Characterization and engineering of photoactivated adenylyl cyclases.

Cyclic nucleoside monophosphates (cNMP) serve as universal second messengers in signal transduction across prokaryotes and eukaryotes. As signaling often relies on transiently formed microdomains of elevated second messenger concentration, means to precisely perturb the spatiotemporal dynamics of cNMPs are uniquely poised for the interrogation of the underlying physiological processes. Optogenetics appears particularly suited as it affords light-dependent, accurate control in time and space of diverse cellular processes. Several sensory photoreceptors function as photoactivated adenylyl cyclases (PAC) and hence serve as light-regulated actuators for the control of intracellular levels of 3', 5'-cyclic adenosine monophosphate. To characterize PACs and to refine their properties, we devised a test bed for the facile analysis of these photoreceptors. Cyclase activity is monitored in bacterial cells via expression of a fluorescent reporter, and programmable illumination allows the rapid exploration of multiple lighting regimes. We thus probed two PACs responding to blue and red light, respectively, and observed significant dark activity for both. We next engineered derivatives of the red-light-sensitive PAC with altered responses to light, with one variant, denoted DdPAC, showing enhanced response to light. These PAC variants stand to enrich the optogenetic toolkit and thus facilitate the detailed analysis of cNMP metabolism and signaling.

mem-iLID, a fast and economic protein purification method.

Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system improved light-induced dimer (iLID), which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the POI, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.

The lipolytic activity of LipJ, a stress-induced enzyme, is regulated by its C-terminal adenylate cyclase domain.

Aim: The confirmation of lipolytic activity and role of Rv1900c in the Mycobacterium physiology Methods:rv1900c/N-terminus domain (rv1900NT) were cloned in pET28a/Escherichia coli, purified by affinity chromatography and characterized. Results: A zone of clearance on tributyrin-agar and activity with pNP-decanoate confirmed the lipolytic activity of Rv1900c. The Rv1900NT demonstrated higher enzyme specific activity, Vmax and kcat, but Rv1900c was more thermostable. The lipolytic activity of Rv1900c decreased in presence of ATP. Mycobacterium smegmatis expressed rv1900c/rv1900NT-altered colony morphology, growth, cell surface properties and survival under stress conditions. The effect was more prominent with Rv1900NT as compared with Rv1900c. Conclusion: The study confirmed the lipolytic activity of Rv1900c and suggested its regulation by the adenylate cyclase domain and role in the intracellular survival of bacteria.

Lay abstract Tuberculosis (TB) remains the top contagious/infectious killer in the world. It is caused by the bacteria Mycobacterium tuberculosis. The bacteria resides/replicates in the immune cell that normally has to eradicate infectious microorganisms. Though the treatment of TB is available, the emergence of drug-resistant bacteria is of major concern. The treatment of drug-resistant TB has been reported to be more difficult due to lengthy and complex treatment regimens. Therefore, there is an urgent need for new and better drugs to treat TB/drug-resistant TB. For this purpose understanding the role of each protein in the physiology of mycobacteria is required. Lipids play a critical role in the intracellular survival of this pathogen in the host. Our study demonstrated that LipJ supported the intracellular survival of bacteria. Therefore, it could be a potential drug target.

Identification of sea urchin sperm adenylate cyclase.

Calmodulin (CaM) affinity chromatography of a detergent extract of sea urchin sperm yielded approximately 20 major proteins. One of these proteins, of Mr 190,000, was purified and used to immunize rabbits. After absorption with living sperm, the serum reacted monospecifically on one- and two-dimensional Western immunoblots with the Mr 190,000 protein. The anti-190-kD serum inhibited 94% of the adenylate cyclase (AC) activity of the CaM eluate. An immunoaffinity column removed 95% of the AC activity, and the purified (but inactive) Mr 190,000 protein was eluted from the column. The antiserum also inhibited 23% of the activity of bovine brain CaM-sensitive AC and 90% of the activity of horse sperm CaM-sensitive AC. These data support the hypothesis that the Mr 190,000 protein is sea urchin sperm AC. Although this AC bound to CaM, it was not possible to demonstrate directly a Ca2+ or CaM sensitivity. However, two CaM antagonists, calmidazolium and chlorpromazine, both inhibited AC activity, and the inhibition was released by added CaM, suggesting the possibility of regulation of this AC by CaM. Indirect immunofluorescence showed the Mr 190,000 protein to be highly concentrated on only the proximal half of the sea urchin sperm flagellum. This asymmetric localization of AC may be important to its function in flagellar motility. This is the first report of the identification of an AC from animal spermatozoa.

