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Scott A Waldman - One of the best experts on this subject based on the ideXlab platform.
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guanylyl Cyclases and signaling by cyclic gmp
Pharmacological Reviews, 2000Co-Authors: Kimberly A Lucas, Stephanie Schulz, Giovanni Pitari, Shiva Kazerounian, Inez Ruizstewart, Jason Y Park, Kenneth P Chepenik, Scott A WaldmanAbstract:Guanylyl Cyclases are a family of enzymes that catalyze the conversion of GTP to cGMP. The family comprises both membrane-bound and soluble isoforms that are expressed in nearly all cell types. They are regulated by diverse extracellular agonists that include peptide hormones, bacterial toxins, and free radicals, as well as intracellular molecules, such as calcium and adenine nucleotides. Stimulation of guanylyl Cyclases and the resultant accumulation of cGMP regulates complex signaling cascades through immediate downstream effectors, including cGMP-dependent protein kinases, cGMP-regulated phosphodiesterases, and cyclic nucleotide-gated ion channels. Guanylyl Cyclases and cGMP-mediated signaling cascades play a central role in the regulation of diverse (patho)physiological processes, including vascular smooth muscle motility, intestinal fluid and electrolyte homeostasis, and retinal phototransduction. Topics addressed in this review include the structure and chromosomal localization of the genes for guanylyl Cyclases, structure and function of the members of the guanylyl Cyclase family, molecular mechanisms regulating enzymatic activity, and molecular sequences coupling ligand binding to catalytic activity. A brief overview is presented of the downstream events controlled by guanylyl Cyclases, including the effectors that are regulated by cGMP and the role that guanylyl Cyclases play in cell physiology and pathophysiology.
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the guanylyl Cyclase family of natriuretic peptide receptors
Vitamins and Hormones Series, 1997Co-Authors: Stephanie Schulz, Scott A WaldmanAbstract:Guanylyl Cyclases are cytoplasmic and membrane-associated enzymes that catalyze the conversion of GTP to cyclic GMP, an intracellular signaling molecule. Molecular cloning has identified a multigene family encoding both soluble and particulate forms of the enzymes. Diffusible agents such as nitric oxide and carbon monoxide activate the soluble guanylyl Cyclases. The particulate members of the family share a characteristic domain arrangement, with a single transmembrane span separating a variable extracellular ligand-binding domain from a conserved intracellular regulatory and Cyclase catalytic domain. Seven members of the particulate guanylyl Cyclase family have been identified, and they include the receptors for natriuretic peptides and Escherichia coli heat-stable enterotoxin. Recently, animal models have been developed to study the role of natriuretic peptides and their guanylyl Cyclase-coupled receptors in renal and cardiovascular physiology.
Lonny R Levin - One of the best experts on this subject based on the ideXlab platform.
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pharmacological modulation of the co2 hco3 ph calcium and atp sensing soluble adenylyl Cyclase
Pharmacology & Therapeutics, 2018Co-Authors: Shakarr V Wiggins, Lonny R Levin, Clemens Steegborn, Jochen BuckAbstract:Cyclic AMP (cAMP), the prototypical second messenger, has been implicated in a wide variety of (often opposing) physiological processes. It simultaneously mediates multiple, diverse processes, often within a single cell, by acting locally within independently-regulated and spatially-restricted microdomains. Within each microdomain, the level of cAMP will be dependent upon the balance between its synthesis by adenylyl Cyclases and its degradation by phosphodiesterases (PDEs). In mammalian cells, there are many PDE isoforms and two types of adenylyl Cyclases; the G protein regulated transmembrane adenylyl Cyclases (tmACs) and the CO2/HCO3-/pH-, calcium-, and ATP-sensing soluble adenylyl Cyclase (sAC). Discriminating the roles of individual cyclic nucleotide microdomains requires pharmacological modulators selective for the various PDEs and/or adenylyl Cyclases. Such tools present an opportunity to develop therapeutics specifically targeted to individual cAMP dependent pathways. The pharmacological modulators of tmACs have recently been reviewed, and in this review, we describe the current status of pharmacological tools available for studying sAC.
