Temperature and pressure limits of guanosine monophosphate self-assemblies.
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ABSTRACT:
SUBMITTER: Gao M
PROVIDER: S-EPMC5574928 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Concentration and temperature dependent studies of the circular dichroism of dianionic guanosine 5'-monophosphate (5'-GMP), where the cation was Na(+), K(+), or Rb(+) ion, were done to obtain information regarding the nature of the self-assembled 5'-GMP species in aqueous solution, including G-quartets and other structures. Concentrations in the 0.05-0.85 M range and temperatures in the 5-50 °C range were used. At the highest concentrations and 5 °C, Na(2)(5'-GMP) and K(2)(5'-GMP) formed a cholesteric phase, but Rb(2)(5'-GMP) did not. Evidence for antiparallel base stacking (stacked with opposite polarity; head to head) was observed for Rb(2)(5'-GMP) but not for the Na(+) or K(+) salts. This structure, believed to be G-quartets, had a melting temperature of 15 °C and dissociated into a second associated species as the temperature increased. The latter was present to the greatest extent at ∼40 °C, and it is characterized by a prominent negative CD band at 306 nm, which may be indicative of an X-DNA type of structure (an expanded G-quartet) or base-stacked monomers or dimers. The same negative band appeared at 310 nm in the CD spectra of K(2)(5'-GMP) and Na(2)(5'-GMP) but was much less intense in the latter case. K(2)(5'-GMP) also formed a noncholesteric phase containing at least two different species, one more stable at low temperatures and the other more stable at higher temperatures, similar to Rb(2)(5'-GMP). (1)H NMR spectroscopy was used to assist in the interpretation of the CD spectra.
| S-EPMC3131790 | biostudies-literature
Project description:This study addresses the hypothesis that renal interstitial (RI) cGMP, a modulator of pressure-natriuresis, exerts its effect through a relationship with renal interstitial hydrostatic pressure (RIHP). Increasing renal perfusion pressure in Sprague-Dawley rats led to increases in RIHP (5.2+/-0.6 to 10.9+/-1.6 mm Hg; P<0.01), urine sodium excretion (0.062+/-0.009 to 0.420+/-0.068 micromol/min per gram; P<0.01), and RI cGMP (3.5+/-0.8 to 9.5+/-1.7 fmol/min; P<0.01), and these effects were blocked by partial renal decapsulation. Infusion of cGMP into the RI compartment of decapsulated animals restored natriuresis (0.067+/-0.010 to 0.310+/-0.061 micromol/min per gram; P<0.01). These changes were independent of changes in glomerular filtration rate . Artificially increasing RIHP in normotensive animals increased RI cGMP (4.1+/-0.6 to 6.9+/-0.7 fmol/min; P<0.01) and urine sodium excretion (0.071+/-0.013 to 0.179+/-0.039 micromol/min per gram; P<0.05). Coinfusion of organic anion transport-inhibitor probenecid, or soluble guanylyl cyclase inhibitor 1-H(1,2,4) oxadiazolo-(4,2)quinoxalin-1-one, abolished these effects. Infusion of cGMP into the RI compartment of normotensive animals increased RIHP (6.7+/-0.4 to 10.3+/-0.9 mm Hg; P<0.001). Exogenous RI cGMP delivery did not affect total, cortical, or medullary renal blood flow. These studies suggest that extracellular RI cGMP is required for the natriuresis observed after increases in renal perfusion pressure and RIHP and that cGMP acts via a tubule mechanism. The results support an intrarenal positive-feedback loop wherein RI cGMP increases RIHP, which, in turn, increases RI cGMP, contributing to the reinforcement of pressure-natriuresis.
| S-EPMC3057527 | biostudies-literature
Project description:cGMP functions as an extracellular (paracrine) messenger acting at the renal proximal tubule and is an important modulator of pressure-natriuresis (P-N). The signaling pathway activated by cGMP in the tubule cell basolateral membrane remains unknown. We hypothesized that renal interstitial microinfusion of cGMP (50 nmol/kg per minute) or P-N would be accompanied by increased renal protein levels of phospho-Src (Tyr 416) and that the natriuresis would be decreased by Src inhibition. Renal interstitial cGMP-induced natriuresis was blocked by Src inhibitor PP2 (2.0±0.4 versus 0.5±0.01 μEq/g per minute; P<0.001). The inactive analog of PP2, PP3, had no effect on cGMP-induced natriuresis. SU6656, another Src inhibitor, also inhibited cGMP-induced natriuresis (2.0±0.4 versus 1.02±0.01 μEq/g per minute; P<0.001). Renal interstitial cGMP infusion increased phospho-Src protein levels 5.6-fold at 15 minutes and 6.8-fold at 30 minutes compared with vehicle infusion but returned toward basal levels after 60 minutes. PP2 also blunted P-N (3.1±0.1 versus 1.1±0.3 μEq/g per minute; P<0.01) despite a similar increase in blood pressure. PP3 had no effect on P-N. Phospho-Src protein levels increased during P-N in vehicle- (1.8-fold) and PP3-treated (2.1-fold) groups compared with the sham-operated group. PP2 blocked the pressure-induced increase in renal phospho-Src protein levels. PP2 had no effect on renal hemodynamics but decreased both fractional excretion of Na(+) and lithium. Both extracellular cGMP and increased renal perfusion pressure increased renal phospho-Src protein levels and induced natriuresis in an Src-dependent manner, demonstrating that Src is an important downstream signaling molecule for extracellular cGMP-induced natriuresis.
