Cyclic Cytidine Monophosphate Research Articles

Structural and functional characterization of cyclic pyrimidine-regulated anti-phage system

3’,5’-cyclic uridine monophosphate (cUMP) and 3’,5’-cyclic cytidine monophosphate (cCMP) have been established as bacterial second messengers in the phage defense system, named pyrimidine cyclase system for anti-phage resistance (Pycsar). This system consists of a pyrimidine cyclase and a cyclic pyrimidine receptor protein. However, the molecular mechanism underlying cyclic pyrimidine synthesis and recognition remains unclear. Herein, we determine the crystal structures of a uridylate cyclase and a cytidylate cyclase, revealing the conserved residues for cUMP and cCMP production, respectively. In addition, a distinct zinc-finger motif of the uridylate cyclase is identified to confer substantial resistance against phage infections. Furthermore, structural characterization of cUMP receptor protein PycTIR provides clear picture of specific cUMP recognition and identifies a conserved N-terminal extension that mediates PycTIR oligomerization and activation. Overall, our results contribute to the understanding of cyclic pyrimidine-mediated bacterial defense.

Cyclic CMP and cyclic UMP mediate bacterial immunity against phages

The cyclic pyrimidines 3',5'-cyclic cytidine monophosphate (cCMP) and 3',5'-cyclic uridine monophosphate (cUMP) have been reported in multiple organisms and cell types. As opposed to the cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP), which are second messenger molecules with well-established regulatory roles across all domains of life, the biological role of cyclic pyrimidines has remained unclear. Here we report that cCMP and cUMP are second messengers functioning in bacterial immunity against viruses. We discovered a family of bacterial pyrimidine cyclase enzymes that specifically synthesize cCMP and cUMP following phage infection and demonstrate that these molecules activate immune effectors that execute an antiviral response. A crystal structure of a uridylate cyclase enzyme from this family explains the molecular mechanism of selectivity for pyrimidines as cyclization substrates. Defense systems encoding pyrimidine cyclases, denoted here Pycsar (pyrimidine cyclase system for antiphage resistance), are widespread in prokaryotes. Our results assign clear biological function to cCMP and cUMP as immunity signaling molecules in bacteria.

Microanalytical study of sub-nanoliter samples by capillary electrophoresis – mass spectrometry with 100 % injection efficiency

A method is presented for conducting fast capillary electrophoresis (CE) with ultrasmall sample volumes (<10 nL) and subsequent determination of target analytes by time-of-flight mass spectrometry (TOF-MS). Furthermore, an approach towards preconcentration by solvent evaporation of samples as small as 500 nL is shown. Ultrasmall samples of biologically important cyclic nucleotides (cyclic guanosine monophosphate, cyclic adenosine monophosphate, cyclic cytidine monophosphate) served as model compounds. Sample volumes as small as 500 pL were used to perform sample uptake and CE injections by capillary batch injection (CBI). This resulted in detection limits between 2 and 5 fmol. The fast rate of sample evaporation prevents the operation of nano-/subnano-liter samples under room temperature conditions. A CBI-CE-TOF-MS setup incorporating a Peltier element and a thermostat was used for the handling of very small samples. In combination with tapered fused silica injection capillaries and the high-precision guiding system of the CBI setup, nearly 100 % efficient injections of ultrasmall samples into funnelled separation capillaries could be managed. The overall system allowed for high-throughput CE separations from densely arranged sample droplets or vials with RSD of peak heights ranging from 6 to 8 % (n = 5). CE-TOF-MS separations of mixtures of cyclic nucleotides were carried out in capillaries as short as 20 cm (25 μm ID) in less than 55 s with high separation efficiency. We report details of the setup and optimizations with respect to etching of capillaries (tapering and funnelling), sample uptake and injection as well as separation of the model compounds. Moreover, the capabilities of the experimental setup for preconcentration of target analytes by solvent evaporation are described. Preconcentration factors of up to 100 could be achieved. This approach was successfully used for the determination of the cyclic nucleotide cGMP in (spiked) human urine samples.

