemolysin moiety of ACT does not reach the cytosolic entry of the MHC class I pathway.
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PMID:Delivery of CD8(+) T-cell epitopes into major histocompatibility complex class I antigen presentation pathway by Bordetella pertussis adenylate cyclase: delineation of cell invasive structures and permissive insertion sites. 1060 95

The Bordetella pertussis adenylate cyclase toxin-hemolysin (ACT or CyaA) is a multifunctional protein. It forms small cation-selective channels in target cell and lipid bilayer membranes and it delivers into cell cytosol the amino-terminal adenylate cyclase (AC) domain, which catalyzes uncontrolled conversion of ATP to cAMP and causes cell intoxication. Here, we demonstrate that membrane translocation of the AC domain into cells is selectively dissociated from ACT membrane insertion and channel formation when a helix-breaking proline residue is substituted for glutamate 509 (Glu-509) within a predicted transmembrane amphipathic alpha-helix. Neutral substitutions of Glu-509 had little effect on toxin activities. In contrast, charge reversal by lysine substitutions of the Glu-509 or of the adjacent Glu-516 residue reduced the capacity of the toxin to translocate the AC domain across membrane and enhanced significantly its specific hemolytic activity and channel forming capacity in lipid bilayer membranes. Combination of the E509K and E516K mutations in a single molecule further exacerbated hemolytic and channel forming activity and ablated translocation of the AC domain into cells. The lysine substitutions strongly decreased the cation selectivity of the channels, indicating that Glu-509 and Glu-516 are located within or close to the membrane channel. These results suggest that the structure including glutamate residues 509 and 516 is critical for AC membrane translocation and channel forming activity of ACT.
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PMID:An amphipathic alpha-helix including glutamates 509 and 516 is crucial for membrane translocation of adenylate cyclase toxin and modulates formation and cation selectivity of its membrane channels. 1060 20

In eukaryotes, transcriptional regulation on stimulation of the adenylate cyclase signaling pathway is mediated by a family of cyclic AMP-responsive nuclear factors, including CREB, CREM, and ATF-1. These factors contain the basic domain/leucine zipper motifs and bind as dimers to cAMP-responsive elements (CREs). The activation function of CRE-binding proteins is modulated by phosphorylation by several kinases and is mediated by coactivators such as CBP and p300. Activation might also be independent of CBP and phosphorylation in some specific cell types, such as male germ cells, wherein the protein ACT confers a powerful activation function to CREM. The inducible cAMP early repressor (ICER) protein is the only inducible member of this family. The induction of this powerful repressor is likely to be important for the transient nature of cAMP-induced gene expression. CRE-binding proteins have been found to play an important role in the physiology of the pituitary gland, in regulating spermatogenesis, in the response to circadian rhythms, and in the molecular basis of memory.
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PMID:Transcriptional regulation by cyclic AMP-responsive factors. 1069 14

Calmodulin-dependent adenylate cyclase toxin (ACT or CyaA) of Bordetella pertussis requires calcium ions for target cell binding, formation of hemolytic channels, and delivery of its enzyme component into cells. We examined the effect of calcium and calmodulin on toxin interaction with planar lipid bilayers. While calmodulin binding did not affect the properties of CyaA channels, addition of calcium ions and toxin to the same side of the membrane caused a steep increase of the channel-forming capacity of CyaA. The calcium effect was highly specific, since among other divalent cations only strontium caused some CyaA activity enhancement. The minimal stimulatory concentration of calcium ions ranged from 0.6 to 0.8 mM, depending on the ionic strength of the aqueous phase. Half-maximal channel activity of CyaA was observed at 2-4 mM, and saturation was reached at 10 mM calcium concentration, respectively. The unit size of single CyaA channels, assessed as single-channel conductance, was not affected by calcium ions, while the frequency of CyaA channel formation strongly depended on calcium concentration. The calcium effect was abrogated upon deletion of the RTX repeats of the toxin, suggesting that binding of calcium ions to the repeats modulates the propensity of CyaA to form membrane channels.
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PMID:Channel formation in model membranes by the adenylate cyclase toxin of Bordetella pertussis: effect of calcium. 1283 59

