Visualization of oxidized guanine nucleotides accumulation in living cells with split MutT.

Cancer cells produce vast quantities of reactive oxygen species, leading to the accumulation of toxic nucleotides as 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP). The human MTH1 protein catalyzes the hydrolysis of 8-oxo-dGTP, and cancer cells are dependent on MTH1 for their survival. MTH1 inhibitors are possible candidates for a class of anticancer drugs; however, a reliable screening system using live cells has not been developed. Here we report a visualization method for 8-oxo-dGTP and its related nucleotides in living cells. Escherichia coli MutT, a functional homologue of MTH1, is divided into the N-terminal (1-95) and C-terminal (96-129) parts (Mu95 and 96tT, respectively). Mu95 and 96tT were fused to Ash (assembly helper tag) and hAG (Azami Green), respectively, to visualize the nucleotides as fluorescent foci formed upon the Ash-hAG association. The foci were highly increased when human cells expressing Ash-Mu95 and hAG-96tT were treated with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-dGTP. The foci formation by 8-oxo-dG(TP) was strikingly enhanced by the MTH1 knockdown. Moreover, known MTH1 inhibitors and oxidizing reagents also increased foci. This is the first system that visualizes damaged nucleotides in living cells, provides an excellent detection method for the oxidized nucleotides and oxidative stress, and enables high throughput screening for MTH1 inhibitors.

Extracellular Guanosine and Guanine Nucleotides Decrease Viability of Human Breast Cancer SKBR-3 Cells.

Though the physiological effects of adenosine and adenine nucleotides on purinergic receptors in cancer cells have been well studied, the influence of extracellular guanosine and guanine nucleotides on breast cancer cells remains unclear. Here, we show that extracellular guanosine and guanine nucleotides decrease the viability and proliferation of human breast cancer SKBR-3 cells. Treatment with guanosine or guanine nucleotides increased mitochondrial production of reactive oxygen species (ROS), and modified the cell cycle. Guanosine-induced cell death was suppressed by treatment with adenosine or the equilibrium nucleoside transporter (ENT) 1/2 inhibitor dipyridamole, but was not affected by adenosine receptor agonists or antagonists. These results suggest that guanosine inhibits adenosine uptake through ENT1/2, but does not antagonize adenosine receptors. In contrast, guanosine triphosphate (GTP)-induced cell death was suppressed not only by adenosine and dipyridamole, but also by the A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), suggesting that GTP-induced cell death is mediated in part by an antagonistic effect on adenosine A1 receptor. Thus, both guanosine and GTP induce apoptosis of breast cancer cells, but via at least partially different mechanisms.

Azathioprine-induced vanishing bile duct syndrome: The value of early thiopurine metabolism assessment.

About 15% to 28% of patients treated with thiopurines experienced adverse drug reactions, such as haematological and hepatic toxicities. Some of these related to the polymorphic activity of the thiopurine S-methyltransferase (TPMT), the key detoxifying enzyme of thiopurine metabolism. We report here a case of thiopurine-induced ductopenia with a comprehensive pharmacological analysis on thiopurine metabolism. A 34-year-old woman, with a medical history of severe systemic lupus erythematosus with recent introduction of azathioprine therapy, presented with mild fluctuating transaminase blood levels consistent with a hepatocellular pattern, which evolved to a cholestatic pattern over the next weeks. A blood thiopurine metabolite assay revealed low 6-thioguanine nucleotides (6-TGN) level and a dramatically increased 6-methylmercaptopurine ribonucleotides (6-MMPN) level, together with an unfavourable [6-MMPN:6-TGN] metabolite ratio and a high TPMT activity. After a total of about 6 months of thiopurine therapy, a transjugular liver biopsy revealed a ductopenia, and azathioprine discontinuation led to further clinical improvement. In line with previous reports from the literature, our case supports the fact that ductopenia is a rare adverse drug reaction of azathioprine. The mechanism of reaction is unknown but may involve high 6-MMPN blood level, due to unusual thiopurine metabolism (switched metabolism). Early therapeutic drug monitoring with measurement of 6-TGN and 6-MMPN blood levels may help physicians to identify patients at risk of similar duct injury.

STING activation in alveolar macrophages and group 2 innate lymphoid cells suppresses IL-33-driven type 2 immunopathology.

