Identification of a novel protein with guanylyl cyclase activity in Arabidopsis thaliana

Guanylyl cyclases (GCs) catalyze the formation of the second messenger guanosine 3',5'-cyclic monophosphate (cGMP) from guanosine 5'-triphosphate (GTP). While many cGMP-mediated processes in plants have been reported, no plant molecule with GC activity has been identified. When the Arabidopsis thaliana genome is queried with GC sequences from cyanobacteria, lower and higher eukaryotes no unassigned proteins with significant similarity are found. However, a motif search of the A. thaliana genome based on conserved and functionally assigned amino acids in the catalytic center of annotated GCs returns one candidate that also contains the adjacent glycine-rich domain typical for GCs. In this molecule, termed AtGC1, the catalytic domain is in the N-terminal part. AtGC1 contains the arginine or lysine that participates in hydrogen bonding with guanine and the cysteine that confers substrate specificity for GTP. When AtGC1 is expressed in Escherichia coli, cell extracts yield >2.5 times more cGMP than control extracts and this increase is not nitric oxide dependent. Furthermore, purified recombinant AtGC1 has Mg(2+)-dependent GC activity in vitro and >3 times less adenylyl cyclase activity when assayed with ATP as substrate in the absence of GTP. Catalytic activity in vitro proves that AtGC1 can function either as a monomer or homo-oligomer. AtGC1 is thus not only the first functional plant GC but also, due to its unusual domain organization, a member of a new class of GCs.     

Modulation of rat guanylate cyclase activity in vitro by chemical carcinogens.

The nucleotide cyclic GMP has been reported to be involved in cell proliferation and malignant transformation. Nitroso chemical carcinogens activate the enzyme guanylate cyclase (EC 4.6.1.2) which catalyzes the production of cyclic GMP. The present investigation demonstrates that compounds from other major classes of carcinogens including (1) alpha-halo ethers (chloromethyl methyl ether); (2) aromatic amines (benzidine and B-naphthylamine); (3) polycyclic hydrocarbons (1,2-benzanthracene and acridine); (4) azo dyes (p-dimethylaminoazobenzene), and (5) aflatoxins (B1, B2, G1, G2) produced a striking and significant inhibition of guanylate cyclase over a general concentration range of 0.5-13 mmol/1 in a variety of tissues. Some of the nitrosamides which increase guanylate cyclase activity, increase DNA synthesis whereas carcinogens which decrease guanylate cyclase activity inhibit DNA or RNA synthesis suggesting a relationship between cyclic GMP, DNA synthesis, and chemical carcinogenesis.     

Characterization of a novel monoclonal antibody that senses nitric oxide-dependent activation of soluble guanylate cyclase.

Two monoclonal antibodies (mAbs) against bovine lung soluble guanylate cyclase (sGC) were prepared and characterized. mAb 3221 recognized both the alpha- and beta-subunits of sGC and had greater binding affinity to the enzyme in the presence of NO. mAb 28131 recognized only the beta-subunit and its affinity did not change with NO. Neither mAb cross-reacted with particulate GC. Cultured Purkinje cells from rats were treated with S-nitroso-N-acetylpenicillamine, an NO donor, and examined by immunocytochemical methods. The immunoreactivity associated with mAb 3221 increased with the cGMP content in a crude extract of cerebellum and the NO2 generated in the culture medium increased.     

Melatonin enhances guanylate cyclase activity in a variety of tissues

Summary The objective of the present investigation was to determine if melatonin at physiological concentrations might have part of its mechanism of action through enhancement of guanylate cyclase (E.C.4.6.1.2) activity. Melatonin enhanced guanylate cyclase activity two-three fold in rat anterior pituitary, thyroid, testis, ovary, liver and small intestine at the 1 nanomolar concentration. Some stimulation of hepatic guanylate cyclase activity by melatonin was seen at concentrations as low as 1 picomolar. There was no stimulation of guanylate cyclase activity at concentrations below 1 picomolar. Maximal enhancement of guanylate cyclase activity was seen at the 1 nanomolar concentration of melatonin with no further enhancement being observed with increasing the concentration to the micromolar range. Thus, the data in the present investigation indicates that at concentrations at which melatonin is known to cause physiological effects, melatonin does cause an enhancement of the activity of the guanylate cyclase-cyclic GMP system.     