Bordetella pertussis secretes an adenylate cyclase toxin (CyaA or ACT) that targets primarily cells expressing the alphaMbeta2 integrin (CD11b/CD18) receptor. This toxin can deliver its N-terminal catalytic AC domain (400 amino acid residues) into the cytosol directly across the cytoplasmic membrane. Various heterologous CD8+, as well as CD4+ T-cell epitopes have been engineered into genetically detoxified CyaA and the resulting toxoids were successfully used as vectors for delivery of inserted epitopes into antigen-presenting cells. Upon processing and presentation, these recombinant CyaAs trigger specific MHC class I and/or class II-restricted T-cell responses both in vitro and in vivo.
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PMID:The adenylate cyclase toxin from Bordetella pertussis--a novel promising vehicle for antigen delivery to dendritic cells. 1514 33

The Bordetella adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) forms cation-selective membrane channels and delivers into the cytosol of target cells an adenylate cyclase domain (AC) that catalyzes uncontrolled conversion of cellular ATP to cAMP. Both toxin activities were previously shown to depend on post-translational activation of proCyaA to CyaA by covalent palmitoylation of the internal Lys983 residue (K983). CyaA, however, harbors a second RTX acylation site at residue Lys860 (K860), and the role of K860 acylation in toxin activity is unclear. We produced in E. coli the CyaA-K860R and CyaA-K983R toxin variants having the Lys860 and Lys983 acylation sites individually ablated by arginine substitutions. When examined for capacity to form membrane channels and to penetrate sheep erythrocytes, the CyaA-K860R acylated on Lys983 was about 1 order of magnitude more active than CyaA-K983R acylated on Lys860, although, in comparison to intact CyaA, both monoacylated constructs exhibited markedly reduced activities in erythrocytes. Channels formed in lipid bilayers by CyaA-K983R were importantly less selective for cations than channels formed by CyaA-K860R, intact CyaA, or proCyaA, showing that, independent of its acylation status, the Lys983 residue may play a role in toxin structures that determine the distribution of charged residues at the entry or inside of the CyaA channel. While necessary for activity on erythrocytes, acylation of Lys983 was also sufficient for the full activity of CyaA on CD11b+ J774A.1 monocytes. In turn, acylation of Lys860 alone did not permit toxin activity on erythrocytes, while it fully supported the high-affinity binding of CyaA-K983R to the toxin receptor CD11b/CD18 and conferred on CyaA-K983R a reduced but substantial capacity to penetrate and kill the CD11b+ cells. This is the first evidence that acylation of Lys860 may play a role in the biological activity of CyaA, even if redundant to the acylation of Lys983.
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PMID:Acylation of lysine 860 allows tight binding and cytotoxicity of Bordetella adenylate cyclase on CD11b-expressing cells. 1617 90

Bordetella adenylate cyclase toxin-hemolysin (CyaA, AC-Hly, or ACT) permeabilizes cell membranes by forming small cation-selective (hemolytic) pores and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Both AC delivery and pore formation were previously shown to involve a predicted amphipathic alpha-helix(502-522) containing a pair of negatively charged Glu(509) and Glu(516) residues. Another predicted transmembrane alpha-helix(565-591) comprises a Glu(570) and Glu(581) pair. We examined the roles of these glutamates in the activity of CyaA. Substitutions of Glu(516) increased specific hemolytic activity of CyaA by two different molecular mechanisms. Replacement of Glu(516) by positively charged lysine residue (E516K) increased the propensity of CyaA to form pores, whereas proline (E516P) or glutamine (E516Q) substitutions extended the lifetime of open single pore units. All three substitutions also caused a drop of pore selectivity for cations. Substitutions of Glu(570) and Glu(581) by helix-breaking proline or positively charged lysine residue reduced (E570K, E581P) or ablated (E570P, E581K) AC membrane translocation. Moreover, E570P, E570K, and E581P substitutions down-modulated also the specific hemolytic activity of CyaA. In contrast, the E581K substitution enhanced the hemolytic activity of CyaA 4 times, increasing both the frequency of formation and lifetime of toxin pores. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu(570) in ion filtering inside or close to pore mouth. The pairs of glutamate residues in the predicted transmembrane segments of CyaA thus appear to play a key functional role in membrane translocation and pore-forming activities of CyaA.
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PMID:Segments crucial for membrane translocation and pore-forming activity of Bordetella adenylate cyclase toxin. 1734 46

