High-level expression of the colicin A lysis protein

Summary Two plasmids that overproduce the colicin A lysis protein, Cal, are described. Plasmid AT1 was constructed by a deletion in the colicin A operon, which placed thecal gene near a truncatedcaa gene in such a way that both gene products were synthesized at high levels following induction. Plasmid Ck4 was constructed by insertion of thecal gene downstream from thetac promoter of an expression vector. Overproduction of Cal was obtained after mitomycin C induction of pAT1 cells and after IPTG induction of pCK4 cells. The kinetics of Cal synthesis were examined with [³⁵S] methionine and [2-³H] glycerol inlpp orlpp ⁺ host strains. Each of the steps of the lipid modification and maturation pathway of Cal was demonstrated. The modified precursor form of overproduced Cal was not chased as efficiently as when it is produced in pColA cells. After treatment with globomycin, a significant amount of this modified precursor form accumulated and was degraded with time into smaller acylated proteins, but without release of the signal peptide. Release of cellular proteins and quasi-lysis were observed after about 1 hour of induction for cells containing either plasmid. In addition, in Cal-overproducing cells, the rate of quasi-lysis was increased but not its extent. InpldA cells, quasi-lysis was reduced but not abolished. Lethality of the Cal induction in the overproducing cells was in the same range as that in wild-type cells.     

Role of calmodulin in the effect of guanyl nucleotides on rat hippocampal adenylate cyclase: involvement of adenosine and opiates

Abstract: The adenylate cyclase activity of rat hippocampal plasma membranes can be stimulated by vaso-active intestinal polypeptide (VIP). Low concentrations (10⁻⁹ to 10⁻⁷M) of 5'-guanylyl-imido diphosphate (GppNHp) evoke a transient inhibition of the enzyme, which is followed by stimulation with increasing GppNHp concentrations (10⁻⁶ to 10⁻⁴M). Inclusion of ethyleneglycol - bis - (β - aminoethylether) - N,N' - tetraacetic acid (EGTA) during incubation abolishes the GppNHp inhibition while preserving GppNHp activation. The stimulation induced by GppNHp is amplified by VIP, but the inhibition is unaffected. Adenosine analogs and opiates are inhibitory ligands in the presence of GTP, and their effects can be reversed by the appropiate receptor antagonists, 3-isobutyl-1-methylxanthine and naloxone. Treatment of membranes with trypsin abolishes the GppNHp-induced inhibition without affecting the GppNHp stimulation. The inhibition induced by GppNHp is also abolished by EGTA treatment followed by washing, which coincides wtih a reduction in the adenosine- and opiate-mediated, GTP-dependent inhibition. The GppNHp inhibition can be restored in EGTA-treated but not in trypsin-treated membranes by addition of calcium-calmodulin but not by Ca²⁺ or Mg²⁺. Calcium-calmodulindepleted membranes lack calcium stimulation as well as GppNHp-induced inhibition, whereas untreated membranes and calcium-calmodulin-depleted membranes plus exogenous calcium-calmodulin showed calcium stimulation and GppNHp inhibition. These results suggest that calmodulin is involved in both Ca²⁺ stimulation and guanine nucleotide-mediated inhibition of rat hippocampal adenylate cyclase.     

Properties of Detergent-Dispersed Adenylate Cyclase from Cerebral Cortex. Presence of an Inhibitor Protein

Abstract: Adenylate cyclase was solubilized from washed paniculate fraction of rabbit cerebral cortex with the nonionic detergent Lubrol 12A9 and subjected to either gel filtration on Ultrogel AcA 34 or chromatography on DEAE Bio-Gel A. By both procedures the enzyme was resolved into two components, one insensitive to guanyl 5'-yl imidodiphosphate [Gpp(NH)p] and NaF but stimulated by Ca²⁺ and calmodulin, and another that was sensitive to Gpp(NH)p and NaF but relatively insensitive to Ca²⁺ and calmodulin. The data support the possibility that two independent forms of adenylate cyclase exist in cerebral cortex, one regulated by guanine nucleotide regulatory protein and another by Ca²⁺-calmodulin. Fractions containing the guanylnucleotide-sensitive activity were found to contain a factor that inhibited basal and Ca²⁺-stimulated adenylate cyclase in the Ca²⁺-sensitive fraction. The inhibitor was inactivated by heating at 60°C and by incubation with trypsin. Inhibition was not time-dependent, and it was not due to destruction of cAMP by phosphodiesterase or of ATP by ATPase. Inhibitory action was not reversed by calmodulin and therefore it does not appear to be a calmodulin binding protein. Sucrose density gradient sedimentation indicated a sedimentation coefficient of 4S for the inhibitor; by this technique it co-sedimented with the adenylate cyclase sensitive to Gpp(NH)p and NaF.     

