Guanylyl cyclase sees the light.
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ABSTRACT:
SUBMITTER: Tesmer JJ
PROVIDER: S-EPMC2776402 | biostudies-literature | 2008 Oct
REPOSITORIES: biostudies-literature
Publications
Cyclic guanosine monophosphate (cGMP) is a critical second messenger that regulates cardiovascular function and vision in humans. Two recent papers, including one in BMC Structural Biology, have revealed atomic structures of the enzymes that catalyze the synthesis of cGMP providing new clues about the molecular basis of substrate specificity and allosteric regulation in nucleotide cyclases. ...[more]
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Project description:BackgroundMicrobial rhodopsins vary in their chemical properties, from light sensitive ion transport to different enzymatic activities. Recently, a novel family of two-component Cyclase (rhod)opsins (2c-Cyclop) from the green algae Chlamydomonas reinhardtii and Volvox carteri was characterized, revealing a light-inhibited guanylyl cyclase (GC) activity. More genes similar to 2c-Cyclop exist in algal genomes, but their molecular and physiological functions remained uncharacterized.ResultsChlamyopsin-5 (Cop5) from C. reinhardtii is related to Cr2c-Cyclop1 (Cop6) and can be expressed in Xenopus laevis oocytes, but shows no GC activity. Here, we exchanged parts of Cop5 with the corresponding ones of Cr2c-Cyclop1. When exchanging the opsin part of Cr2c-Cyclop1 with that of Cop5, we obtained a bi-stable guanylyl cyclase (switch-Cyclop1) whose activity can be switched by short light flashes. The GC activity of switch-Cyclop1 is increased for hours by a short 380 nm illumination and switched off (20-fold decreased) by blue or green light. switch-Cyclop1 is very light-sensitive and can half-maximally be activated by ~ 150 photons/nm2 of 380 nm (~ 73 J/m2) or inhibited by ~ 40 photons/nm2 of 473 nm (~ 18 J/m2).ConclusionsThis engineered guanylyl cyclase is the first light-switchable enzyme for cGMP level regulation. Light-regulated cGMP production with high light-sensitivity is a promising technique for the non-invasive investigation of the effects of cGMP signaling in many different tissues.
| S-EPMC8006352 | biostudies-literature
Project description: Not available
| S-EPMC2884595 | biostudies-literature
Project description:Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC ?1?1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides.
| S-EPMC3726482 | biostudies-literature
Project description:The nitric oxide/soluble guanylyl cyclase (NO-sGC) signaling pathway regulates the cardiovascular, neuronal, and gastrointestinal systems. Impaired sGC signaling can result in disease and system-wide organ failure. This review seeks to examine the redox control of sGC through heme and cysteine regulation while discussing therapeutic drugs that target various conditions. Heme regulation involves mechanisms of insertion of the heme moiety into the sGC protein, the molecules and proteins that control switching between the oxidized (Fe3+) and reduced states (Fe2+), and the activity of heme degradation. Modifications to cysteine residues by S-nitrosation on the α1 and β1 subunits of sGC have been shown to be important in sGC signaling. Moreover, redox balance and localization of sGC is thought to control downstream effects. In response to altered sGC activity due to changes in the redox state, many therapeutic drugs have been developed to target decreased NO-sGC signaling. The importance and relevance of sGC continues to grow as sGC dysregulation leads to numerous disease conditions.
| S-EPMC5916318 | biostudies-literature
Project description:BackgroundThe green algae Chlamydomonas reinhardtii and Volvox carteri are important models for studying light perception and response, expressing many different photoreceptors. More than 10 opsins were reported in C. reinhardtii, yet only two-the channelrhodopsins-were functionally characterized. Characterization of new opsins would help to understand the green algae photobiology and to develop new tools for optogenetics.ResultsHere we report the characterization of a novel opsin family from these green algae: light-inhibited guanylyl cyclases regulated through a two-component-like phosphoryl transfer, called "two-component cyclase opsins" (2c-Cyclops). We prove the existence of such opsins in C. reinhardtii and V. carteri and show that they have cytosolic N- and C-termini, implying an eight-transmembrane helix structure. We also demonstrate that cGMP production is both light-inhibited and ATP-dependent. The cyclase activity of Cr2c-Cyclop1 is kept functional by the ongoing phosphorylation and phosphoryl transfer from the histidine kinase to the response regulator in the dark, proven by mutagenesis. Absorption of a photon inhibits the cyclase activity, most likely by inhibiting the phosphoryl transfer. Overexpression of Vc2c-Cyclop1 protein in V. carteri leads to significantly increased cGMP levels, demonstrating guanylyl cyclase activity of Vc2c-Cyclop1 in vivo. Live cell imaging of YFP-tagged Vc2c-Cyclop1 in V. carteri revealed a development-dependent, layer-like structure at the immediate periphery of the nucleus and intense spots in the cell periphery.ConclusionsCr2c-Cyclop1 and Vc2c-Cyclop1 are light-inhibited and ATP-dependent guanylyl cyclases with an unusual eight-transmembrane helix structure of the type I opsin domain which we propose to classify as type Ib, in contrast to the 7 TM type Ia opsins. Overexpression of Vc2c-Cyclop1 protein in V. carteri led to a significant increase of cGMP, demonstrating enzyme functionality in the organism of origin. Fluorescent live cell imaging revealed that Vc2c-Cyclop1 is located in the periphery of the nucleus and in confined areas at the cell periphery.