Activation of cerebral guanylate cyclase by nitric oxide.

Mouse cerebral guanylate cyclase was activated by catalase in the presence of sodium azide (NaN₃), which is known to form catalase-NO complex, while nitrosamines and nitric oxide (NO gas) were capable of activating cerebral guanylate cyclase in the absence of catalase. The activation of guanylate cyclase by NaN₃-catalase or nitrosamines was markedly inhibited by ferrohemoglobin which has a high affinity for NO, but not by ferrihemoglobin. These data suggest that NO or NO containing compounds may activate guanylate cyclase, whereas ferrohemoglobin may exhibit an inhibitory effect on the activation of guanylate cyclase, possibly by interacting with NO or NO containing compounds.     

Receptor-mediated regulation of guanylate cyclase activity in spermatozoa

Two peptides, speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2), which activate sperm respiration and motility and elevate cyclic GMP concentrations in a species-specific manner, were tested for effects on guanylate cyclase activity. The guanylate cyclase of sea urchin spermatozoa is a glycoprotein and it is localized entirely on the plasma membrane. When intact sea urchin sperm cells were incubated with the appropriate peptide for time periods as short as 5 s and subsequently homogenized in detergent, guanylate cyclase activity was found to be as low as 10% of the activity of cells not treated with peptide. The peptides showed complete species specificity and analogues of one peptide (speract) caused decreases in enzyme activity coincident with their receptor binding properties. The peptides did not inhibit enzyme activity when added after detergent solubilization of the enzyme. When detergent-solubilized spermatozoa were incubated at 22 degrees C, guanylate cyclase activity declined in previously nontreated cells to the peptide-treated level. The rate of decline was dependent on temperature and protein concentration. When spermatozoa were first incubated with 32P, the decrease in guanylate cyclase activity was accompanied by a shift in the apparent molecular weight of a major plasma membrane protein (160,000-150,000) and a loss of 32P label from the 160,000 band. Other agents (Monensin A, NH4Cl) which were capable of stimulating sperm respiration and motility also caused decreases of guanylate cyclase activity when added to intact but not detergent-solubilized spermatozoa. The maximal decrease in guanylate cyclase activity occurred 5-10 min after addition of these agents. The enzyme response to Monensin A required extracellular Na+ suggestive that the ionophore caused the effect on guanylate cyclase activity by virtue of its ability to catalyze Na+/H+ exchange. These studies demonstrate that guanylate cyclase activity of sperm cells can be altered by the specific interaction of egg-associated peptides with their plasma membrane receptors.     

Identification of a novel heteroglycan-interacting protein, HIP 1.3, from Arabidopsis thaliana

Plastidial degradation of transitory starch yields mainly maltose and glucose. Following the export into the cytosol, maltose acts as donor for a glucosyl transfer to cytosolic heteroglycans as mediated by a cytosolic transglucosidase (DPE2; EC 2.4.1.25) and the second glucosyl residue is liberated as glucose. The cytosolic phosphorylase (Pho2/PHS2; EC 2.4.1.1) also interacts with heteroglycans using the same intramolecular sites as DPE2. Thus, the two glucosyl transferases interconnect the cytosolic pools of glucose and glucose 1-phosphate. Due to the complex monosaccharide pattern, other heteroglycan-interacting proteins (HIPs) are expected to exist.Identification of those proteins was approached by using two types of affinity chromatography. Heteroglycans from leaves of Arabidopsis thaliana (Col-0) covalently bound to Sepharose served as ligands that were reacted with a complex mixture of buffer-soluble proteins from Arabidopsis leaves. Binding proteins were eluted by sodium chloride. For identification, SDS-PAGE, tryptic digestion and MALDI-TOF analyses were applied. A strongly interacting polypeptide (approximately 40 kDa; designated as HIP1.3) was observed as product of locus At1g09340. Arabidopsis mutants deficient in HIP1.3 were reduced in growth and contained heteroglycans displaying an altered monosaccharide pattern. Wild type plants express HIP1.3 most strongly in leaves. As revealed by immuno fluorescence, HIP1.3 is located in the cytosol of mesophyll cells but mostly associated with the cytosolic surface of the chloroplast envelope membranes. In an HIP1.3-deficient mutant the immunosignal was undetectable. Metabolic profiles from leaves of this mutant and wild type plants as well were determined by GC-MS. As compared to the wild type control, more than ten metabolites, such as ascorbic acid, fructose, fructose bisphosphate, glucose, glycine, were elevated in darkness but decreased in the light. Although the biochemical function of HIP1.3 has not yet been elucidated, it is likely to possess an important function in the central carbon metabolism of higher plants.     

