Showing papers on "Cyclase published in 1994"

Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells

Abstract: Pathogenic bacteria of the genus Yersinia release in vitro a set of antihost proteins called Yops. Upon infection of cultured epithelial cells, extracellular Yersinia pseudotuberculosis transfers YopE across the host cell plasma membrane. To facilitate the study of this translocation process, we constructed a recombinant Yersinia enterocolitica strain producing YopE fused to a reporter enzyme. As a reporter, we selected the calmodulin-dependent adenylate cyclase of Bordetella pertussis and we monitored the accumulation of cyclic AMP (cAMP). Since bacteria do not produce calmodulin, cyclase activity marks the presence of hybrid enzyme in the cytoplasmic compartment of the eukaryotic cell. Infection of a monolayer of HeLa cells by the recombinant Y. enterocolitica strain led to a significant increase of cAMP. This phenomenon was dependent not only on the integrity of the Yop secretion pathway but also on the presence of YopB and/or YopD. It also required the presence of the adhesin YadA at the bacterial surface. In contrast, the phenomenon was not affected by cytochalasin D, indicating that internalization of the bacteria themselves was not required for the translocation process. Our results demonstrate that Y. enterocolitica is able to transfer hybrid proteins into eukaryotic cells. This system can be used not only to study the mechanism of YopE translocation but also the fate of the other Yops or even of proteins secreted by other bacterial pathogens.

The Arabidopsis GA1 locus encodes the cyclase ent-kaurene synthetase A of gibberellin biosynthesis.

Abstract: The first committed step in the gibberellin (GA) biosynthetic pathway is the conversion of geranylgeranyl pyrophosphate (GGPP) through copalyl pyrophosphate (CPP) to ent-kaurene catalyzed by ent-kaurene synthetases A and B. The ga1 mutants of Arabidopsis are gibberellin-responsive male-sterile dwarfs. Biochemical studies indicate that biosynthesis of GAs in the ga1 mutants is blocked prior to the synthesis of ent-kaurene. The GA1 locus was cloned previously using the technique of genomic subtraction. Here, we report the isolation of a nearly full-length GA1 cDNA clone from wild-type Arabidopsis. This cDNA clone encodes an active protein and is able to complement the dwarf phenotype in ga1-3 mutants by Agrobacterium-mediated transformation. In Escherichia coli cells that express both the Arabidopsis GA1 gene and the Erwinia uredovora gene encoding GGPP synthase, CPP was accumulated. This result indicates that the GA1 gene encodes the enzyme ent-kaurene synthetase A, which catalyzes the conversion of GGPP to CPP. Subcellular localization of the GA1 protein was studied using 35S-labeled GA1 protein and isolated pea chloroplasts. The results showed that the GA1 protein is imported into and processed in pea chloroplasts in vitro.

Cloning and functional characterization of a third pituitary adenylate cyclase-activating polypeptide receptor subtype expressed in insulin-secreting cells

Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide belonging to the vasoactive intestinal polypeptide/glucagon/secretin family. It is widely distributed in the body, and a variety of biological actions have been reported. PACAP exerts its biological effects by binding to specific receptors that are coupled to GTP-binding proteins. Recent studies have shown that there is a family of PACAP receptors (PACAPRs), and two members of this family have been identified. We report here the cloning, functional expression, and tissue distribution of a third PACAPR subtype, designated PACAPR-3. The cDNA encoding PACAPR-3 has been isolated from a mouse insulin-secreting beta-cell line MIN6 cDNA library. Mouse PACAPR-3 is a protein of 437 amino acids that has 50% and 51% identity with rat PACAP type I and type II receptors, respectively. Expression of recombinant mouse PACAPR-3 in mammalian cells shows that it binds to vasoactive intestinal polypeptide as well as PACAP-38 and -27, with a slightly higher affinity for PACAP-38, and is positively coupled to adenylate cyclase. The expression of PACAPR-3 in Xenopus oocytes indicates that calcium-activated chloride currents are evoked by PACAP and vasoactive intestinal polypeptide, suggesting that PACAPR-3 can also be coupled to phospholipase C. RNA blot analysis studies reveal that PACAPR-3 mRNA is expressed at high levels in MIN6, at moderate levels in pancreatic islets and other insulin-secreting cell lines, HIT-T15 and RINm5F, as well as in the lung, brain, stomach, and colon, and at low levels in the heart. Furthermore, insulin secretion from MIN6 cells is significantly stimulated by PACAP-38. These results suggest that the diverse biological effects of PACAP are mediated by a family of structurally related proteins and that PACAPR-3 participates in the regulation of insulin secretion.