| S-EPMC3117057 | biostudies-literature
Project description:BACKGROUND: Cyclic guanosine monophosphate (cGMP) is the common second messenger for the cardiovascular effects of nitric oxide (NO) and natriuretic peptides, such as atrial or brain natriuretic peptide, which activate the soluble and particulate forms of guanylyl cyclase, respectively. However, natriuretic peptides and NO donors exert different effects on cardiac and vascular smooth muscle function. We therefore tested whether these differences are due to an intracellular compartmentation of cGMP and evaluated the role of phosphodiesterase (PDE) subtypes in this process. METHODS AND RESULTS: Subsarcolemmal cGMP signals were monitored in adult rat cardiomyocytes by expression of the rat olfactory cyclic nucleotide-gated (CNG) channel alpha-subunit and recording of the associated cGMP-gated current (ICNG). Atrial natriuretic peptide (10 nmol/L) or brain natriuretic peptide (10 nmol/L) induced a clear activation of ICNG, whereas NO donors (S-nitroso-N-acetyl-penicillamine, diethylamine NONOate, 3-morpholinosydnonimine, and spermine NO, all at 100 micromol/L) had little effect. The ICNG current was strongly potentiated by nonselective PDE inhibition with isobutyl methylxanthine (100 micromol/L) and by the PDE2 inhibitors erythro-9-(2-hydroxy-3-nonyl)adenine (10 micromol/L) and Bay 60-7550 (50 nmol/L). Surprisingly, sildenafil, a PDE5 inhibitor, produced a dose-dependent increase of I(CNG) activated by NO donors but had no effect (at 100 nmol/L) on the current elicited by atrial natriuretic peptide. CONCLUSIONS: These results indicate that in rat cardiomyocytes (1) the particulate cGMP pool is readily accessible at the plasma membrane, whereas the soluble pool is not; and (2) PDE5 controls the soluble but not the particulate pool, whereas the latter is under the exclusive control of PDE2. Differential spatiotemporal distributions of cGMP may therefore contribute to the specific effects of natriuretic peptides and NO donors on cardiac function.
| S-EPMC1877795 | biostudies-literature
Project description:The hierarchical process of guanosine (G) self-assembly, leading in aqueous solution and in the presence of metal cations to the formation of G-quadruplexes, represents an intriguing topic both for the biological correlation with telomerase activity and for the nano-technological applications, as demonstrated by the current measured in a quadruplex wire 100 nm long. Similar to G-rich DNA sequences and G-oligonucleotides, the guanosine 5'-monophosphate (GMP) self-aggregates in water to form quadruplexes. However, due to the absence of a covalent axial backbone, this system can be very useful to understand the chemical-physical conditions that govern the guanosine supramolecular aggregation. We have then investigated by in-solution Synchrotron Small Angle X-ray Scattering technique the role of different cations in promoting the quadruplex formation as a function of concentration and temperature. Results show how potassium, with its peculiar biological traits, favours the G-quadruplex elongation process in respect to other cations (Na + , but also NH 4 + and Li + ), determining the longest particles in solution. Moreover, the formation and the elongation of G-quadruplexes have been demonstrated to be controlled by both GMP concentration and excess cation content, even if they specifically contribute to these processes in different ways. The occurrence of condensed liquid crystalline phases was also detected, proving that excess cations play also unspecific effects on the effective charges on the G-quadruplex surface.
| S-EPMC7221663 | biostudies-literature
Project description:Chronic hepatitis B virus (HBV) infection is a major cause of chronic liver disease and cancer worldwide. The mechanisms of viral genome sensing and the evasion of innate immune responses by HBV infection are still poorly understood. Recently, the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) was identified as a DNA sensor. In this study, we investigated the functional role of cGAS in sensing HBV infection and elucidate the mechanisms of viral evasion. We performed functional studies including loss-of-function and gain-of-function experiments combined with cGAS effector gene expression profiling in an infectious cell culture model, primary human hepatocytes, and HBV-infected human liver chimeric mice. Here, we show that cGAS is expressed in the human liver, primary human hepatocytes, and human liver chimeric mice. While naked relaxed-circular HBV DNA is sensed in a cGAS-dependent manner in hepatoma cell lines and primary human hepatocytes, host cell recognition of viral nucleic acids is abolished during HBV infection, suggesting escape from sensing, likely during packaging of the genome into the viral capsid. While the hepatocyte cGAS pathway is functionally active, as shown by reduction of viral covalently closed circular DNA levels in gain-of-function studies, HBV infection suppressed cGAS expression and function in cell culture models and humanized mice. Conclusion: HBV exploits multiple strategies to evade sensing and antiviral activity of cGAS and its effector pathways.