| S-EPMC6284317 | biostudies-literature
Project description:Genomic and behavioral investigations were performed to determine the effects of a mutation in a Drosophila soluble guanylyl cyclase gene. A mutant DGCalpha1[3] third chromosome was crossed into a natural rover (for[R]) or natural sitter (for[s]) genetic background. (See Osborne et al. 1997; PMID: 9242616.) First instar larvae were collected and grown on 60mm Petri plates containing 10 mL of food until mid-third instar. (Approximate density was 3 animals per mL food). Larvae were collected and washed quickly with distilled water and were flash frozen in liquid nitrogen. Co-reared larvae were tested for behavioural effects. Four independent collections were made for each of the two conditions (Rover_DGCalpha1[3] or sitter_DGCalpha1[3]). Keywords = Drosophila Keywords = foraging Keywords = behavior Keywords = cGMP Keywords = guanylyl cyclase Keywords = genetic background Keywords: other
2005-05-01 | GSE2136 | GEO
Project description:Guanylyl cyclase activating protein 1 (GCAP1), after substitution of Ca(2+) by Mg(2+) in its EF-hands, stimulates photoreceptor guanylyl cyclase, RetGC1, in response to light. We inactivated metal binding in individual EF-hands of GCAP1 tagged with green fluorescent protein to assess their role in GCAP1 binding to RetGC1 in co-transfected HEK293 cells. When expressed alone, GCAP1 was uniformly distributed throughout the cytoplasm and the nuclei of the cells, but when co-expressed with either fluorescently tagged or non-tagged RetGC1, it co-localized with the cyclase in the membranes. The co-localization did not occur when the C-terminal portion of RetGC1, containing its regulatory and catalytic domains, was removed. Mutations that preserved Mg(2+) binding in all three metal-binding EF-hands did not affect GCAP1 association with the cyclase in live cells. Locking EF-hand 4 in its apo-conformation, incapable of binding either Ca(2+) or Mg(2+), had no effect on GCAP1 association with the cyclase. In contrast to EF-hand 4, inactivation of EF-hand 3 reduced the efficiency of the co-localization, and inactivation of EF-hand 2 drastically suppressed GCAP1 binding to the cyclase. These results directly demonstrate that metal binding in EF-hand 2 is crucial for GCAP1 attachment to RetGC1, and that in EF-hand 3 it is less critical, although it enhances the efficiency of the GCAP1 docking on the target enzyme. Metal binding in EF-hand 4 has no role in the primary attachment of GCAP1 to the cyclase, and it only triggers the activator-to-inhibitor functional switch in GCAP1.
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Project description:Cyclic GMP (cGMP) is an important second messenger in eukaryotes. It is formed by guanylyl cyclases (GCs), members of the nucleotidyl cyclases class III, which also comprises adenylyl cyclases (ACs) from most organisms. To date, no structures of eukaryotic GCs are available, and all bacterial class III proteins were found to be ACs. Here we describe the biochemical and structural characterization of the class III cyclase Cya2 from cyanobacterium Synechocystis PCC6803. Cya2 shows high specificity for GTP versus ATP, revealing it to be the first bacterial GC, and sequence similarity searches indicate that GCs are also present in other bacteria. The crystal structure of Cya2 provides first structural insights into the universal GC family. Structure and mutagenesis studies show that a conserved glutamate, assisted by an interacting lysine, dominates substrate selection by forming hydrogen bonds to the substrate base. We find, however, that a second residue involved in substrate selection has an unexpected sterical role in GCs, different from its hydrogen bonding function in the related ACs. The structure identifies a tyrosine that lines the guanine binding pocket as additional residue contributing to substrate specificity. Furthermore, we find that substrate specificity stems from faster turnover of GTP, rather than different affinities for GTP and ATP, implying that the specificity-determining interactions are established after the binding step.
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| S-EPMC7294094 | biostudies-literature
Project description:A new Dictyostelium discoideum cyclase gene was identified that encodes a protein (sGC) with 35% similarity to mammalian soluble adenylyl cyclase (sAC). Gene disruption of sGC has no effect on adenylyl cyclase activity and results in a >10-fold reduction in guanylyl cyclase activity. The scg- null mutants show reduced chemotactic sensitivity and aggregate poorly under stringent conditions. With Mn(2+)/GTP as substrate, most of the sGC activity is soluble, but with the more physiological Mg(2+)/GTP the activity is detected in membranes and stimulated by GTPgammaS. Unexpectedly, orthologues of sGC and sAC are present in bacteria and vertebrates, but absent from Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae.
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