Structure-activity relationship of adrenomedullin, a novel vasodilatory peptide, in cultured rat vascular smooth muscle cells

Abstract: Vascular smooth muscle cells (VSMC) from rat aorta possess specific receptors for a novel potent vasorelaxant peptide, adrenomedullin (AM). To elucidate its receptor coupling to guanine nucleotide-binding stimulatory protein and the structural requirement of the AM molecule to its vascular receptors, we have studied the effects of guanine nucleotides on [125I]human (h) AM binding and adenylate cyclase activity in cultured rat VSMC, and the effects of various synthetic hAM analogs on [125I]hAM binding and the cAMP response. Guanosine 5'-O-(3-thiotriphosphate) dose dependently inhibited [125I]hAM binding to rat VSMC membranes. hAM stimulated adenylate cyclase activity, and its effect was additive with GTP. hAM-induced cAMP formation was abrogated by pretreatment with cholera toxin, but not by that with pertussis toxin. Intact hAM-(1-52)-NH2 and N-terminal truncated derivatives [hAM-(13-52)-NH2, hAM-(16-52)-NH2] almost equally inhibited [125I]hAM binding and stimulated cAMP formation, whereas removal of C-terminal Tyr52 residue [hAM-(1-51)-NH2] remarkably decreased receptor-binding activity and the cAMP response. The effects of hAM-(1-52)-OH, hAM-(1-51)-OH, and a linear hAM analog ([carbamoylmethyl-Cys16,21]hAM-NH2) were far less potent on receptor binding and the cAMP response than that of hAM-(1-52)-NH2. The C-terminal fragment [hAM-(33-52)-NH2] and the N-terminal fragment [hAM-(1-10)-OH] had neither receptor-binding nor adenylate cyclase activity. hAM-(22-52)-NH2 had no agonistic effect, but showed an antagonistic effect on the hAM-induced cAMP response. These data suggest that vascular AM receptors are functionally coupled to adenylate cyclase via guanine nucleotide-binding stimulatory protein. Studies of the structure-activity relationship of hAM revealed that the cyclic structure formed by the disulfide bridge and amidation of the C-terminal residue of the AM molecule are critical for receptor binding and subsequent cAMP generation and suggest that the C-terminal fragment hAM-(22-52)-NH2 may be an antagonist for vascular AM receptors.

Internal Lysine Palmitoylation in Adenylate Cyclase Toxin from Bordetella pertussis

Abstract: A number of bacterial protein toxins, including adenylate cyclase (AC) toxin from Bordetella pertussis, require the product of an accessory gene in order to express their biological activities. In this study, mass spectrometry was used to demonstrate that activated, wild-type AC toxin was modified by amide-linked palmitoylation on the epsilon-amino group of lysine 983. This modification was absent from a mutant in which the accessory gene had been disrupted. A synthetic palmitoylated peptide corresponding to the tryptic fragment (glutamine 972 to arginine 984) that contained the acylation blocked AC toxin-induced accumulation of adenosine 3',5'-monophosphate, whereas the non-acylated peptide had no effect.

Cross-talk between cyclooxygenase and nitric oxide pathways: prostaglandin E2 negatively modulates induction of nitric oxide synthase by interleukin 1.

Abstract: The inflammatory cytokine interleukin 1 beta (IL-1 beta) induces both cyclooxygenase (COX) and nitric oxide synthase (NOS) with increases in the release of prostaglandin (PG) and nitric oxide (NO) by mesangial cells. Recently, activation of the COX enzyme by NO has been described. However, the effects of COX products (PGs) on the NO pathway have not been fully clarified. Thus we determined the effect of COX inhibition and exogenous PGs on NO production and NOS induction in rat mesangial cells. A COX inhibitor, indomethacin, enhanced IL-1 beta-induced steady-state level of the inducible NOS (iNOS) mRNA and nitrite production. The effect of indomethacin was dose dependently reversed by the replacement of endogenous PGE2 with exogenous PGE2, which is the predominant product of the COX pathway in rat mesangial cells. In contrast to PGE2, a stable analog of PGI2, carba prostacyclin, enhanced IL-1 beta-induced iNOS mRNA levels and nitrite production. Forskolin, an activator of the adenylate cyclase, mimicked the effect of carba prostacyclin but not PGE2. These data suggest that (i) endogenous PGE2 downregulates iNOS induction, (ii) this inhibitory effect of PGE2 on iNOS induction is not mediated by activation of adenylate cyclase, and (iii) exogenous PGI2 stimulates COX induction possibly by activation of adenylate cyclase.