Adjuvant and protective properties of native and recombinant Bordetella pertussis adenylate cyclase toxin preparations in mice

Bordetella pertussis produces a cell-invasive adenylate cyclase toxin which is synthesised from the cyaA gene as an inactive protoxin that is post-translationally activated by the product of the cyaC gene. Purified active and inactive CyaA proteins were prepared from B. pertussis or from recombinant Escherichia coli expressing both cyaA and cyaC genes or the cyaA gene alone. respectively. In addition, a hybrid toxin (Hyb2) in which an internal region of CyaA had been replaced with the analogous region from the leukotoxin (LktA) of Pasteurella haemolytica, and which had low cell-invasive activity, was also prepared from E. coli expressing the cyaC gene. The CyaA preparations showed no evidence of toxicity in a mouse weight-gain test. Active toxin preparations were protective in mice against intranasal challenge with wild-type B. pertussis, as evidenced by lung:body weight ratios and bacterial numbers in the lungs, which were comparable to those in mice given whole-cell DPT vaccine. Hyb2 was not as protective as active CyaA and inactive CyaA preparations were not protective. Active CyaA, when co-administered with ovalbumin (OA), had a marked adjuvant effect on the anti-OA IgG antibody response which was not as apparent with inactive CyaA preparations. Similarly, active CyaA stimulated a greater anti-CyaA response than the inactive form.     

Synthesis and functioning of the colicin E1 lysis protein: comparison with the colicin A lysis protein.

The colicin E1 lysis protein, CelA, was identified as a 3-kDa protein in induced cells of Escherichia coli K-12 carrying pColE1 by pulse-chase labeling with either [35S]cysteine or [3H]lysine. This 3-kDa protein was acylated, as shown by [2-3H]glycerol labeling, and seemed to correspond to the mature CelA protein. The rate of modification and processing of CelA was different from that observed for Cal, the colicin A lysis protein. In contrast to Cal, no intermediate form was detected for CelA, no signal peptide accumulated, and no modified precursor form was observed after globomycin treatment. Thus, the rate of synthesis would not be specific to lysis proteins. Solubilization in sodium dodecyl sulfate of the mature forms of both CelA and Cal varied similarly at the time of colicin release, indicating a change in lysis protein structure. This particular property would play a role in the mechanism of colicin export. The accumulation of the signal peptide seems to be a factor determining the toxicity of the lysis proteins since CelA provoked less cell damage than Cal. Quasi-lysis and killing due to CelA were higher in degP mutants than in wild-type cells. They were minimal in pldA mutants.     

The acylated precursor form of the colicin A lysis protein is a natural substrate of the DegP protease.

The acylated precursor form of the colicin A lysis protein (pCalm) is specifically cleaved by the DegP protease into two acylated fragments of 6 and 4.5 kilodaltons (kDa). This cleavage was observed after globomycin treatment, which inhibits the processing of pCalm into mature colicin A lysis protein (Cal) and the signal peptide. The cleavage took place in lpp, pldA, and wild-type strans carrying plasmids which express the lysis protein following SOS induction and also in cells containing a plasmid which expresses it under the control of the tac promoter. Furthermore, the DegP protease was responsible for the production of two acylated Cal fragments of 3 and 2.5 kDa in cells carrying plasmids which overproduce the Cal protein, without treatment with globomycin. DegP could also cleave the acylated precursor form of a mutant Cal protein containing a substitution in he amino-terminal portion of the protein, but not that of a mutant Cal containing a frameshift mutation in its carboxyl-terminal end. The functions of Cal in causing protein release, quasi-lysis, and lethality were increased in degP41 cells, suggesting that mature Cal was produced in higher amounts in the mutant than in the wild type. These effects were limited in cells deficient in phospholipase A. Interactions between the DegP protease and phospholipase A were suggested by the characteristics of degP pldA double mutants.     