Adenylyl cyclase amino acid sequence: possible channel- or transporter-like structure.

Complementary DNA's that encode an adenylyl cyclase were isolated from a bovine brain library. Most of the deduced amino acid sequence of 1134 residues is divisible into two alternating sets of hydrophobic and hydrophilic domains. Each of the two large hydrophobic domains appears to contain six transmembrane spans. Each of the two large hydrophilic domains contains a sequence that is homologous to a single cytoplasmic domain of several guanylyl cyclases; these sequences may represent nucleotide binding sites. An unexpected topographical resemblance between adenylyl cyclase and various plasma membrane channels and transporters was observed. This structural complexity suggests possible, unappreciated functions for this important enzyme.

The effect of posttranslational modifications on the interaction of Ras2 with adenylyl cyclase.

Ras proteins undergo a series of posttranslational modifications that are critical for their cellular function. These modifications are necessary to anchor Ras proteins to the membrane. Yeast Ras2 proteins were purified with various degrees of modification and examined for their ability to activate their effector, adenylyl cyclase. The farnesylated intermediate form of Ras2 had more than 100 times higher affinity for adenylyl cyclase than for the unprocessed form. The subsequent palmitoylation reaction had little effect. In contrast, palmitoylation was required for efficient membrane localization of the Ras2 protein. These results indicate the importance of farnesylation in the interaction of Ras2 with its effector.

Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor.

Spermatozoa undergo a poorly understood activation process induced by bicarbonate and mediated by cyclic adenosine 3',5'-monophosphate (cAMP). It has been assumed that bicarbonate mediates its effects through changes in intracellular pH or membrane potential; however, we demonstrate here that bicarbonate directly stimulates mammalian soluble adenylyl cyclase (sAC) activity in vivo and in vitro in a pH-independent manner. sAC is most similar to adenylyl cyclases from cyanobacteria, and bicarbonate regulation of cyclase activity is conserved in these early forms of life. sAC is also expressed in other bicarbonate-responsive tissues, which suggests that bicarbonate regulation of cAMP signaling plays a fundamental role in many biological systems.

Structure-function relationships in Escherichia coli adenylate cyclase.

Class I adenylate cyclases are found in gamma- and delta-proteobacteria. They play central roles in processes such as catabolite repression in Escherichia coli or development of full virulence in pathogens such as Yersinia enterocolitica and Vibrio vulnificus. The catalytic domain (residues 2-446) of the adenylate cyclase of E. coli was overexpressed and purified. It displayed a V(max) of 665 nmol of cAMP x mg(-1) x min(-1) and a K(m) of 270 microM. Titration of the metal cofactor Mg(2+) against the substrate ATP showed a requirement for free metal ions in addition to the MgATP complex, suggesting a two-metal-ion mechanism as is known for class II and class III adenylate cyclases. Twelve residues which are essential for catalysis were identified by mutagenesis of a total of 20 polar residues conserved in all class I adenylate cyclases. Five essential residues (Ser(103), Ser(113), Asp(114), Asp(116) and Trp(118)) were part of a region which is found in all members of the large DNA polymerase beta-like nucleotidyltransferase superfamily. Alignment of the E. coli adenylate cyclase with the crystal structure of a distant member of the superfamily, archaeal tRNA CCA-adding enzyme, suggested that Asp(114) and Asp(116) are the metal-cofactor-ion-binding residues. The S103A mutant had a 17-fold higher K(m) than wild-type, demonstrating its important role in substrate binding. In comparison with the tRNA CCA-adding enzyme, Ser(103) of the E. coli adenylate cyclase apparently binds the gamma-phosphate group of ATP. Consistent with this function, the S103A mutation caused a marked reduction of discrimination between ATP- and ADP- or AMP-derived inhibitors.