The bifunctional adenylate cyclase toxin (ACT or CyaA) of Bordetella pertussis invades target cells via transport through the cytoplasmic membrane. The membrane potential represents thereby an important factor for the uptake in vivo. Previous studies demonstrated that adenylate cyclase (AC) delivery into cells requires a negative membrane potential inside the cells. The results of lipid bilayer experiments with ACT presented here indicated that two different types of pore-like structures are formed by ACT dependent on the orientation of the electrical potential across the membranes. Pore formation at a positive potential at the cis side of the membranes, the side of the addition of the toxin, was fast and its conductance had a defined size, whereas at negative potential the pores were not defined, had a reduced pore-forming activity and a very short lifetime. Fluctuations inserted at positive potentials showed asymmetric current-voltage relationships for positive and negative voltages. Positive potentials at the cis side resulted in an increasing current, whereas at negative potentials the current decreased or remained at a constant level. Calcium ions enhanced the voltage dependence of the ACT pores when they were added to the cis side. The single-pore conductance was strongly affected by the variation of the pH value and increased in 1M KCl with increasing pH from about 4 pS at pH 5 to about 60 pS at pH 9. The ion selectivity remained unaffected by pH. Experiments with ACT mutants revealed, that the adenylate cyclase (AC) and repeat (RT) domains were not involved in voltage and pH sensing.
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PMID:Pore formation by the Bordetella adenylate cyclase toxin in lipid bilayer membranes: role of voltage and pH. 1797 30

Bordetella pertussis adenylate cyclase (AC) toxin-hemolysin (Hly) (CyaA, ACT, or AC-Hly) is a cytotoxin of the RTX (repeat in toxin) family. It delivers into target cells an AC domain that catalyzes uncontrolled conversion of ATP to cAMP, a key signaling molecule subverting phagocyte functions. CyaA utilizes a heavily N-glycosylated beta(2) integrin receptor CD11b/CD18 (alpha(M)beta(2), Mac-1, or CR3). We show that deglycosylation of cell surface proteins by glycosidase treatment, or inhibition of protein N-glycosylation by tunicamycin, ablates CyaA binding and penetration of CD11b-expressing cells. Furthermore, binding of CyaA to cells was strongly inhibited in the presence of free saccharides occurring as building units of integrin oligosaccharide complex, whereas saccharides absent from integrin oligosaccharide chains failed to inhibit CyaA binding to CD11b/CD18-expressing cells. CyaA, hence, selectively recognized sugar residues of N-linked oligosaccharides of integrins. Moreover, glycosylation of CD11a/CD18, another receptor of the beta(2) integrin family, was also essential for cytotoxic action of other RTX cytotoxins, the leukotoxin of Aggregatibacter actinomycetemcomitans (LtxA) and the Escherichia coli alpha-Hly (HlyA). These results show that binding and killing of target cells by CyaA, LtxA, and HlyA depends on recognition of N-linked oligosaccharide chains of beta(2) integrin receptors. This sets a new paradigm for action of RTX cytotoxins.
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PMID:RTX cytotoxins recognize beta2 integrin receptors through N-linked oligosaccharides. 1837 64


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