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glucose and glp 1 stimulate camp production via distinct adenylyl Cyclases in ins 1e insulinoma cells
The Journal of General Physiology, 2008Co-Authors: Lavoisier S Ramos, Jochen Buck, Margarita Kamenetsky, Jonathan H. Zippin, Lonny R LevinAbstract:In β cells, both glucose and hormones, such as GLP-1, stimulate production of the second messenger cAMP, but glucose and GLP-1 elicit distinct cellular responses. We now show in INS-1E insulinoma cells that glucose and GLP-1 produce cAMP with distinct kinetics via different adenylyl Cyclases. GLP-1 induces a rapid cAMP signal mediated by G protein–responsive transmembrane adenylyl Cyclases (tmAC). In contrast, glucose elicits a delayed cAMP rise mediated by bicarbonate, calcium, and ATP-sensitive soluble adenylyl Cyclase (sAC). This glucose-induced, sAC-dependent cAMP rise is dependent upon calcium influx and is responsible for the glucose-induced activation of the mitogen-activated protein kinase (ERK1/2) pathway. These results demonstrate that sAC-generated and tmAC-generated cAMP define distinct signaling cascades.
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soluble adenylyl Cyclase mediates nerve growth factor induced activation of rap1
Journal of Biological Chemistry, 2006Co-Authors: Alexander M Stessin, Jochen Buck, Kenneth C Hess, Margarita Kamenetsky, Jonathan H. Zippin, Lonny R LevinAbstract:Nerve growth factor (NGF) and the ubiquitous second messenger cyclic AMP (cAMP) are both implicated in neuronal differentiation. Multiple studies indicate that NGF signals to at least a subset of its targets via cAMP, but the link between NGF and cAMP has remained elusive. Here, we have described the use of small molecule inhibitors to differentiate between the two known sources of cAMP in mammalian cells, bicarbonate- and calcium-responsive soluble adenylyl Cyclase (sAC) and G protein-regulated transmembrane adenylyl Cyclases. These inhibitors, along with sAC-specific small interfering RNA, reveal that sAC is uniquely responsible for the NGF-elicited rise in cAMP and is essential for the NGF-induced activation of the small G protein Rap1 in PC12 cells. In contrast and as expected, transmembrane adenylyl Cyclase-generated cAMP is responsible for Rap1 activation by the G protein-coupled receptor ligand PACAP (pituitary adenylyl Cyclase-activating peptide). These results identify sAC as a mediator of NGF signaling and reveal the existence of distinct pathways leading to cAMP-dependent signal transduction.
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A Novel Mechanism for Adenylyl Cyclase Inhibition from the Crystal Structure of Its Complex with Catechol Estrogen
The Journal of biological chemistry, 2005Co-Authors: Clemens Steegborn, Jochen Buck, Kenneth C Hess, Tatiana N Litvin, Austin B. Capper, Ronald Taussig, Lonny R LevinAbstract:Catechol estrogens are steroid metabolites that elicit physiological responses through binding to a variety of cellular targets. We show here that catechol estrogens directly inhibit soluble adenylyl Cyclases and the abundant trans-membrane adenylyl Cyclases. Catechol estrogen inhibition is non-competitive with respect to the substrate ATP, and we solved the crystal structure of a catechol estrogen bound to a soluble adenylyl Cyclase from Spirulina platensis in complex with a substrate analog. The catechol estrogen is bound to a newly identified, conserved hydrophobic patch near the active center but distinct from the ATP-binding cleft. Inhibitor binding leads to a chelating interaction between the catechol estrogen hydroxyl groups and the catalytic magnesium ion, distorting the active site and trapping the enzyme substrate complex in a non-productive conformation. This novel inhibition mechanism likely applies to other adenylyl Cyclase inhibitors, and the identified ligand-binding site has important implications for the development of specific adenylyl Cyclase inhibitors.