2'3'-cGAMP is known as a nonclassical second messenger and small immune modulator that possesses potent antitumor and antiviral activities via inducing the stimulator of IFN genes-mediated (STING-mediated) signaling pathway. However, its function in regulating type 2 immune responses remains unknown. Therefore, we sought to determine a role of STING activation by 2'3'-cGAMP in type 2 inflammatory reactions in multiple mouse models of eosinophilic asthma. We discovered that 2'3'-cGAMP administration strongly attenuated type 2 lung immunopathology and airway hyperreactivity induced by IL-33 and a fungal allergen, Aspergillus flavus. Mechanistically, upon the respiratory delivery, 2'3'-cGAMP was mainly internalized by alveolar macrophages, in which it activated the STING/IFN regulatory factor 3/type I IFN signaling axis to induce the production of inhibitory factors containing IFN-α, which blocked the IL-33-mediated activation of group 2 innate lymphoid (ILC2) cells in vivo. We further demonstrated that 2'3'-cGAMP directly suppressed the proliferation and function of both human and mouse ILC2 cells in vitro. Taken together, our findings suggest that STING activation by 2'3'-cGAMP in alveolar macrophages and ILC2 cells can negatively regulate type 2 immune responses, implying that the respiratory delivery of 2'3'-cGAMP might be further developed as an alternative strategy for treating type 2 immunopathologic diseases such as eosinophilic asthma.

Effect of Ras-guanine nucleotide release factor 1-mediated H-Ras/ERK signaling pathway on glioma.

OBJECTIVE: To investigate the function of Ras-guanine nucleotide release factor 1 (Ras-GRF1) in glioma through mediating H-Ras/ERK signaling pathway. METHODS: Ras-GRF1, H-Ras, K-Ras and N-Ras expressions in glioma and normal brain tissues were detected via Immunohistochemistry. Glioma cells (U87 cells, U251 cells and primary human glioma cells) were transfected with Ras-GRF1 siRNA, H-Ras siRNA and/or Ras-GRF1 lentivirus activation particles. Then, the following aspects were evaluated: cell proliferation by MTT assay, clonogenic ability by the plate clone formation experiment, cell migration and invasion by Wound-healing and Transwell assays, and cell apoptosis by Annexin-V-FITC/PI staining. The protein expressions were measured by Western blotting. Subcutaneous and orthotopic mouse models of glioma were conducted to determine the role of Ras-GRF1 in glioma tumorigenesis. RESULTS: Ras-GRF1, H-Ras, K-Ras and N-Ras expressions were upregulated in the glioma tissues, which were correlated with the WHO grade of glioma. Besides, Ras-GRF1 expression was positively related to H-Ras expression. Ras-GRF1 siRNA could reduce the expression of H-Ras and p-ERK/ERK in glioma cell. H-Ras siRNA inhibited the proliferation, clone formation, migration and invasion, and enhance the apoptosis of glioma cells, which, however, were reversed by Ras-GRF1 lentivirus activation particles. In vivo experiments also revealed that Ras-GRF1 shRNA reduced the volume and weight of the tumors in the nude mice, with down-regulations of H-Ras and p-ERK/ERK. CONCLUSION: Ras-GRF1 was upregulated in glioma tissues and correlated with its malignancy and prognosis. Silencing Ras-GRF1, through mediating H-Ras/ERK pathway, may suppress the growth and metastasis of glioma.

hGBP1 Coordinates Chlamydia Restriction and Inflammasome Activation through Sequential GTP Hydrolysis.

Human guanylate binding protein 1 (hGBP1) belongs to the dynamin superfamily of GTPases and conveys host defense against intracellular bacteria and parasites. During infection, hGBP1 is recruited to pathogen-containing vacuoles, such as Chlamydia trachomatis inclusions, restricts pathogenic growth, and induces the activation of the inflammasome pathway. hGBP1 has a unique catalytic activity to hydrolyze guanosine triphosphate (GTP) to guanosine monophosphate (GMP) in two consecutive cleavage steps. However, the functional significance of this activity in host defense remains elusive. Here, we generate a structure-guided mutant that specifically abrogates GMP production, while maintaining fast cooperative GTP hydrolysis. Complementation experiments in human monocytes/macrophages show that hGBP1-mediated GMP production is dispensable for restricting Chlamydia trachomatis growth but is necessary for inflammasome activation. Mechanistically, GMP is catabolized to uric acid, which in turn activates the NLRP3 inflammasome. Our study demonstrates that the unique enzymology of hGBP1 coordinates bacterial growth restriction and inflammasome signaling.