Cytidylyl cyclase activity of bacterial exotoxins

3',5'-Cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) are key second messengers for a wide variety of mammalian cellular processes. The natural occurrence of a third cyclic nucleotide, 3',5'-cyclic cytidine monophosphate (cCMP) had been discussed very controversially 30–35 years ago [1-3], but later, cCMP was identified unambiguously in various mammalian tissues [4] indicating cCMP to be a novel second messenger with potential importance in regulation of cell growth, proliferation, tissue development and modulation of immune responses [5]. However, the precise identity of the cCMP-forming enzyme in mammalian cells is still unclear. Here, for the first time, we provide evidence for cytidylyl cyclase (CC) activity of purified bacterial exotoxins. Bacillus anthracis and Bordetella pertussis, the causative bacteria of anthrax disease and whooping cough, respectively, secrete the adenylyl cyclase(AC) toxins edema factor (EF) and CyaA, weakening immune responses through massive cAMP production and, thereby, promoting the pathogenesis of the infections [6]. We found that both toxins also possess cytidylyl cyclase activity, resulting in the conversion of CTP to cCMP. In our CC activity assay, the radioactively labeled substrate [α-32P]CTP is converted to [32P]cCMP which is quantified by liquid scintillation. Upon incubation of the toxins with [α-32P]CTP, [32P]cCMP is produced in a linear manner over time. As [32P]cCMP production depends on the endogenous toxin activator protein calmodulin, as physiological pH promotes the reaction and as potent AC inhibitors show high potency on EF and CyaA, catalysis results from specific CC activity. Michaelis-Menten kinetics of EF CC activity yielded Km = 13 ± 3 μM and Vmax = 8 ± 1 s-1. When CTP consumption and cCMP formation were monitored by HPLC, 100 μM CTP were converted to cCMP by 20 nM EF within 1 h. In the presence of heat-inactivated enzyme, no cCMP was formed. The identity of cCMP was confirmed by co-eluting standard cCMP in HPLC experiments and by mass spectrometry methods. Based on these findings and the fact that cCMP inhibits host immune responses [7,8], we propose that the bacterial exotoxins edema factor (EF) and CyaA generate cCMP, resulting in increased infection severity. The molecular targets of cCMP remain to be determined.

Expression of wild-type and mutant bovine pancreatic ribonuclease A in Escherichia coli.

Wild-type ribonuclease A and five mutants thereof have been expressed in Escherichia coli as fusion proteins by using a T7 expression system. The five mutants are C[65-72]S, C[40-95]S, C[58-110]S, C[26-84]S, and K41G. The expressed fusion protein formed inclusion bodies which were then cleaved by factor Xa. The cleaved ribonuclease A was isolated as unfolded (sulfonated), soluble protein which was subsequently folded. This expression system can be used to produce mutants of ribonuclease A in yields suitable for folding and structural studies. All four native three-disulfide mutants exhibited enzymatic activity (5-30%), although only two were thermally stable at room temperature, demonstrating that no single native disulfide bond is essential for folding. The K41G mutant was enzymatically inactive with cyclic cytidine monophosphate as substrate.

Cytidylate cyclase activity: identification of cytidine 3',5'-cyclic monophosphate and four novel cytidine cyclic phosphates as biosynthetic products from cytidine triphosphate.

Identification of cytidine 3',5'-cyclic monophosphate (cyclic CMP) as one of the products resulting from the incubation of dialysed cell-free preparations from rat brain, liver and kidney with cytidine 5'-triphosphate (CTP) is described. The non-acidic precipitable products after incubation of the tissue preparations with unlabelled, with 14C-single labelled, and with 14C- and 32P-dual labelled CTP were examined by thin-layer chromatography and high-pressure liquid chromatography, isotopic ratio determination, UV absorbance spectrophotometry, selective hydrolysis with nucleotidase, phosphodiesterase and acid, and by fast atom bombardment mass spectrometry with mass-analysed ion kinetic energy spectrum scanning. In addition to cyclic CMP and unchanged CTP, the products of the reaction were found to include cytidine monophosphate (CMP) and cytidine diphosphate (CDP) together with four novel cytidine compounds identified as cytidine 3',5'-cyclic pyrophosphate, cytidine 2'-monophosphate 3',5'-cyclic monophosphate, cytidine 2'-O-aspartyl-3',5'-cyclic monophosphate and cytidine 2'-O-glutamyl-3',5'-cyclic monophosphate. The evidence presented constitutes conclusive proof of the natural occurrence of cytidylate cyclase activity; the four novel cytidine cyclic phosphates described provide a feasible explanation of the discrepancies in previous reports which have led to the controversy which exists concerning the existence of cytidylate cyclase activity.

The analysis of enzyme progress curves by numerical differentiation, including competitive product inhibition and enzyme reactivation

A new method for analyzing steady-state enzyme kinetic data is presented. The technique, which is based on the numerical differentiation of the complete reaction curve, has several advantages over initial velocity and integrated Michaelis-Menten equation methods. The differentiated data are fit to the differential equation describing the appropriate kinetic scheme. This approach is particularly valuable in cases of strong competitive product inhibition and of changing concentrations of active enzyme. The method assumes a reversible reaction and is applicable to a very wide variety of steady-state kinetic schemes. A particular advantage of this approach over integrated methods is that it is independent of [S 0] and hence of errors in [S 0]. The combination of complete progress curve and computer analysis makes this approach very efficient with respect to both time and materials. Running on an IBM PC XT or equivalent microcomputer with an 8087 coprocessor, the analyses are very fast, the complete process usually being complete in a minute or two. The utility of the technique is demonstrated by application to both simulated and real data. We show that the differentiation of the progress curve for the ribonuclease-catalyzed hydrolysis of 2′, 3′-cyclic cytidine monophosphate reveals strong product inhibition by 3′-CMP, and this product inhibition accounts for the large discrepancies reported in the literature for the value of K m for this substrate. The method was also applied to determine the rate of reactivation of β-lactamase which had been reversibly inactivated by cloxacillin. Since large numbers of data points are required for the numerical differentiation the method has become practical only with the advent of computer-acquired data systems.