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Purification of soluble adenylyl Cyclase.
Methods in enzymology, 2002Co-Authors: Jochen Buck, Meeghan L Sinclair, Lonny R LevinAbstract:Cyclic AMP (cAMP), the nearly universal second messenger, is synthesized by a broad family of adenylyl Cyclases (ACs). Mammals have at least nine isoforms of transmembrane-spanning adenylyl Cyclases (tmACs). These differ in their expression patterns, and their catalytic activities are differentially regulated by G proteins and other signaling molecules. Mammals also possess a cylosolic form of AC. Originally detected solely in soluble extracts derived from mammalian testis, the catalytic domains of soluble AC (sAC) are more closely related to (cyano)bacterial adenylyl Cyclases than to the hormone-responsive, G-protein-regulated mammalian tmACs. Molecular isolation of the sAC gene revealed this form of mammalian adenylyl Cyclase to be more similar to (cyano) bacterial adenylyl Cyclases than to the more widely studied, hormone-responsive. Future studies of sAC will place it into the context of the mammalian tmACs, and will provide a greater understanding of the cellular role of cAMP.
Alfred G. Gilman - One of the best experts on this subject based on the ideXlab platform.
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interactions of forskolin and atp with the cytosolic domains of mammalian adenylyl Cyclase
Journal of Biological Chemistry, 1997Co-Authors: Carmen W. Dessauer, Tracy T Scully, Alfred G. GilmanAbstract:Fragments of the two cytoplasmic domains of mammalian adenylyl Cyclases can be synthesized independently (and abundantly) as soluble proteins; Gsalpha- and forskolin-stimulated enzymatic activity is restored upon their mixture. We have utilized this system to characterize the interactions of adenylyl Cyclase with forskolin and its substrate, ATP. In the presence of Gsalpha, adenylyl Cyclase is activated in response to occupation of only one forskolin-binding site. A single binding site for forskolin was identified by equilibrium dialysis; its Kd (0.1 microM) corresponds to the EC50 for enzyme activation. The affinity of forskolin for adenylyl Cyclase is greatly reduced in the absence of Gsalpha ( approximately 40 microM). Binding of forskolin to the individual cytoplasmic domains of the enzyme was not detected. A single binding site for the ATP analog, alpha,beta-methylene ATP (Ap(CH2)pp), was also detected by equilibrium dialysis. Such binding was not observed with the individual domains. Binding of Ap(CH2)pp was unaffected by P-site inhibitors of adenylyl Cyclase. A modified P-loop sequence located near the carboxyl terminus of adenylyl Cyclase has been implicated in ATP binding. Mutation of the conserved, non-glycine residues within this region caused no significant changes in the Km for ATP or the Ki for Ap(CH2)pp. It thus seems unlikely that this region is part of the active site. However, a mutation in the C1 domain (E518A) causes a 10-fold decrease in the binding affinity for Ap(CH2)pp. This residue and the active site of the enzyme may lie at the interface between the two cytosolic domains.
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purification and characterization of a soluble form of mammalian adenylyl Cyclase
Journal of Biological Chemistry, 1996Co-Authors: Carmen W. Dessauer, Alfred G. GilmanAbstract:An engineered, soluble form of mammalian adenylyl Cyclase has been expressed in Escherichia coli and purified by three chromatographic steps. The enzyme utilizes one molecule of ATP to synthesize one molecule of cyclic AMP and pyrophosphate at a maximal specific activity of 12.8 µmol/min/mg, corresponding to a turnover number of 720 min−1. Although devoid of membrane spans, the enzyme displays all of the regulatory properties that are common to mammalian adenylyl Cyclases. It is activated synergistically by Gsα and forskolin and is inhibited by adenosine (P-site) analogs with kinetic patterns that are identical to those displayed by the native enzymes. The purified enzyme is also inhibited directly by the G protein βγ subunit complex. After adenovirus-mediated expression in adenylyl Cyclase-deficient HC-1 cells, the enzyme can be stimulated synergistically by Gs-coupled receptors and forskolin.