Highly sensitive and rapid determination of azathioprine metabolites in whole blood lysate by liquid chromatography-tandem mass spectrometry.

Individualized therapy involves genetic test of drug metabolism, which provides information about the initial dose and therapeutic drug monitoring for adjusting the subsequent dose. Consequently, toxic side effects are expected to be minimized and therapeutic effects to be maximized. In this study, an ultra-performance liquid chromatography tandem mass spectrometry method that was specific, accurate and sensitive was developed to simultaneously determine azathioprine two metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methyl-mercaptopurine riboside (6-MMPr) in the whole blood lysate. We precipitated the sample by trifluoroacetic acid under the protection of dithiothreitol, with 6-MMPr and 6-TGN being hydrolyzed to produce 6-methymercaptopurine and 6-thioguanine. In the chromatographic separation, Waters ACQUITY BEH C18 (2.1 × 100 mm, 1.7 µm) chromatographic column was applied and gradient elution was conducted with 0.02 mol/L ammonium acetate buffer (which contains 0.3% formic acid) and acetonitrile at a flow rate of 0.4 ml/min. Tandem mass spectrometry in multiple reaction monitoring mode was applied for detection via electrospray ionization source in positive ionization mode. The analyzing process lasted for no more than 2 min. The calibration curve for each metabolite fitted a least squares model (weighed 1/X) from 1.25 to 5000 ng/ml (r2 > 0.99). The ion pairs were detected as 6-MMP m/z 167.07 â†’ 152.15, 6-TG m/z 168.06 â†’ 134.13, and internal standard m/z 171.07 â†’ 137.14. Under the guidance of FDA guidelines for bioanalytical method validation, we carried out validation and obtained satisfactory results. The method was successfully utilized for monitoring the concentrations of each metabolite from 65 affected patients who had received azathioprine maintenance therapy and achieved optimal results.

Nucleotide second messengers in Streptomyces.

Antibiotic producing Streptomyces sense and respond to environmental signals by using nucleotide second messengers, including (p)ppGpp, cAMP, c-di-GMP and c-di-AMP. As summarized in this review, these molecules are important message carriers that coordinate the complex Streptomyces morphological transition from filamentous growth to sporulation along with the secondary metabolite production. Here, we provide an overview of the enzymes that make and break these second messengers and suggest candidates for (p)ppGpp and cAMP enzymes to be studied. We highlight the target molecules that bind these signalling molecules and elaborate individual functions that they control in the context of Streptomyces development. Finally, we discuss open questions in the field, which may guide future studies in this exciting research area.

Disruption of flavin homeostasis in isolated rat liver mitochondria.

It has been shown that spontaneous release of non-covalent flavins (from flavoenzymes) begins after isolation of mitochondria from rat liver, which is hydrolyzed to riboflavin. This process is stopped by 1 mM EDTA in the incubation medium. In the presence of NADH, deflavinization of flavoproteins leads to formation of superoxide by at least of three processes. The first of these occurs in complex I as a result of the spontaneous release of FMN from the active center. This process is inhibited by adenosine and guanosine phosphates, as well as NAD, but amplified by nicotinamide. The second process is associated with enzymatic hydrolysis of FAD and FMN to riboflavin; it is blocked by EDTA, AMP, NA, NAD. The third process is associated with non-enzymatic hydrolysis of FAD by iron ions in matrix; it is blocked by EDTA and AMP.

Identification of YdhV as the First Molybdoenzyme Binding a Bis-Mo-MPT Cofactor in Escherichia coli.