Bleached pigment activates transduction in isolated rods of the salamander retina.

Abstract: 1. We have used suction electrode recording together with rapid steps into Li+ solution and 0.5 mM IBMX solution to estimate the rates of the guanylyl phosphodiesterase (PDE) and guanylyl cyclase in isolated rods of the salamander, Ambystoma tigrinum. 2. We show that both the PDE and cyclase velocities are accelerated by steady background light. The steady velocities of both enzymes appear to be saturating functions of background intensity. 3. Bleaching also accelerates both the PDE and cyclase. This effect is maintained long after the bleaching stimulus is removed (up to 2 h) and is reversed only if the photopigment is regenerated with exogenous chromophore. 4. The estimated steady-state PDE and cyclase velocities appear to be linear functions of the amount of pigment bleached, as if each bleached pigment molecule activated the transduction cascade with the same probability and gain. 5. The effectiveness of bleached pigment in activating transduction is only 10(-6) to 10(-7) times that of activated rhodopsin (Rh*), but this is sufficient after large bleaches to produce an 'equivalent background' excitation of the rod, which is probably responsible, at least in part, for bleaching desensitization.

Engineered biosynthesis of novel polyketides: influence of a downstream enzyme on the catalytic specificity of a minimal aromatic polyketide synthase

Abstract: To identify the minimum set of polyketide synthase (PKS) components required for in vivo biosynthesis of aromatic polyketides, combinations of genes encoding subunits of three different aromatic PKSs--act from Streptomyces coelicolor A3(2) (an actinorhodin producer), fren from Streptomyces roseofulvus (a frenolicin and nanaomycin producer), and tcm from Streptomyces glaucescens (a tetracenomycin producer)--were expressed in a recently developed Streptomyces host-vector system. The "minimal" components (ketosynthase/putative acyltransferase, chain length-determining factor, and acyl carrier protein) were produced with and without a functional polyketide ketoreductase and/or cyclase, and the polyketide products of these recombinant strains were structurally characterized. Several previously identified polyketides were isolated in addition to two previously unidentified polyketides, dehydromutactin and SEK 15b, described here. The results proved that the act cyclase is not required for the biosynthesis of several aberrantly cyclized products that have been previously reported. They are also consistent with earlier conclusions that the minimal PKS controls chain length as well as the regiospecificity of the first cyclization and that it can do so in the absence of both a ketoreductase and a cyclase. However, the ability of the minimal tcm PKS to synthesize two different singly cyclized intermediates suggests that it is unable to accurately control the course of this reaction by itself. In the presence of a downstream enzyme, the flux through one branch of the cyclization pathway increases relative to the other. We propose that these alternative specificities may be due to the ability of downstream enzymes to associate with the minimal PKS and to selectively inhibit a particular branch of the cyclization pathway.

Distinct cellular localization of mRNAs for three subtypes of prostaglandin E receptor in kidney

Abstract: Distribution of the mRNAs for three subtypes of prostaglandin E (PGE) receptors in the mouse kidney was investigated by in situ hybridization. The mRNA for EP1 subtype, which is coupled to Ca2+ mobilization, was specifically localized to the collecting ducts from the cortex to the papilla. The mRNA for EP2 subtype, which is linked to stimulation of adenylate cyclase, was localized to the glomeruli. The mRNA for EP3 subtype, which is coupled to inhibition of adenylate cyclase, was located densely in the tubules in the outer medulla and in the distal tubules in the cortex. These results exhibit distinct cellular localization of three subtypes of PGE receptor in the kidney and suggest that PGE2 exerts multiple functions via these subtypes expressed in different segments of the nephron.

Structural and functional characterization of the rod outer segment membrane guanylate cyclase.

Abstract: In the vertebrate photoreceptor cell, rod outer segment (ROS) is the site of visual signal-transduction process, and a pivotal molecule that regulates this process is cyclic GMP. Cyclic GMP controls the cationic conductance into the ROS, and light causes a decrease in the conductance by activating hydrolysis of the cyclic nucleotide. The identity of the granylate cyclase (ROS-GC) that synthesizes this pool of cyclic GMP is unknown. We now report the cloning, expression and functional characterization of a DNA from bovine retina that encodes ROS-GC.