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construction of a soluble adenylyl Cyclase activated by gs alpha and forskolin
Science, 1995Co-Authors: Weijen Tang, Alfred G. GilmanAbstract:A soluble adenylyl Cyclase was constructed by linkage of portions of the cytosolic domains of the mammalian type I and type II enzymes. The soluble enzyme was stimulated by both forskolin and the alpha subunit of the heterotrimeric guanine nucleotide-binding protein (G protein) Gs (Gs alpha). Expression of the construct complemented the catabolic defect in a strain of Escherichia coli that is deficient in adenylyl Cyclase activity. The active, approximately 60-kilodalton enzyme accumulated in the cytoplasmic fraction of E. coli to yield activities in excess of 1 nanomole per minute per milligram of protein. The two sets of transmembrane helices of mammalian adenylyl Cyclases are thus not necessary for the catalytic or the most characteristic regulatory activities of the enzyme. This system may be useful for both genetic and biochemical analysis of G protein-regulated adenylyl Cyclases.
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Cloning and expression of a widely distributed (type IV) adenylyl Cyclase
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Boning Gao, Alfred G. GilmanAbstract:Abstract We have cloned and expressed a cDNA that encodes a widely distributed form of mammalian adenylyl Cyclase (EC 4.6.1.1). Although those adenylyl Cyclases described previously have a rather narrow tissue distribution, this enzyme (type IV) is apparently synthesized in a variety of peripheral tissues and in the central nervous system. The protein resembles the other adenylyl Cyclases in its proposed structure. It most resembles the type II adenylyl Cyclase described in the preceding paper [Feinstein, P. G., Schrader, K. A., Bakalyar, H. A., Tang, W.-J., Krupinski, J., Gilman, A. G. & Reed, R. R. (1991) Proc. Natl. Acad. Sci. USA 88, 10173-10177] in its amino acid sequence, lack of response to calmodulin, and synergistic activation by a combination of the Gs alpha subunit and the G-protein beta gamma subunit complex.
David W. Christianson - One of the best experts on this subject based on the ideXlab platform.
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discovery of the cryptic function of terpene Cyclases as aromatic prenyltransferases
Nature Communications, 2020Co-Authors: Guangkai Bian, David W. Christianson, Takahiro Mori, Corey J Herbstgervasoni, Stephen A Shinsky, Anwei Hou, Minjian Huang, Shu Cheng, Zixin Deng, Ikuro AbeAbstract:Catalytic versatility is an inherent property of many enzymes. In nature, terpene Cyclases comprise the foundation of molecular biodiversity as they generate diverse hydrocarbon scaffolds found in thousands of terpenoid natural products. Here, we report that the catalytic activity of the terpene Cyclases AaTPS and FgGS can be switched from Cyclase to aromatic prenyltransferase at basic pH to generate prenylindoles. The crystal structures of AaTPS and FgGS provide insights into the catalytic mechanism of this cryptic function. Moreover, aromatic prenyltransferase activity discovered in other terpene Cyclases indicates that this cryptic function is broadly conserved among the greater family of terpene Cyclases. We suggest that this cryptic function is chemoprotective for the cell by regulating isoprenoid diphosphate concentrations so that they are maintained below toxic thresholds.
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Structure of 2-methylisoborneol synthase from Streptomyces coelicolor and implications for the cyclization of a noncanonical C-methylated monoterpenoid substrate.