The oxidoreductase YdhV in Escherichia coli has been predicted to belong to the family of molybdenum/tungsten cofactor (Moco/Wco)-containing enzymes. In this study, we characterized the YdhV protein in detail, which shares amino acid sequence homology with a tungsten-containing benzoyl-CoA reductase binding the bis-W-MPT (for metal-binding pterin) cofactor. The cofactor was identified to be of a bis-Mo-MPT type with no guanine nucleotides present, which represents a form of Moco that has not been found previously in any molybdoenzyme. Our studies showed that YdhV has a preference for bis-Mo-MPT over bis-W-MPT to be inserted into the enzyme. In-depth characterization of YdhV by X-ray absorption and electron paramagnetic resonance spectroscopies revealed that the bis-Mo-MPT cofactor in YdhV is redox active. The bis-Mo-MPT and bis-W-MPT cofactors include metal centers that bind the four sulfurs from the two dithiolene groups in addition to a cysteine and likely a sulfido ligand. The unexpected presence of a bis-Mo-MPT cofactor opens an additional route for cofactor biosynthesis in E. coli and expands the canon of the structurally highly versatile molybdenum and tungsten cofactors.

The Roc-COR tandem domain of leucine-rich repeat kinase 2 forms dimers and exhibits conventional Ras-like GTPase properties.

The Parkinson's disease (PD)-causative leucine-rich repeat kinase 2 (LRRK2) belongs to the Roco family of G-proteins comprising a Ras-of-complex (Roc) domain followed by a C-terminal of Roc (COR) domain in tandem (called Roc-COR domain). Two prokaryotic Roc-COR domains have been characterized as 'G proteins activated by guanine nucleotide-dependent dimerization' (GADs), which require dimerization for activation of their GTPase activity and bind guanine nucleotides with relatively low affinities. Additionally, LRRK2 Roc domain in isolation binds guanine nucleotides with relatively low affinities. As such, LRRK2 GTPase domain was predicted to be a GAD. Herein, we describe the design and high-level expression of human LRRK2 Roc-COR domain (LRRK2 Roc-COR). Biochemical analyses of LRRK2 Roc-COR reveal that it forms homodimers, with the C-terminal portion of COR mediating its dimerization. Furthermore, it co-purifies and binds Mg2+ GTP/GDP at 1 : 1 stoichiometry, and it hydrolyzes GTP with Km  and kcat  of 22 nM and 4.70 × 10-4  min-1 ,  respectively. Thus, even though LRRK2 Roc-COR forms GAD-like homodimers, it exhibits conventional Ras-like GTPase properties, with high-affinity binding of Mg2+ -GTP/GDP and low intrinsic catalytic activity. The PD-causative Y1699C mutation mapped to the COR domain was previously reported to reduce the GTPase activity of full-length LRRK2. In contrast, this mutation induces no change in the GTPase activity, and only slight perturbations in the secondary structure contents of LRRK2 Roc-COR. As this mutation does not directly affect the GTPase activity of the isolated Roc-COR tandem, it is possible that the effects of this mutation on full-length LRRK2 occur via other functional domains. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

The role of tubulin-tubulin lattice contacts in the mechanism of microtubule dynamic instability.

Microtubules form from longitudinally and laterally assembling tubulin α-ß dimers. The assembly induces strain in tubulin, resulting in cycles of microtubule catastrophe and regrowth. This 'dynamic instability' is governed by GTP hydrolysis that renders the microtubule lattice unstable, but it is unclear how. We used a human microtubule nucleating and stabilizing neuronal protein, doublecortin, and high-resolution cryo-EM to capture tubulin's elusive hydrolysis intermediate GDP•Pi state, alongside the prehydrolysis analog GMPCPP state and the posthydrolysis GDP state with and without an anticancer drug, Taxol. GTP hydrolysis to GDP•Pi followed by Pi release constitutes two distinct structural transitions, causing unevenly distributed compressions of tubulin dimers, thereby tightening longitudinal and loosening lateral interdimer contacts. We conclude that microtubule catastrophe is triggered because the lateral contacts can no longer counteract the strain energy stored in the lattice, while reinforcement of the longitudinal contacts may support generation of force.

Luminescence determination of microRNAs based on the use of terbium(III) sensitized with an enzyme-activated guanine-rich nucleotide.