Role of DegP protease on levels of various forms of colicin A lysis protein

Abstract The total amount of the colicin A lysis protein produced by cells grown in rich medium was analysed by immunoblotting. The intermediate forms of synthesis of this small lipoprotein were present in the cells at any time of induction, confirming that processing and maturation of colicin A lysis protein are slow and incomplete processes. The level of these various forms varied according to the time of induction, the growth conditions, the producing strain and the plasmid carrying the cal gene. It depended mainly on the presence in the producing strain of a degP gene which encodes the DegP protease. According to growth conditions, the DegP protease hydrolysed either a part or the total amount of the acylated precursor form. In some cases, a protease(s) other than DegP seemed to act on either form(s) of the colicin A lysis protein.     

Islet-activating protein discriminates the antilipolytic mechanism of insulin from that of other antilipolytic compounds

In vivo administration of islet-activating protein to rats resulted in an increase in fat cell lipolysis in vitro, which was associated with almost complete resistance of adipocytes towards the antilipolytic effects of N6-phenylisopropyladenosine, prostaglandin E2 and nicotinic acid. Concomitantly, the inhibitory effects of these compounds on adenylate cyclase activity in membranes were impaired. In contrast, the antilipolytic action of insulin was not only preserved, but even augmented in cells from rats treated with islet-activating protein. The data suggest that insulin exerts its antilipolytic effects via mechanisms which are different from those involved in the effects of prostaglandin E2, N6-phenylisopropyladenosine and nicotinic acid.     

Evidence That Calmodulin Binding to the Dopamine D2 Receptor Enhances Receptor Signaling

The Ca²⁺ sensor calmodulin (CaM) regulates numerous proteins involved in G protein-coupled receptor (GPCR) signaling. CaM binds directly to some GPCRs, including the dopamine D₂ receptor. We confirmed that the third intracellular loop of the D₂ receptor is a direct contact point for CaM binding using coimmunoprecipitation and a polyHis pull-down assay, and we determined that the D₂-like receptor agonist 7-OH-DPAT increased the colocalization of the D₂ receptor and endogenous CaM in both 293 cells and in primary neostriatal cultures. The N-terminal three or four residues of D₂-IC3 were required for the binding of CaM; mutation of three of these residues in the full-length receptor (I210C/K211C/I212C) decreased the coprecipitation of the D₂ receptor and CaM and also significantly decreased D₂ receptor signaling, without altering the coupling of the receptor to G proteins. Taken together, these findings suggest that binding of CaM to the dopamine D₂ receptor enhances D₂ receptor signaling.     

In vitro activation of the Saccharomyces cerevisiae Ras/adenylate cyclase system by glucose and some of its analogues

Using crude membrane preparations of Saccharomyces cerevisiae, we have demonstrated that glucose and glucose analogues which are not efficiently phosphorylated activate the guanine nucleotide-dependent adenylate cyclase in vitro. The activation appears to be mediated by the Ras proteins. Moreover, data are presented indicating that glucose and its analogues activate adenylate cyclase by stimulating the exchange of guanine nucleotides at its regulatory component. Thus, it has been possible to show the action of a physiological effector on the nucleotide exchange reaction in a member of the ras superfamily.     

In vitro activation of the Saccharomyces cerevisiae Ras/adenylate cyclase system by glucose and some of its analogues

Using crude membrane preparations of Saccharomyces cerevisiae, we have demonstrated that glucose and glucose analogues which are not efficiently phosphorylated activate the guanine nucleotide-dependent adenylate cyclase in vitro. The activation appears to be mediated by the Ras proteins. Moreover, data are presented indicating that glucose and its analogues activate adenylate cyclase by stimulating the exchange of guanine nucleotides at its regulatory component. Thus, it has been possible to show the action of a physiological effector on the nucleotide exchange reaction in a member of the ras superfamily.     