Biochemistry, 2012Co-Authors: Mustafa Köksal, Wayne Kw Chou, David E Cane, David W. ChristiansonAbstract:The crystal structure of 2-methylisoborneol synthase (MIBS) from Streptomyces coelicolor A3(2) has been determined in complex with substrate analogues geranyl-S-thiolodiphosphate and 2-fluorogeranyl diphosphate at 1.80 and 1.95 A resolution, respectively. This terpenoid Cyclase catalyzes the cyclization of the naturally occurring, noncanonical C-methylated isoprenoid substrate, 2-methylgeranyl diphosphate, to form the bicyclic product 2-methylisoborneol, a volatile C11 homoterpene alcohol with an earthy, musty odor. While MIBS adopts the tertiary structure of a class I terpenoid Cyclase, its dimeric quaternary structure differs from that previously observed in dimeric terpenoid Cyclases from plants and fungi. The quaternary structure of MIBS is nonetheless similar in some respects to that of dimeric farnesyl diphosphate synthase, which is not a Cyclase. The structures of MIBS complexed with substrate analogues provide insights regarding differences in the catalytic mechanism of MIBS and the mechanisms of ...
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structure and mechanism of the diterpene Cyclase ent copalyl diphosphate synthase
Nature Chemical Biology, 2011Co-Authors: Mustafa Köksal, Robert M Coates, Reuben J Peters, David W. ChristiansonAbstract:The structure of ent-copalyl diphosphate synthase reveals three α-helical domains (α, β and γ), as also observed in the related diterpene Cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in ent-copalyl diphosphate synthase but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene Cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.
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taxadiene synthase structure and evolution of modular architecture in terpene biosynthesis
Nature, 2011Co-Authors: Mustafa Köksal, Rodney Croteau, Robert M Coates, David W. ChristiansonAbstract:The first step in the biosynthesis of the anticancer compound Taxol (paclitaxel) and many other natural C20 diterpenes is the cyclization of an isoprenoid, catalysed by taxadiene synthase. The X-ray crystal structure of this enzyme from the Pacific yew has now been determined. Its C-terminal catalytic domain binds and activates the substrate in a manner seen in class I terpenoid Cyclases, but the N-terminal domain and a third 'insertion' domain adopt the fold of a class II terpenoid Cyclase. This suggests that this enzyme could be the ancestral progenitor of all terpenoid Cyclases. The first X-ray crystal structure of a diterpene Cyclase is reported — this enzyme, taxadiene synthase, catalyses the cyclization of an isoprenoid in the first committed step of the biosynthesis of the cancer chemotherapeutic drug Taxol. The C-terminal catalytic domain binds and activates the substrate in a manner seen in class I terpenoid Cyclases, but the N-terminal domain and a third 'insertion' domain together adopt the fold of a class II terpenoid Cyclase. It is proposed that this enzyme could be the ancestral progenitor of all terpenoid Cyclases. With more than 55,000 members identified so far in all forms of life, the family of terpene or terpenoid natural products represents the epitome of molecular biodiversity. A well-known and important member of this family is the polycyclic diterpenoid Taxol (paclitaxel), which promotes tubulin polymerization1 and shows remarkable efficacy in cancer chemotherapy2. The first committed step of Taxol biosynthesis in the Pacific yew (Taxus brevifolia)3 is the cyclization of the linear isoprenoid substrate geranylgeranyl diphosphate (GGPP) to form taxa-4(5),11(12)diene4, which is catalysed by taxadiene synthase5. The full-length form of this diterpene Cyclase contains 862 residues, but a roughly 80-residue amino-terminal transit sequence is cleaved on maturation in plastids6. We now report the X-ray crystal structure of a truncation variant lacking the transit sequence and an additional 27 residues at the N terminus, hereafter designated TXS. Specifically, we have determined structures of TXS complexed with 13-aza-13,14-dihydrocopalyl diphosphate (1.82 A resolution) and 2-fluorogeranylgeranyl diphosphate (2.25 A resolution). The TXS structure reveals a modular assembly of three α-helical domains. The carboxy-terminal catalytic domain is a class I terpenoid Cyclase, which binds and activates substrate GGPP with a three-metal ion cluster. The N-terminal domain and a third ‘insertion’ domain together adopt the fold of a vestigial class II terpenoid Cyclase. A class II Cyclase activates the isoprenoid substrate by protonation instead of ionization, and the TXS structure reveals a definitive connection between the two distinct Cyclase classes in the evolution of terpenoid biosynthesis.