A method is reported for the fluorometric quantitation of microRNA. It is making use of a luminescent probe deribed from terbium(III) ion whose fluorescence is sensitized with a guanine-rich (G-rich) nucleotide. The probe has a large Stokes' shift and strong and sharp emission bands. The assay relies on the wide substrate specificity of terminal deoxynucleotidyl transferase (TdTase), which catalyzes the formation of long G-rich nucleotides when using microRNA primer as a trigger to start the polymerization. The addition of Tb(III) induces the formation of a G-quadruplex from the G-rich nucleotide, and this strongly enhances the green fluorescence of Tb(III) (peaking at 545 nm upon photoexcitation at 290 nm). Specifically, microRNA-21 was chosen as the analyte. The fluorescence intensity of Tb(III) increases linearly in the 1 pM to 1 nM microRNA concentration range, and the detection limit is as low as 0.11 pM. The method can distinguish between family members of microRNA and performs excellently even when applied to extracts of cancer cells. Graphical abstract A fluorometric technique is reported for the determination of microRNA. It is based on signal enhancement based on the sensitization of terbium(III) via a guanine-rich nucleotide sequence. Klenow Fragment exo- (KFexo-) generates DNA sequence at the 3'-OH of microRNA, and terminal deoxynucleotidyl transferase (TdTase) catalyzes the formation of long G-rich nucleotides.

A nucleotide-controlled conformational switch modulates the activity of eukaryotic IMP dehydrogenases.

Inosine-5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme for nucleotide metabolism and cell proliferation. Despite IMPDH is the target of drugs with antiviral, immunosuppressive and antitumor activities, its physiological mechanisms of regulation remain largely unknown. Using the enzyme from the industrial fungus Ashbya gossypii, we demonstrate that the binding of adenine and guanine nucleotides to the canonical nucleotide binding sites of the regulatory Bateman domain induces different enzyme conformations with significantly distinct catalytic activities. Thereby, the comparison of their high-resolution structures defines the mechanistic and structural details of a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity of eukaryotic IMPDHs. Remarkably, retinopathy-associated mutations lie within the mechanical hinges of the conformational change, highlighting its physiological relevance. Our results expand the mechanistic repertoire of Bateman domains and pave the road to new approaches targeting IMPDHs.

Identification of an RNase that preferentially cleaves A/G nucleotides.

Ribonucleases play an important role in the RNA metabolism which is critical for the localization, stability and function of mature RNA transcripts. More and more ribonucleases were discovered in recent years with the progress of technology. In the present study, we found that the uncharacterized C19orf43, a novel interacting protein of human telomerase RNA (hTR), digested T7 transcribed RNA, total cellular RNA and RNA oligos but not DNA. Thus we named this new RNase as hTRIR (human telomerase RNA interacting RNase). Genetic analysis showed that hTRIR is conserved among eukaryotic species and widely expressed in different cell lines. The RNase activity of hTRIR works in a broad temperature and pH range while divalent cations are not required. The conserved C-terminus of C19orf43 is necessary for its activity. Finally, we found that hTRIR cleaves all four unpaired RNA nucleotides from 5' end or 3' end with higher efficiency for purine bases, which suggested that hTRIR is an exoribonuclease. Taken together, our study showed the first evidence of the novel function of hTRIR in vitro, which provides clue to study the regulatory mechanism of hTR homeostasis in vivo.

Trichomonas vaginalis NTPDase and ecto-5'-nucleotidase hydrolyze guanine nucleotides and increase extracellular guanosine levels under serum restriction.

Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5'-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5'-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5'-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final accumulation of the nucleoside. The transcript levels of the five TvNTPDases gene sequences were analyzed by qRT-PCR and the highest gene expressions were found for TvNTPDase 2 and 4. The extracellular guanosine uptake was observed as (13C)GTP nucleotide into parasite DNA and it was lower than that observed for adenosine, labeled as (13C)ATP. These findings indicate the T. vaginalis preference for adenosine uptake and the accumulation of guanosine in the extracellular milieu, corroborating with HPLC data. Our data demonstrate, for the first time, the cascade of guanine nucleotides in T. vaginalis and open possibilities on the study of guanine-related purines other than the classical intracellular activity of G proteins for signal transduction.

A study of the utility of azathioprine metabolite testing in myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune neuromuscular disorder characterised by fatigable voluntary skeletal muscle weakness. The underlying pathogenesis is complex involving adaptive autoimmune responses. Azathioprine remains a first line broad acting immunosuppressant for MG. Due to varied clinical responses to azathioprine we aimed to investigate the relationship between azathioprine metabolites and symptom control. Mild correlations between Quantitative Myasthenia Gravis Score (QMG) vs. 6-thioguanine nucleotides (R=-0.317 p=0.186) and QMG vs. lymphocyte count (R=0.402 p=0.08) were found. Azathioprine metabolite measurement should be considered in MG patients with; pancytopenia, deranged liver function or recurrent infections.