Changes in type VI adenylyl cyclase isoform expression correlate with a decreased capacity for cAMP generation in the aging ventricle.

Abstract: We investigated the developmental regulation of the beta-adrenergic receptor-Gs-adenylyl cyclase pathway in myocardial membranes from fetal, neonatal, adult, and mature adult rats by measuring the density of the beta-adrenergic receptor and the activities of the stimulatory guanine nucleotide-binding protein Gs and the adenylyl cyclase enzyme. Total beta-adrenergic receptor content (in femtomoles per milligram protein) was greatest in the fetal (124.4 +/- 20.5 fmol/mg) and neonatal (122.3 +/- 16.1 fmol/mg) stages and gradually decreased in the adult (90.9 +/- 8.0 fmol/mg) and mature adult (70.0 +/- 9.6 fmol/mg) stages. An equivalent pattern was seen for adenylyl cyclase activity: the basal activity of the effector enzyme or that measured in the presence of 0.1 mmol/L isoproterenol with 0.1 mmol/L Gpp(NH)p, 10 mmol/L NaF, or 0.05 mmol/L forskolin was greater in the fetus and the neonate than in the adult and the mature adult. These data suggested that decreased stimulation of the catalytic unit by Gs could be the underlying cause of diminished adenylyl cyclase activity with aging. However, quantification of Gs by reconstitution into S49 cyc- membranes (in picomoles cAMP per microgram for 10 minutes) demonstrated no significant decrease during development from fetus (1.55 +/- 0.1 pmol/microgram) to neonate (1.9 +/- 0.5 pmol/microgram) and subsequent aging to adult (2.6 +/- 0.2 pmol/micrograms) and mature adult (1.9 +/- 0.2 pmol/microgram). When Northern blot analysis was used to characterize the relative amounts of mRNA coding for Gs alpha, no significant differences were seen among the developmental stages studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and Characterization of the Gene Encoding 2,3-Oxidosqualene- Lanosterol Cyclase from Saccharomyces cerevisiae

Abstract: The ERG7 gene encoding oxidosqualene-lanosterol cyclase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] from Saccharomyces cerevisiae has been cloned by genetic complementation of a cyclase-deficient erg7 strain. The DNA sequence of this gene has been determined and found to contain an open reading frame of 2196 nt (including stop codon) that encodes a predicted protein of 731 amino acids. The predicted molecular mass of the S. cerevisiae cyclase, 83.4 kDa, is similar to the predicted molecular masses of the oxidosqualene-lanosterol cyclase from Candida albicans and the oxidosqualene-cycloartenol cyclase from Arabidopsis thaliana, as well as to the molecular masses assigned to vertebrate oxidosqualene-lanosterol cyclases; however, it is substantially larger than the molecular mass assigned to purified S. cerevisiae cyclase. At the level of DNA and predicted amino acid sequences, the S. cerevisiae and C. albicans cyclases share 56% and 63% identity, respectively. Tryptophan and tyrosine residues are unusually abundant in the predicted amino acid sequences of (oxido)-squalene cyclases, leading to a hypothesis that electron-rich aromatic side chains from these residues are essential features of cyclase active sites.

Capacitative Ca2+ entry regulates Ca2+-sensitive adenylyl cyclases

Abstract: A number of the currently described adenylyl cyclase species can be regulated by Ca2+ in the submicromolar concentration range in in vitro assays. The regulatory significance of these observations hinges on whether a physiological elevation in intracellular Ca2+ can regulate these cyclase activities in intact cells. However, achieving a physiological elevation in cytosolic Ca2+ is complicated by the fact that hormonal increases in cytosolic Ca2+ can be accompanied by additional effects, such as liberation of beta gamma-subunits of G-proteins and activation of protein kinase C, which can have disparate type-specific effects on cyclase activities. Therefore we have devised a strategy based on capacitative Ca2+ entry to show that, when types I and VI adenylyl cyclase are expressed in human embryonic kidney 293 cells, they are stimulated and inhibited respectively by Ca2+ entry. Blockade of Ca2+ entry by La3+ ions blocks the effects of Ca2+ entry on cyclic AMP synthesis. These studies establish that adenylyl cyclases deemed to be sensitive to Ca2+ in in vitro assays can be regulated by physiological Ca2+ entry, and therefore, such cyclases are poised to respond to changes in intracellular Ca2+ in tissues in which they are expressed.