Decreased adenylate cyclase responsiveness of transformed cells correlates with the presence of a viral transforming protein

The adenylate cyclase responsiveness of transformed fibroblastic and epithelial cell lines to forskolin, fluoride, guanine nucleotides and cholera toxin was reduced compared to their parental counterparts. This phenomenon was observed in lines transformed by either RNA or DNA tumor viruses, and in the case of polyoma virus, coincided with the expression of middle T antigen. The data suggest that decreased responsiveness of adenylate cyclase to non-hormone activators is a general consequence of viral transformation and may be related to viral regulation of protein kinase activity.     

Truncated carboxyl-terminal fragments of beta-amyloid precursor protein are processed to amyloid beta-proteins 40 and 42

We previously showed that beta-amyloid precursor protein (APP) is cleaved not only in the middle of the membrane (gamma-cleavage) but also at novel cleavage sites close to the membrane/cytoplasmic boundary (epsilon-cleavage), releasing APP intracellular domains (AICDs) 49-99 and 50-99. To learn more about the relationship between gamma- and epsilon-cleavage, C-terminally truncated carboxyl-terminal fragments (CTFs) of APP, especially CTFs1-48 and 1-49 (the postulated products that are generated by epsilon-cleavage), were transiently expressed in CHO cells. Most importantly, the cells expressing CTF1-49 secreted predominantly amyloid beta-protein (Abeta) 40, while those expressing CTF1-48 secreted preferentially Abeta42. This supports our assumption that epsilon-cleavage precedes Alphabeta production and that preceding epsilon-cleavage determines the preference for the final Abeta species. The gamma-secretase inhibitors, L-685,458 and DAPT, suppressed Abeta production from CTF1-49. Regarding Abeta production from CTF1-48, L-685,458 suppressed it, but DAPT failed to do so. A dominant negative mutant of presenilin 1 suppressed the production of Abeta40 and 42 from both CTFs1-48 and 1-49. These data should shed significant light into the mechanism of Abeta production.     

Hypoxia Increases the Cyclic AMP Content of the Cat Carotid Body In Vitro

The cyclic AMP content of cat carotid bodies in vitro measured with a radioimmunoassay under control conditions (PO2: 230 torr) was 0.79 +/- 0.10 pmol/carotid body (n = 10). Lowering medium PO2 to 20 torr for 2 min significantly increased cyclic AMP content to 1.13 +/- 0.14 pmol/carotid body (n = 10). This increase was inhibited neither by propranolol (34 microM) nor by propranolol plus haloperidol (27 microM). Inhibition of the cyclic nucleotide phosphodiesterase with 1-methyl-3-isobutylxanthine (0.8 mM) provoked a fast and large increase in cyclic AMP during both control and hypoxic conditions. The cyclic AMP increase induced by hypoxia was still observed when extracellular Ca2+ was absent. Inhibition of the adenylate cyclase by N-(cis-2-phenylcyclopentyl)azacyclotridecan-2-imine hydrochloride (MDL 12330A; 20-1,000 microM) under zero-Ca2+ conditions irreversibly inhibited the cyclic AMP increase produced by hypoxia. Similarly, inhibition of the Ca2(+)-calmodulin complex by trifluoperazine (0.2 mM) or calmidazolium (R 24571; 50-200 microM) prevented the cyclic AMP response. These results suggest that cyclic AMP may be involved in the PO2-sensing mechanism of the carotid body. Hypoxia appears to activate adenylate cyclase directly and independent of any hormone-receptor interactions.     

A molecular genetic approach to the functioning of the immunity protein to colicin A

Summary A plasmid (pColAF1), derived from pColA, and lacking the region encoding Cai (colicin A immunity protein) and Cal (colicin A lysis protein) has been constructed. The strains carrying pColAF1 produce normal amounts of colicin A which remains in the cell cytoplasm and does not result in loss of viability. Similar results have also been obtained for transposon insertion mutants lacking Cai. Structure prediction analysis indicates that four peptide regions of Cai might span the cytoplasmic membrane. Since the NH₂-and COOH-terminal regions are charged, this analysis suggests a topology of the 178 residues polypeptide chain in which regions 38 to 70 and 124 to 143 might be exposed at the outer side of the cytoplasmic membrane. With mutants constructed using recombinant DNA techniques, we could demonstrate that the removal of a 30 residue COOH-terminal region, and mutations altering the surface exposed loop comprised of aminoacid residues 124–143 abolish the protecting function of Cai.     