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Project description:Glutamine amidotransferases catalyze the amination of a wide range of molecules using the amide nitrogen of glutamine. The family provides numerous examples for study of multi-active-site regulation and interdomain communication in proteins. Guanosine 5'-monophosphate synthetase (GMPS) is one of three glutamine amidotransferases in de novo purine biosynthesis and is responsible for the last step in the guanosine branch of the pathway, the amination of xanthosine 5'-monophosphate (XMP). In several amidotransferases, the intramolecular path of ammonia from glutamine to substrate is understood; however, the crystal structure of GMPS only hinted at the details of such transfer. Rapid kinetics studies provide insight into the mechanism of the substrate-induced changes in this complex enzyme. Rapid mixing of GMPS with substrates also manifests absorbance changes that report on the kinetics of formation of a reactive intermediate as well as steps in the process of rapid transfer of ammonia to this intermediate. Isolation and use of the adenylylated nucleotide intermediate allowed the study of the amido transfer reaction distinct from the ATP-dependent reaction. Changes in intrinsic tryptophan fluorescence upon mixing of enzyme with XMP suggest a conformational change upon substrate binding, likely the ordering of a highly conserved loop in addition to global domain motions. In the GMPS reaction, all forward rates before product release appear to be faster than steady-state turnover, implying that release is likely rate-limiting. These studies establish the functional role of a substrate-induced conformational change in the GMPS catalytic cycle and provide a kinetic context for the formation of an ammonia channel linking the distinct active sites.
| S-EPMC3859818 | biostudies-literature
Project description:Many bacterial species in nature possess the ability to transition into a sessile lifestyle and aggregate into cohesive colonies, known as biofilms. Within a biofilm, bacterial cells are encapsulated within an extracellular polymeric substance (EPS) comprised of polysaccharides, proteins, nucleic acids, lipids, and other small molecules. The transition from planktonic growth to the biofilm lifecycle provides numerous benefits to bacteria, such as facilitating adherence to abiotic surfaces, evasion of a host immune system, and resistance to common antibiotics. As a result, biofilm-forming bacteria contribute to 65% of infections in humans, and substantially increase the energy and time required for treatment and recovery. Several biofilm specific exopolysaccharides, including cellulose, alginate, Pel polysaccharide, and poly-N-acetylglucosamine (PNAG), have been shown to play an important role in bacterial biofilm formation and their production is strongly correlated with pathogenicity and virulence. In many bacteria the biosynthetic machineries required for assembly of these exopolysaccharides are regulated by common signaling molecules, with the second messenger cyclic di-guanosine monophosphate (c-di-GMP) playing an especially important role in the post-translational activation of exopolysaccharide biosynthesis. Research on treatments of antibiotic-resistant and biofilm-forming bacteria through direct targeting of c-di-GMP signaling has shown promise, including peptide-based treatments that sequester intracellular c-di-GMP. In this review, we will examine the direct role c-di-GMP plays in the biosynthesis and export of biofilm exopolysaccharides with a focus on the mechanism of post-translational activation of these pathways, as well as describe novel approaches to inhibit biofilm formation through direct targeting of c-di-GMP.
| S-EPMC8461298 | biostudies-literature
Project description:Cyclic guanosine monophosphate (cGMP) is an important intracellular second messenger that mediates multiple tissue and cellular responses. The cGMP pathway is a key element in the pathophysiology of the heart and its modulation by drugs such as phosphodiesterase (PDE)-5 inhibitors and guanylate cyclase activators may represent a promising therapeutic approach for acute myocardial infarction, cardiac hypertrophy, heart failure, and doxorubicin cardiotoxicity in patients. In addition, PDE-5 inhibitors may prove to be innovative therapeutic agents for enhancing the chemosensitivity of doxorubicin while providing concurrent cardiac benefit.
| S-EPMC4230443 | biostudies-literature
Project description:Natriuretic peptides (NPs) represent a critical pathway in heart failure (HF). We explored genetic determinants of pharmacodynamic effects of B-type NP (BNP) and changes in plasma cyclic guanosine monophosphate (cGMP) and blood pressure (BP). HF patients (n = 135) received recombinant human BNP (nesiritide) at standard doses, and plasma cGMP levels were measured at baseline and during infusion. We tested the association of 119 single nucleotide polymorphisms (SNPs) in 4 candidate genes (NPR1, NPR2, NPR3, and membrane metallo-endopeptidase (MME)) with the change in cGMP and BP. Gene-based testing for association of genetic variation with endpoints was significant only for MME. Upon individual SNP testing, two loci in MME were associated with ΔcGMP; another (rs16824656) showed association with BP change. In summary, the pharmacodynamic effects of BNP vary substantially in HF patients and are associated with genetic variation in MME. MME genetic variation may be an important determinant of NP-mediated effects in humans.
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