Expression of nitric oxide synthase in rat glomerular mesangial cells mediated by cyclic AMP

Abstract: 1. Treatment of rat mesangial cells with interleukin 1 beta (IL-1 beta) or tumour necrosis factor alpha (TNF alpha) has been shown to induce a macrophage-type of nitric oxide (NO) synthase. Here we report that adenosine 3':5'-cyclic monophosphate (cyclic AMP) is another mediator that triggers induction of NO synthase in mesangial cells. 2. Incubation of mesangial cells with the beta-adrenoceptor agonist, salbutamol, forskolin or cholera toxin, which all activate adenylate cyclase and increase intracellular cyclic AMP concentration, increased nitrite formation in a dose-dependent manner. Likewise, the addition of the membrane-permeable cyclic AMP analogue, N6, 0-2'-dibutyryladenosine 3',5'-phosphate (Bt2 cyclic AMP) or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine enhanced NO synthase activity in a dose-dependent manner. 3. There was a lag period of about 8 h before a significantly enhanced secretion of nitrite could be detected upon exposure of cells to forskolin and for maximal stimulation, forskolin had to be present during the whole incubation period. 4. Treatment of mesangial cells with actinomycin D, cycloheximide or dexamethasone completely suppressed forskolin-stimulated NO-synthase activity, thus demonstrating that transcription and protein synthesis are necessary for nitrite formation. 5. Bt2 cyclic AMP, the most potent inducer of nitrite production, increased NO synthase mRNA levels in mesangial cells in a time- and dose-dependent fashion. Dexamethasone completely inhibited the increase of NO synthase mRNA in response to Bt2 cyclic AMP. 6. Combination of Bt2 cyclic AMP and IL-1 beta or TNF alpha revealed a strong synergy in terms of nitrite formation. Time-course studies indicated that cyclic AMP needed to be increased during the whole period of IL-1 Beta stimulation for maximal nitrite production.7. These observations suggest that cyclic AMP controls NO synthase expression in mesangial cells.Furthermore, the signalling cascades triggered by IL-1 Beta and TNF alpha synergize with the cyclic AMP pathway to stimulate NO synthase activity.

Endothelin ETA-receptors couple to inositol phosphate formation and inhibition of adenylate cyclase in human right atrium.

Abstract: To study signal transduction pathways of endothelin (ET) in human heart, we assessed, in isolated human right atria, the effects of ET-1 and ET-3 on inositol phosphate (IP) formation and on the adenylate cyclase/cyclic AMP system. In right atrial slices, ET-1 (10(-10)-10(-6)M) concentration-dependently increased [3H]IP accumulation and decreased 10-microM isoprenaline-induced or 1-microM forskolin-induced increases in cyclic AMP content. ET-3 was approximately 100 times less potent. The cyclic AMP-decreasing effect of ET-1 (10(-11)-10(-6)M) could also be demonstrated directly in adenylate cyclase assays in right atrial membranes; again, ET-3 was approximately 100 times less potent. We conclude that in human right atrium, ETA receptors couple to two different signal-transduction pathways: IP formation and inhibition of adenylate cyclase.

Glucose repression of fbp1 transcription of Schizosaccharomyces pombe is partially regulated by adenylate cyclase activation by a G protein alpha subunit encoded by gpa2 (git8).

Abstract: In the fission yeast Schizosaccharomyces pombe, genetic studies have identified genes that are required for glucose repression of fbp1 transcription. The git2 gene, also known as cyr1, encodes adenylate cyclase. Adenylate cyclase converts ATP into the second messenger cAMP as part of many eukaryotic signal transduction pathways. The git1, git3, git5, git7, git8 and git10 genes act upstream of adenylate cyclase, presumably encoding an adenylate cyclase activation pathway. In mammalian cells, adenylate cyclase enzymatic activity is regulated by heterotrimeric guanine nucleotide-binding proteins (G proteins). In the budding yeast Saccharomyces cerevisiae, adenylate cyclase enzymatic activity is regulated by monomeric, guanine nucleotide-binding Ras proteins. We show here that git8 is identical to the gpa2 gene that encodes a protein homologous to the alpha subunit of a G protein. Mutations in two additional genes, git3 and git5 are suppressed by gpa2+ in high copy number. Furthermore, a mutation in either git3 or git5 has an additive effect in strains deleted for gpa2 (git8), as it significantly increases expression of an fbp1-lacZ reporter gene. Therefore, git3 and git5 appear to act either in concert with or independently from gpa2 (git8) to regulate adenylate cyclase activity.