Invasive adenylate cyclase toxin of Bordetella pertussis.

Bordetella pertussis produces an adenylate cyclase which is a toxin. The enzyme penetrates eukaryotic cells and, upon activation by host calmodulin, generates high levels of intracellular cAMP; as a result bactericidal functions of immune effector cells are considerably impaired. The toxin is composed of a single polypeptide that possesses both the catalytic and the toxic functions. It penetrates the host cell directly from the plasma membrane and is concomitantly inactivated by a proteolytic degradation.

Receptor-independent sensitization of the adenylyl cylase after chronic treatment with cyclosporine A.

Chronic treatment with cyclosporine A (CyA) is often complicated by severe hypertension. If activation of the beta-adrenergic-receptor-linked adenylyl cyclase (AC) system contributes to hypertension is unresolved. Rats were treated with CyA (20 mg kg(-1) day(-1)) for 7 days. beta-adrenergic, muscarinic, and alpha-adrenergic receptors, G-proteins, and the activity of AC were determined in cardiac and pulmonary plasma membranes. The density of cardiac beta-adrenergic receptors, muscarinic receptors, alpha-adrenergic receptors, G(alphas) and, G(alphai) remained unchanged after treatment with CyA. However, CyA increased the responsiveness of AC to different stimulators. The responsiveness of AC was even more pronounced after solubilization and partial purification, suggesting a direct modulation of the enzyme. These data suggest that CyA modulates the activity of the sympathoadrenergic system by a direct, receptor-independent sensitization of AC, suggesting that this pathway contributes to hypertension in patients treated with CyA.

Molecular, Enzymatic, and Cellular Characterization of Soluble Adenylyl Cyclase From Aquatic Animals.

The enzyme soluble adenylyl cyclase (sAC) is the most recently identified source of the messenger molecule cyclic adenosine monophosphate. sAC is evolutionarily conserved from cyanobacteria to human, is directly stimulated by [Formula: see text] ions, and can act as a sensor of environmental and metabolic CO2, pH, and [Formula: see text] levels. sAC genes tend to have multiple alternative promoters, undergo extensive alternative splicing, be translated into low mRNA levels, and the numerous sAC protein isoforms may be present in various subcellular localizations. In aquatic organisms, sAC has been shown to mediate various functions including intracellular pH regulation in coral, blood acid/base regulation in shark, heart beat rate in hagfish, and NaCl absorption in fish intestine. Furthermore, sAC is present in multiple other species and tissues, and sAC protein and enzymatic activity have been reported in the cytoplasm, the nucleus, and other subcellular compartments, suggesting even more diverse physiological roles. Although the methods and experimental tools used to study sAC are conventional, the complexity of sAC genes and proteins requires special considerations that are discussed in this chapter.

Guanine nucleotide binding regulatory proteins and adenylate cyclase in livers of streptozotocin- and BB/Wor-diabetic rats. Immunodetection of Gs and Gi with antisera prepared against synthetic peptides.

Adenylate cyclase in liver plasma membranes from streptozotocin-diabetic (STZ) or BB/Wor spontaneously diabetic rats showed increased responsiveness to GTP, glucagon, fluoroaluminate, and cholera toxin. Basal or forskolin-stimulated activity was unchanged in STZ rats, but increased in BB/Wor rats. No change in the alpha-subunit of Gi (alpha i) was observed in STZ or BB/Wor rats using pertussis toxin-stimulated [32P]ADP-ribosylation. Immunodetection using antibodies against the COOH-terminal decapeptides of alpha T and alpha i-3 showed no change in alpha i in STZ rats and a slight decrease in BB/Wor rats. Angiotensin II inhibition of hepatic adenylate cyclase was not altered in either diabetic rat. In both models of diabetes, Gs alpha-subunits were increased as measured by cholera toxin-stimulated [32P]-ADP-ribosylation of 43-47.5-kD peptides, reconstitution with membranes from S49 cyc- cells or immunoreactivity using antibodies against the COOH-terminal decapeptide of alpha s. These data indicate that STZ-diabetes increases hepatic Gs but does not change Gi or adenylate cyclase catalytic activity. In contrast, BB/Wor rats show increased hepatic Gs and adenylate cyclase. These changes could explain the increase in hepatic cAMP and related dysfunctions observed in diabetes.

Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method.

We developed a method for immunoaffinity purification of Saccharomyces cerevisiae adenylyl cyclase based on creating a fusion with a small peptide epitope. Using oligonucleotide technology to encode the peptide epitope we constructed a plasmid that expressed the fusion protein from the S. cerevisiae alcohol dehydrogenase promoter ADH1. A monoclonal antibody previously raised against the peptide was used to purify adenylyl cyclase by affinity chromatography. The purified enzyme appeared to be a multisubunit complex consisting of the 200-kilodalton adenylyl cyclase fusion protein and an unidentified 70-kilodalton protein. The purified protein could be activated by RAS proteins. Activation had an absolute requirement for a guanine nucleoside triphosphate.

Mutational mapping of RAS-responsive domains of the Saccharomyces cerevisiae adenylyl cyclase.

Large deletion and small insertion mutations in the adenylyl cyclase gene of Saccharomyces cerevisiae were used to map regions required for activation by RAS protein in vitro. The amino-terminal 605 amino acids were found to be dispensable for responsiveness to RAS protein. All other deletions in adenylyl cyclase destroyed its ability to respond to RAS. Small insertion mutations within the leucine-rich repeat region also prevented RAS responsiveness, while other insertions did not.

Adenylate cyclase in Saccharomyces cerevisiae is a peripheral membrane protein.

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cyclic AMP (cAMP) from ATP, and two RAS polypeptides, which mediate stimulation of cAMP synthesis of guanine nucleotides. By analogy to the mammalian enzyme, models of yeast adenylate cyclase have depicted the enzyme as a membrane protein. We have concluded that adenylate cyclase is only peripherally bound to the yeast membrane, based on the following criteria: (i) substantial activity was found in cytoplasmic fractions; (ii) activity was released from membranes by the addition of 0.5 M NaCl; (iii) in the presence of 0.5 M NaCl, activity in detergent extracts had hydrodynamic properties identical to those of cytosolic or NaCl-extracted enzyme; (iv) antibodies to yeast adenylate cyclase identified a full-length adenylate cyclase in both membrane and cytosol fractions; and (v) activity from both cytosolic fractions and NaCl extracts could be functionally reconstituted into membranes lacking adenylate cyclase activity. The binding of adenylate cyclase to the membrane may have regulatory significance; the fraction of activity associated with the membrane increased as cultures approached stationary phase. In addition, binding of adenylate cyclase to membranes appeared to be inhibited by cAMP. These results indicate the existence of a protein anchoring adenylate cyclase to the membrane. The identity of this protein remains unknown.

Signaling through adenylyl cyclase is essential for hyphal growth and virulence in the pathogenic fungus Candida albicans.

The human fungal pathogen Candida albicans switches from a budding yeast form to a polarized hyphal form in response to various external signals. This morphogenetic switching has been implicated in the development of pathogenicity. We have cloned the CaCDC35 gene encoding C. albicans adenylyl cyclase by functional complementation of the conditional growth defect of Saccharomyces cerevisiae cells with mutations in Ras1p and Ras2p. It has previously been shown that these Ras homologues regulate adenylyl cyclase in yeast. The C. albicans adenylyl cyclase is highly homologous to other fungal adenylyl cyclases but has less sequence similarity with the mammalian enzymes. C. albicans cells deleted for both alleles of CaCDC35 had no detectable cAMP levels, suggesting that this gene encodes the only adenylyl cyclase in C. albicans. The homozygous mutant cells were viable but grew more slowly than wild-type cells and were unable to switch from the yeast to the hyphal form under all environmental conditions that we analyzed in vitro. Moreover, this morphogenetic switch was completely blocked in mutant cells undergoing phagocytosis by macrophages. However, morphogenetic switching was restored by exogenous cAMP. On the basis of epistasis experiments, we propose that CaCdc35p acts downstream of the Ras homologue CaRas1p. These epistasis experiments also suggest that the putative transcription factor Efg1p and components of the hyphal-inducing MAP kinase pathway depend on the function of CaCdc35p in their ability to induce morphogenetic switching. Homozygous cacdc35 Delta cells were unable to establish vaginal infection in a mucosal membrane mouse model and were avirulent in a mouse model for systemic infections. These findings suggest that fungal adenylyl cyclases and other regulators of the cAMP signaling pathway may be useful targets for antifungal drugs.