Identification of novel subunits of the TOM complex from Arabidopsis thaliana

The preprotein translocase of the outer mitochondrial membrane (also called TOM complex) from Arabidopsis thaliana was characterized by Blue-native gel electrophoresis (BN-PAGE) and Electrospray Tandem Mass Spectrometry (ESI-MS/MS). BN-PAGE allows to prepare a very stable 390 kDa complex that includes six different protein types: the 34 kDa translocation pore TOM40, the 21/23 kDa preprotein receptor TOM20, the small TOM component TOM7 and three further subunits of 10, 6.3 and 6.0 kDa. Primary structures of all TOM subunits were elucidated. The 10 kDa subunit represents a truncated version of the TOM22 preprotein receptor and the two 6 kDa proteins represent subunits possibly homologous to fungal TOM6 and TOM5, although sequence conservation is at the borderline of significance. TOM40, TOM7 and one or both of the 6 kDa subunits form a subcomplex of about 100 kDa. The six TOM proteins from Arabidopsis are encoded by 12 genes, at least 11 of which are expressed. While the subunit composition of the TOM complex from fungi, animals and plants is remarkably conserved, the domain structure of individual TOM proteins differs, e.g. acidic domains in TOM22 and the 6 kDa TOM subunits from Arabidopsis are absent. The domain structure of the Arabidopsis TOM complex does not support the so-called ‘acid chain hypothesis’, which explains the translocation of proteins across the outer mitochondrial membrane of mitochondria by the binding of preproteins to acidic protein domains within the TOM complex. Functional implications are discussed.     

Identification of novel phasiRNAs loci on long non-coding RNAs in Arabidopsis thaliana

Long non-coding RNAs (lncRNAs) are the “dark matters”involved in gene regulation with complex mechanisms. However, the functions of most lncRNAs remain to be determined. Our previous work revealed a massive number of degradome-supported cleavage signatures on Arabidopsis lncRNAs. Some of them have been confirmed associated with miRNAs-like sRNAs production, while others without long stem structure remain unexplored. A systematical search for phasiRNAs generating ability of these lncRNAs was conducted. Eight novel small RNA triggered lncRNA-phasiRNA pathways were discovered and three of them were found to be conserved in Arabidopsis, Oryza sativa, Glycine max and Gossypium hirsutum. Besides, Five novel ta-siRNAs derived from these lncRNAs were further identified to be involved in the regulation of plant development, stress responses and aromatic amino acids synthesis. These results substantially expanded the gene regulation mechanisms of lncRNAs.     

Identification of a copper transporter family in Arabidopsis thaliana

Despite copper ions being crucial in proteins participating in plant processes such as electron transport, free-radical elimination and hormone perception and signaling, very little is known about copper inward transport across plant membranes. In this work, a five-member family (COPT1–5) of putative Arabidopsis copper transporters is described. We ascertain the ability of these proteins to functionally complement and transport copper in the corresponding Saccharomyces cerevisiae high-affinity copper transport mutant. The specific expression pattern of the Arabidopsis COPT1–5 mRNA in different tissues was analyzed by RT-PCR. Although all members are ubiquitously expressed, differences in their relative abundance in roots, leaves, stem and flowers have been observed. Moreover, steady-state COPT1 and COPT2 mRNA levels, the members that are most efficacious in complementing the S. cerevisiae high-affinity copper transport mutant, are down-regulated under copper excess, consistent with a role for these proteins in copper transport in Arabidopsis cells.     