Cyclic cytidine monophosphate stimulates DNA synthesis by bovine mammary tissue in vitro

Mammary gland biopsies were taken from midpregnant heifers (n=4), cut into pieces .5 mm thick and 3–5 mm 2 and incubated for 48 hours in Eagle's Minimum Essential Medium containing 0, .1 or 1 μg/ml insulin and 0, 10 −8, 10 −7, 10 −6, 10 −5, or 10 −4 M dibutyryl cyclic 3′, 5′, cytidine monophosphate (dbcCMP). With 0 or .1 μg/ml insulin, dbcCMP decreased incorporation of tritiated thymidine into DNA. Similar declines in DNA synthesis were observed with sodium butyrate, suggesting that the decline was due to the butyrate rather than to a cyclic CMP-specific effect. With 1 μg/ml insulin, dbcCMP increased DNA synthesis. Higher levels of dbcCMP reduced DNA synthesis relative to 10 −6M dbcCMP, as did sodium butyrate. Thus cCMP is capable of stimulating mammary growth.

Ribonuclease inhibitor from human placenta: rapid purification and assay.

The ribonuclease inhibitor from human placenta may be isolated in 65% yield (2.5 mg per placenta) in 2 days. The performance of the affinity chromatography on Sepharose-RNase A has been expedited through adaption of the spectrophotometric assay of ribonuclease toward 2',3'-cyclic cytidine monophosphate to determination of the inhibitor activity. The result of these improvements in procedure is increased availability of the ribonuclease inhibitor for study of its chemical properties (Blackburn, P., and Jailkhani, B.L. (1979) J. Biol. Chem. 254, 12488-12493) and for its inclusion into in vitro systems in which inhibition of mammalian neutral ribonucleases is desired.

Steady-state enzyme kinetics of the pancreatic ribonucleases from five mammalian species

The kinetic parameters K m, κ +2 and κ +2/ K m of the pancreatic ribonucleases (EC 3.1.4.22) from cow, giraffe, horse, rat and lesser rorqual have been determined, using 2′,3′-cyclic cytidine monophosphate and 2′,3′-cyclic uridine monophosphate as substrates. No large differences were found between the activities of the five enzymes. The relative differences between the activities of the five enzymes are mainly due to differences in the rates of hydrolysis and not to differences in the affinities for the substrates.

Amino protons of cytosine. Chemical exchange, rotational exchange, and salt-induced proton magnetic resonance chemical shifts of aqueous 2',3'-cyclic cytidine monophosphate.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAmino protons of cytosine. Chemical exchange, rotational exchange, and salt-induced proton magnetic resonance chemical shifts of aqueous 2',3'-cyclic cytidine monophosphateBruce McConnell and Patricia C. SeawellCite this: Biochemistry 1973, 12, 22, 4426–4434Publication Date (Print):October 1, 1973Publication History Published online1 May 2002Published inissue 1 October 1973https://doi.org/10.1021/bi00746a020RIGHTS & PERMISSIONSArticle Views102Altmetric-Citations25LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

Adrenal cells in tissue culture. 8. Dissociation of the steroidogenic and glycolytic effects of cyclic nucleotides and adrenocorticotropin.

In monolayer cultures of mouse adrenal cortex tumor cells, high concentrations of 3′,5′-cyclic adenosine monophosphate and 3′,5′-cyclic cytidine monophosphate (1.0 to 10.0mm produce steroidogenic responses equivalent to maximally stimulating levels of adrenocorticotropin. 3′,5′-Cyclic guanosine monophosphate and other cyclic nucleotides are not steroidogenic. Although the steroidogenic action of adrenocorticotropin is accompanied by an increased rate of glycolytic activity, the cyclic nucleotides stimulate steroidogenesis without increasing glycolysis. The data suggest that adrenocorticotropin can effect certain alterations in adrenal metabolism by a mechanism which does not involve the adenyl cyclase system.

A ribonuclease from Physarum: Biochemical properties and synthesis in the mitotic cycle

A ribonuclease from the myxomycete Physarum polycephalum has been partially purified and characterized; it is called Physarum ribonuclease I. It is an extracellular enzyme, has a pH optimum of 4.0, is heat labile, stable over a wide range of pH values; and has a molecular weight of about 31 000 daltons, as determined by gel filtration. No metal ions are required for its activity. RNA serves as substrate, while native or denatured DNA are resistant. Poly U and poly A are rapidly degraded to cyclic mononucleotides and slowly to 3′-mononucleotides. Poly C is very slowly degraded to cyclic cytidine monophosphate. Dinucleotide phosphates are rapidly hydrolyzed, except those of the form CpN. After prolonged incubation, RNA is degraded primarily to 3′-AMP, 3′-UMP, 3′-GMP and cyclic 2′,3′-CMP. The enzyme is an endonuclease. It may be useful for sequence studies of RNA, particularly for further partial degradation of T1 fragments. The activity of ribonuclease I was studied in the highly synchronous mitotic cycle of Physarum. Enzyme activity doubles stepwise shortly before the middle of each interphase. This rise depends on protein synthesis de novo.