Bicarbonate-sensing soluble adenylyl cyclase is present in the cell cytoplasm and nucleus of multiple shark tissues.

The enzyme soluble adenylyl cyclase (sAC) is directly stimulated by bicarbonate (HCO3-) to produce the signaling molecule cyclic adenosine monophosphate (cAMP). Because sAC and sAC-related enzymes are found throughout phyla from cyanobacteria to mammals and they regulate cell physiology in response to internal and external changes in pH, CO2, and HCO3-, sAC is deemed an evolutionarily conserved acid-base sensor. Previously, sAC has been reported in dogfish shark and round ray gill cells, where they sense and counteract blood alkalosis by regulating the activity of V-type H+- ATPase. Here, we report the presence of sAC protein in gill, rectal gland, cornea, intestine, white muscle, and heart of leopard shark Triakis semifasciata Co-expression of sAC with transmembrane adenylyl cyclases supports the presence of cAMP signaling microdomains. Furthermore, immunohistochemistry on tissue sections, and western blots and cAMP-activity assays on nucleus-enriched fractions demonstrate the presence of sAC protein in and around nuclei. These results suggest that sAC modulates multiple physiological processes in shark cells, including nuclear functions.

Crystallization of the class IV adenylyl cyclase from Yersinia pestis.

The class IV adenylyl cyclase from Yersinia pestis has been cloned and crystallized in both a triclinic and an orthorhombic form. An amino-terminal His-tagged construct, from which the tag was removed by thrombin, crystallized in a triclinic form diffracting to 1.9 A, with one dimer per asymmetric unit and unit-cell parameters a = 33.5, b = 35.5, c = 71.8 A, alpha = 88.7, beta = 82.5, gamma = 65.5 degrees. Several mutants of this construct crystallized but diffracted poorly. A non-His-tagged native construct (179 amino acids, MW = 20.5 kDa) was purified by conventional chromatography and crystallized in space group P2(1)2(1)2(1). These crystals have unit-cell parameters a = 56.8, b = 118.6, c = 144.5 A, diffract to 3 A and probably have two dimers per asymmetric unit and VM = 3.0 A3 Da(-1). Both crystal forms appear to require pH below 5, complicating attempts to incorporate nucleotide ligands into the structure. The native construct has been produced as a selenomethionine derivative and crystallized for phasing and structure determination.

Multiple forms of brain adenylate cyclase: stimulation by Mn2+.

Mn2+-stimulated adenylate cyclase (ATP pyrophosphate-lyase-(cyclizing), EC 4.6.1.1) activity in detergent solubilized preparations from mouse brain. While NaF-stimulated activity was decreased by both solubilization and storage at 0--4 degrees C, the ability of the enzyme to be stimulated by Mn2+ was maintained for up to one week. By including Mn2+ in the assay of adenylate cyclase in gel fractions after isoelectric focusing, two distinct peaks of enzyme activity (pI1 - 5.8, pI2 = 6.4) were detected, suggesting the existence of more than one type of catalytic subunit in mouse brain cell membranes.

Preparative isoelectric focusing and some properties of solubilized adenylate cyclase from rat brain.

A new procedure for the purification of rat brain adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) is presented. The enzyme solubilized in Lubrol PX was purified either by molecular sieving or by hydrophobic chromatography, followed by a preparative isoelectric focusing step. For this purpose, a new isoelectric focusing technique was developed which allows a good resolution of adenylate cyclase in a short period of time. When resolved by this procedure, the enzyme migrated as a single molecular species with a pI of 6.3. When isoelectric focusing was performed in the presence of EGTA, two distinct peaks of activity could be detected at pI 6.1 and 7.3. This suggests that adenylate cyclase consists of two subunits held together by divalent ions. It is shown that the purified adenylate cyclase has a smaller sedimentation coefficient and is less hydrophobic than the native one. We conclude that the adenylate cyclase containing complex was at least partially disaggregated by this procedure.