Identification, characterization and comparative analysis of a novel chorismate mutase gene in Arabidopsis thaliana

Phenylalanine, tyrosine, and tryptophan have a dual biosynthetic role in plants; they are required for protein synthesis and are also precursors to a number of aromatic secondary metabolites critical to normal development and stress responses. Whereas much has been learned in recent years about the genetic control of tryptophan biosynthesis in Arabidopsis and other plants, relatively little is known about the genetic regulation of phenylalanine and tyrosine synthesis. We have isolated, characterized and determined the expression of Arabidopsis thaliana genes encoding chorismate mutase, the enzyme catalyzing the first committed step in phenylalanine and tyrosine synthesis. Three independent Arabidopsis chorismate mutase cDNAs were isolated by functional complementation of a Saccharomyces cerevisiae mutation. Two of these cDNAs have been reported independently (Eberhard et al., 1993. FEBS 334, 233-236; Eberhard et al., 1996. Plant J. 10, 815-821), but the third (designated CM-3) represents a novel gene. The different organ-specific expression patterns of these cDNAs, their regulation in response to pathogen infiltration, as well as the different enzymatic characteristics of the proteins they encode are also described. Together, these data suggest that each isoform may play a distinct physiological role in coordinating chorismate mutase activity with developmental and environmental signals.     

The interrelationship of somatostatin and guanylate cyclase activity

Summary Somatostatin has been shown to inhibit the release of various polypeptide hormones including insulin, glucagon, gastrin, thyroid stimulating hormone, and growth hormone. The mechanism by which somatostatin inhibits the release of these various polypeptide hormones has not been fully eluciadated. It has been reported that somatostatin increases the level of the second messenger cyclic GMP in rat brain and in the anterior pituitary gland. The present investigation was designed to determine if these responses seen in the anterior pituitary gland and brain were due to activation of guanylate cyclase GTP-pyrophosphate lyase (cyclizing), E.C.4.6.1.2., the enzyme that catalyzes the formation of cyclic GMP. Somatostatin at a concentration of 2 pm enhanced guanylate cyclase activity two-fold in rat cerebrum and anterior pituitary gland. This enhancement of guanylate cyclase activity was also seen in rat liver, pancreas, stomach, and small intestine at the same concentration of somatostatin. Increasing the concentration of somatostatin to 20 m, caused a marked inhibition of guanylate cyclase activity in all these tissues. Dose-response curves done on gastric guanylate cyclase activity revealed that over a concentration range of 2 pm to 0.2 m, somatostatin had a stimulatory effect on guanylate cyclase activity while at concentrations above 10 m somatostatin was inhibitory to guanylate cyclase activity. The biphasic pattern of enhancement of guanylate cyclase activity at lower concentrations of somatostatin and inhibition at higher concentrations may help to explain some of the discrepancies seen with previous investigations with somatostatin, hormone release, and cyclic nucleotide metabolism.     

Modulation of soluble guanylate cyclase activity by phosphorylation

The levels of the cGMP in smooth muscle of the gut reflect continued synthesis by soluble guanylate cyclase (GC) and breakdown by phosphodiesterase 5 (PDE5). Soluble GC is a haem-containing, heterodimeric protein consisting alpha- and beta-subunits: each subunit has N-terminal regulatory domain and a C-terminal catalytic domain. The haem moiety acts as an intracellular receptor for nitric oxide (NO) and determines the ability of NO to activate the enzyme and generate cGMP. In the present study the mechanism by which protein kinases regulate soluble GC in gastric smooth muscle was examined. Sodium nitroprusside (SNP) acting as a NO donor stimulated soluble GC activity and increased cGMP levels. SNP induced soluble GC phosphorylation in a concentration-dependent fashion. SNP-induced soluble GC phosphorylation was abolished by the selective cGMP-dependent protein kinase (PKG) inhibitors, Rp-cGMPS and KT-5823. In contrast, SNP-stimulated soluble GC activity and cGMP levels were significantly enhanced by Rp-cGMPS and KT-5823. Phosphorylation and inhibition of soluble GC were PKG specific, as selective activator of cAMP-dependent protein kinase, Sp-5, 6-DCl-cBiMPS had no effect on SNP-induced soluble GC phosphorylation and activity. The ability of PKG to stimulate soluble GC phosphorylation was demonstrated in vitro by back phosphorylation technique. Addition of purified phosphatase 1 inhibited soluble GC phosphorylation in vitro, and inhibition was reversed by a high concentration (10 microM) of okadaic acid. In gastric smooth muscle cells, inhibition of phosphatase activity by okadaic acid increased soluble GC phosphorylation in a concentration-dependent fashion. The increase in soluble GC phosphorylation inhibited SNP-stimulated soluble GC activity and cGMP formation. The results implied the feedback inhibition of soluble GC activity by PKG-dependent phosphorylation impeded further formation of cGMP.     