Susceptibility of primary human airway epithelial cells to Bordetella pertussis adenylate cyclase toxin in two- and three-dimensional culture conditions

The human pathogen Bordetella pertussis targets the respiratory epithelium and causes whooping cough. Its virulence factor adenylate cyclase toxin (CyaA) plays an important role in the course of infection. Previous studies on the impact of CyaA on human epithelial cells have been carried out using cell lines derived from the airways or the intestinal tract. Here, we investigated the interaction of CyaA and its enzymatically inactive but fully pore-forming toxoid CyaA-AC^– with primary human airway epithelial cells (hAEC) derived from different anatomical sites (nose and tracheo-bronchial region) in two-dimensional culture conditions. To assess possible differences between the response of primary hAEC and respiratory cell lines directly, we included HBEC3-KT in our studies. In comparative analyses, we studied the impact of both the toxin and the toxoid on cell viability, intracellular cAMP concentration and IL-6 secretion. We found that the selected hAEC, which lack CD11b, were differentially susceptible to both CyaA and CyaA-AC^–. HBEC3-KT appeared not to be suitable for subsequent analyses. Since the nasal epithelium first gets in contact with airborne pathogens, we further studied the effect of CyaA and its toxoid on the innate immunity of three-dimensional tissue models of the human nasal mucosa. The present study reveals first insights in toxin–cell interaction using primary hAEC.     

Amino acid sequence and length requirements for assembly and function of the colicin A lysis protein.

The roles of the various parts of the mature colicin A lysis protein (Cal) in its assembly into the envelope and its function in causing "quasi-lysis," the release of colicin A, and the activation of phospholipase A were investigated. By using cassette mutagenesis, many missense mutations were introduced into the highly conserved portion of the lysis protein. In vitro mutagenesis was also used to introduce stop codons after amino acids 16 and 18 and a frameshift mutation at amino acid 17 of the mature Cal sequence. The processing and modification of the mutants were identical to those of the wild type, except for the truncated Cal proteins, which were neither acylated nor processed. Thus, the carboxy-terminal half of Cal must be present (or replaced by another peptide) for the proper processing and assembly of the protein. However, the specific sequence of this region is not required for the membrane-damaging function of the protein. Furthermore, the sequence specificity for even the conserved amino acids of the amino-terminal half of the protein is apparently exceedingly relaxed, since only those mutant Cal proteins in which a highly conserved amino acid has been replaced by a glutamate were impaired in their function.     

S-100b protein regulates the activity of skeletal muscle adenylate cyclase in vitro

We have investigated the effect of the b isoform of S-100 proteins on adenylate cyclase activity of rat skeletal muscle. S-100b inhibits the adenylate cyclase activity in the presence of Mg²⁺ (5.0–50 mM), while it activates the same enzyme in the presence of Ca²⁺ (0.1–1.0 mM) dose-dependently in both cases. S-100b counteracts the stimulatory effect of NaF on adenylate cyclase in the presence of Mg²⁺ and the inhibitory effect of RMI 12330 A in the presence of Ca²⁺.     

Effects of proteolysis on the actions of monovalent cations and 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine on platelet adenylate cyclase

NaCl stimulated the adenylate cyclase activities of human and rabbit platelet particulate fractions prepared in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetate, but inhibited the activities of particulate fractions proteolysed by endogenous Ca2+-activated protease or treatment with alpha-chymotrypsin. Studies with other monovalent cations showed that LiCl had weak effects similar to those of NaCl, whereas KCl inhibited the enzyme in both proteolysed and non-proteolysed preparations. The results suggest that NaCl exerts stimulatory and inhibitory effects through different sites. NaCl potentiated and proteolysis greatly reduced the inhibition of platelet adenylate cyclase by 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (platelet-activating factor).