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crystal structure determination of aristolochene synthase from the blue cheese mold penicillium roqueforti
Journal of Biological Chemistry, 2000Co-Authors: Jonathan M Caruthers, David E Cane, Ilgu Kang, M J Rynkiewicz, David W. ChristiansonAbstract:Abstract The 2.5-A resolution crystal structure of recombinant aristolochene synthase from the blue cheese mold,Penicillium roqueforti, is the first of a fungal terpenoid Cyclase. The structure of the enzyme reveals active site features that participate in the cyclization of the universal sesquiterpene Cyclase substrate, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. Metal-triggered carbocation formation initiates the cyclization cascade, which proceeds through multiple complex intermediates to yield one exclusive structural and stereochemical isomer of aristolochene. Structural homology of this fungal Cyclase with plant and bacterial terpenoid Cyclases, despite minimal amino acid sequence identity, suggests divergence from a common, primordial ancestor in the evolution of terpene biosynthesis.
Stephanie Schulz - One of the best experts on this subject based on the ideXlab platform.
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guanylyl Cyclases and signaling by cyclic gmp
Pharmacological Reviews, 2000Co-Authors: Kimberly A Lucas, Stephanie Schulz, Giovanni Pitari, Shiva Kazerounian, Inez Ruizstewart, Jason Y Park, Kenneth P Chepenik, Scott A WaldmanAbstract:Guanylyl Cyclases are a family of enzymes that catalyze the conversion of GTP to cGMP. The family comprises both membrane-bound and soluble isoforms that are expressed in nearly all cell types. They are regulated by diverse extracellular agonists that include peptide hormones, bacterial toxins, and free radicals, as well as intracellular molecules, such as calcium and adenine nucleotides. Stimulation of guanylyl Cyclases and the resultant accumulation of cGMP regulates complex signaling cascades through immediate downstream effectors, including cGMP-dependent protein kinases, cGMP-regulated phosphodiesterases, and cyclic nucleotide-gated ion channels. Guanylyl Cyclases and cGMP-mediated signaling cascades play a central role in the regulation of diverse (patho)physiological processes, including vascular smooth muscle motility, intestinal fluid and electrolyte homeostasis, and retinal phototransduction. Topics addressed in this review include the structure and chromosomal localization of the genes for guanylyl Cyclases, structure and function of the members of the guanylyl Cyclase family, molecular mechanisms regulating enzymatic activity, and molecular sequences coupling ligand binding to catalytic activity. A brief overview is presented of the downstream events controlled by guanylyl Cyclases, including the effectors that are regulated by cGMP and the role that guanylyl Cyclases play in cell physiology and pathophysiology.
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the guanylyl Cyclase family of natriuretic peptide receptors
Vitamins and Hormones Series, 1997Co-Authors: Stephanie Schulz, Scott A WaldmanAbstract:Guanylyl Cyclases are cytoplasmic and membrane-associated enzymes that catalyze the conversion of GTP to cyclic GMP, an intracellular signaling molecule. Molecular cloning has identified a multigene family encoding both soluble and particulate forms of the enzymes. Diffusible agents such as nitric oxide and carbon monoxide activate the soluble guanylyl Cyclases. The particulate members of the family share a characteristic domain arrangement, with a single transmembrane span separating a variable extracellular ligand-binding domain from a conserved intracellular regulatory and Cyclase catalytic domain. Seven members of the particulate guanylyl Cyclase family have been identified, and they include the receptors for natriuretic peptides and Escherichia coli heat-stable enterotoxin. Recently, animal models have been developed to study the role of natriuretic peptides and their guanylyl Cyclase-coupled receptors in renal and cardiovascular physiology.