Inhibition of soluble guanylate cyclase activity by citrate

Soluble guanylate cyclase activity from guinea pig heart is inhibited by increasing concentrations of sodium citrate. The Ki value was found to be 2.83 +/- 0.05 mM in the presence of 3 mM Mn2+ and 0.6 mM GTP. Citrate acts by lowering Vmax and increasing the apparent values of Km for GTP and K0.5 for Mn2+ and Mg2+. The soluble guanylate cyclase, activated by sodium nitroprusside, was also inhibited by citrate. This inhibitory action of citrate was not restricted to soluble guanylate cyclase activity of the heart and has been demonstrated also in the supernatant of lung, liver, diencephalon and in the homogenate of blood platelets. Since citrate is known to be an important intermediate of metabolism, its intracellular concentration may be also of relevance for guanylate cyclase activity.     

Identification of residues crucially involved in soluble guanylate cyclase activation

The ubiquitous heterodimeric nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in various signal transduction pathways. Binding of NO takes place at the prosthetic heme moiety at the N-terminus of the beta(1)-subunit of sGC. The induced structural changes lead to an activation of the catalytic C-terminal domain of the enzyme and to an increased conversion of GTP into the second messenger cyclic GMP (cGMP). In the present work we selected and substituted different residues of the sGC heme-binding pocket based on a sGC homology model. The generated sGC variants were tested in a cGMP reporter cell for their effect on the enzyme activation by heme-dependent (NO, BAY 41-2272) stimulators and heme-independent (BAY 58-2667) activators. The use of these experimental tools allows the enzyme's heme content to be explored in a non-invasive manner. Asp(44), Asp(45) and Phe(74) of the beta(1)-subunit were identified as being crucially important for functional enzyme activation. beta(1)Asp(45) may serve as a switch between different conformational states of sGC and point to a possible mechanism of action of the heme dependent sGC stimulator BAY 41-2272. Furthermore, our data shows that the activation profile of beta(1)IIe(145) Tyr is unchanged compared to the native enzyme, suggesting that Tyr(145) does not confer the ability to distinguish between NO and O(2). In summary, the present work further elucidated intramolecular mechanisms underlying the NO- and BAY 41-2272-mediated sGC activation and raises questions regarding the postulated role of Tyr(145) for ligand discrimination.     

Regulation of activity of purified guanylate cyclase from liver that is unresponsive to nitric oxide.

Guanylate cyclase was purified from rat liver supernatant. Electrophoresis under denaturing conditions revealed one major peptide of Mr approx. 69 000. On the basis of the Stokes radius (4.7 nm) and S20,w (6.4S), the calculated Mr value of the native enzyme was 133 000, i.e. it is apparently a homodimer. Kinetics of inactivation by diamide (which was reversible with dithiothreitol) suggested that oxidation of a single class of thiol sites was involved. In the absence of other additions, cyclase activity assayed with Mn2+ was over 7 times that assayed with Mg2+; maximal effects were observed with approx. 5 mM of each (with 1 mM-GTP). The purified enzyme was markedly activated by nitrosylhaemoglobin. Relative activation was much greater in assays with Mg2+ than with Mn2+, although maximal activities were similar. When assayed with Mg2+, the enzyme exhibited a single Km (0.35 mM) for GTP; with Mn2+, plots of 1/v versus 1/[S] were non-linear. Activator or nitrosylhaemoglobin increased Vmax, but did not alter Km in the presence of either Mg2+ or Mn2+. The enzyme was inhibited by Na3VO4, Na2WO4 and Na2B4O7. Reduction from VV to VIV abolished the inhibitory effect of vanadate. Na2B4O7 (2 mM) inhibited activity with Mn2+, but not with Mg2+. In assays with Mg2+, but not with Mn2+, FMN, NAD+ and NADH (each 0.5 mM) inhibited activation by protoporphyrin IX and nitrosylhaemoglobin. Rotenone (0.6 mM) inhibited activity with protoporphyrin IX to a greater extent than with nitrosylhaemoglobin. Methylene Blue (1 mM) inhibited activation by nitrosylhaemoglobin, protoporphyrin IX and activator. It appears that this enzyme purified from rat liver lacks haem (and perhaps other components) required for activation by NO, and it should be particularly useful for elucidating the mechanism of action of NO, protoporphyrin IX and other activators.