A role for adenine nucleotides in the sensing mechanism to purine starvation in Leishmania donovani.

Purine salvage by Leishmania is an obligatory nutritional process that impacts both cell viability and growth. Previously, we have demonstrated that the removal of purines in culture provokes significant metabolic changes that enable Leishmania to survive prolonged periods of purine starvation. In order to understand how Leishmania sense and respond to changes in their purine environment, we have exploited several purine pathway mutants, some in which adenine and guanine nucleotide metabolism is uncoupled. While wild type parasites grow in any one of a variety of naturally occurring purines, the proliferation of these purine pathway mutants requires specific types or combinations of exogenous purines. By culturing purine pathway mutants in high levels of extracellular purines that are either permissive or non-permissive for growth and monitoring for previously defined markers of the adaptive response to purine starvation, we determined that adaptation arises from a surveillance of intracellular purine nucleotide pools rather than from a direct sensing of the extracellular purine content of the environment. Specifically, our data suggest that perturbation of intracellular adenine-containing nucleotide pools provides a crucial signal for inducing the metabolic changes necessary for the long-term survival of Leishmania in a purine-scarce environment.

NO-Mediated [Ca2+]cyt Increases Depend on ADP-Ribosyl Cyclase Activity in Arabidopsis.

Cyclic ADP ribose (cADPR) is a Ca(2+)-mobilizing intracellular second messenger synthesized from NAD by ADP-ribosyl cyclases (ADPR cyclases). In animals, cADPR targets the ryanodine receptor present in the sarcoplasmic/endoplasmic reticulum to promote Ca(2+) release from intracellular stores to increase the concentration of cytosolic free Ca(2+) in Arabidopsis (Arabidopsis thaliana), and cADPR has been proposed to play a central role in signal transduction pathways evoked by the drought and stress hormone, abscisic acid, and the circadian clock. Despite evidence for the action of cADPR in Arabidopsis, no predicted proteins with significant similarity to the known ADPR cyclases have been reported in any plant genome database, suggesting either that there is a unique route for cADPR synthesis or that a homolog of ADPR cyclase with low similarity might exist in plants. We sought to determine whether the low levels of ADPR cyclase activity reported in Arabidopsis are indicative of a bona fide activity that can be associated with the regulation of Ca(2+) signaling. We adapted two different fluorescence-based assays to measure ADPR cyclase activity in Arabidopsis and found that this activity has the characteristics of a nucleotide cyclase that is activated by nitric oxide to increase cADPR and mobilize Ca(2.)

Guanine nucleotide induced conformational change of Cdc42 revealed by hydrogen/deuterium exchange mass spectrometry.

Cdc42 regulates pathways related to cell division. Dysregulation of Cdc42 can lead to cancer, cardiovascular diseases and neurodegenerative diseases. GTP induced activation mechanism plays an important role in the activity and biological functions of Cdc42. P-loop, Switch I and Switch II are critical regions modulating the enzymatic activity of Cdc42. We applied amide hydrogen/deuterium exchange coupled with liquid chromatography mass spectrometry (HDXMS) to investigate the dynamic changes of apo-Cdc42 after GDP, GTP and GMP-PCP binding. The natural substrate GTP induced significant decreases of deuteration in P-loop and Switch II, moderate changes of deuteration in Switch I and significant changes of deuteration in the α7 helix, a region far away from the active site. GTP binding induced similar effects on H/D exchange to its non-hydrolysable analog, GMP-PCP. HDXMS results indicate that GTP binding blocked the solvent accessibility in the active site leading to the decrease of H/D exchange rate surrounding the active site, and further triggered a conformational change resulting in the drastic decrease of H/D exchange rate at the remote α7 helix. Comparing the deuteration levels in three activation states of apo-Cdc42, Cdc42-GDP and Cdc42-GMP-PCP, the apo-Cdc42 has the most flexible structure, which can be stabilized by guanine nucleotide binding. The rates of H/D exchange of Cdc42-GDP are between the GMP-PCP-bound and the apo form, but more closely to the GMP-PCP-bound form. Our results show that the activation of Cdc42 is a process of conformational changes involved with P-loop, Switch II and α7 helix for structural stabilization.