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Cell Reports Apr 2023Programmed cell suicide of infected bacteria, known as abortive infection (Abi), serves as an immune defense strategy to prevent the propagation of bacteriophage...
Programmed cell suicide of infected bacteria, known as abortive infection (Abi), serves as an immune defense strategy to prevent the propagation of bacteriophage viruses. Many Abi systems utilize bespoke cyclic nucleotide immune messengers generated upon infection to mobilize cognate death effectors. Here, we identify a family of bacteriophage nucleotidyltransferases (NTases) that synthesize competitor cyclic dinucleotide (CDN) ligands and inhibit TIR NADase effectors activated via a linked STING CDN sensor domain (TIR-STING). Through a functional screen of NTase-adjacent phage genes, we uncover candidate inhibitors of cell suicide induced by heterologous expression of tonically active TIR-STING. Among these, we demonstrate that a virus MazG-like nucleotide pyrophosphohydrolase, Atd1, depletes the starvation alarmone (p)ppGpp, revealing a potential role for the alarmone-activated host toxin MazF as an executioner of TIR-driven Abi. Phage NTases and counterdefenses like Atd1 preserve host viability to ensure virus propagation and represent tools to modulate TIR and STING immune responses.
Topics: Bacteria; Bacteriophages; Dinucleoside Phosphates; Guanosine Pentaphosphate; Immunity; Nucleotides; Nucleotidyltransferases
PubMed: 36952342
DOI: 10.1016/j.celrep.2023.112305 -
Biophysical Journal Jun 2022Heme has been shown to have a crucial role in the signal transduction mechanism of the facultative photoheterotrophic bacterium Rhodobacter sphaeroides. It interacts...
Heme has been shown to have a crucial role in the signal transduction mechanism of the facultative photoheterotrophic bacterium Rhodobacter sphaeroides. It interacts with the transcriptional regulatory complex AppA/PpsR, in which AppA and PpsR function as the antirepressor and repressor, respectively, of photosynthesis gene expression. The mechanism, however, of this interaction remains incompletely understood. In this study, we combined electron paramagnetic resonance (EPR) spectroscopy and Förster resonance energy transfer (FRET) to demonstrate the ligation of heme in PpsR with a proposed cysteine residue. We show that heme binding in AppA affects the fluorescent properties of the dark-adapted state of the protein, suggesting a less constrained flavin environment compared with the absence of heme and the light-adapted state. We performed ultrafast transient absorption measurements in order to reveal potential differences in the dynamic processes in the full-length AppA and its heme-binding domain alone. Comparison of the CO-binding dynamics demonstrates a more open heme pocket in the holo-protein, qualitatively similar to what has been observed in the CO sensor RcoM-2, and suggests a communication path between the blue-light-using flavin (BLUF) and sensing containing heme instead of cobalamin (SCHIC) domains of AppA. We have also examined quantitatively the affinity of PpsR to bind to individual DNA fragments of the puc promoter using fluorescence anisotropy assays. We conclude that oligomerization of PpsR is initially triggered by binding of one of the two DNA fragments and observe a ∼10-fold increase in the dissociation constant K for DNA binding upon heme binding to PpsR. Our study provides significant new insight at the molecular level on the regulatory role of heme that modulates the complex transcriptional regulation in R. sphaeroides and supports the two levels of heme signaling, via its binding to AppA and PpsR and via the sensing of gases like oxygen.
Topics: Bacterial Proteins; Dinucleoside Phosphates; Flavins; Flavoproteins; Gene Expression Regulation, Bacterial; Heme; Repressor Proteins; Rhodobacter sphaeroides
PubMed: 35488435
DOI: 10.1016/j.bpj.2022.04.031 -
Journal of the American Chemical Society Oct 2021Triazole linkages (TLs) are mimics of the phosphodiester bond in oligonucleotides with applications in synthetic biology and biotechnology. Here we report the...
Triazole linkages (TLs) are mimics of the phosphodiester bond in oligonucleotides with applications in synthetic biology and biotechnology. Here we report the RuAAC-catalyzed synthesis of a novel 1,5-disubstituted triazole (TL) dinucleoside phosphoramidite as well as its incorporation into oligonucleotides and compare its DNA polymerase replication competency with other TL analogues. We demonstrate that TL has superior replication kinetics to these analogues and is accurately replicated by polymerases. Derived structure-biocompatibility relationships show that linker length and the orientation of a hydrogen bond acceptor are critical and provide further guidance for the rational design of artificial biocompatible nucleic acid backbones.
Topics: Catalysis; DNA; DNA-Directed DNA Polymerase; Dinucleoside Phosphates; Molecular Mimicry; Triazoles
PubMed: 34546729
DOI: 10.1021/jacs.1c08057 -
Journal of the National Cancer Institute Oct 2019Age is one of the strongest predictors of cancer, chronic disease, and mortality, but biological responses to aging differ among people. Epigenetic DNA modifications...
BACKGROUND
Age is one of the strongest predictors of cancer, chronic disease, and mortality, but biological responses to aging differ among people. Epigenetic DNA modifications have been used to estimate "biological age," which may be a useful predictor of disease risk. We tested this hypothesis for breast cancer.
METHODS
Using a case-cohort approach, we measured baseline blood DNA methylation of 2764 women enrolled in the Sister Study, 1566 of whom subsequently developed breast cancer after an average of 6 years. Using three previously established methylation-based "clocks" (Hannum, Horvath, and Levine), we defined biological age acceleration for each woman by comparing her estimated biological age with her chronological age. Hazard ratios and 95% confidence intervals for breast cancer risk were estimated using Cox regression models. All statistical tests were two-sided.
RESULTS
Each of the three clocks showed that biological age acceleration was statistically significantly associated with increased risk of developing breast cancer (5-year age acceleration, Hannum's clock: hazard ratio [HR] = 1.10, 95% confidence interval [CI] = 1.00 to 1.21, P = .04; Horvath's clock: HR = 1.08, 95% CI = 1.00 to 1.17, P = .04; Levine's clock: HR = 1.15, 95% CI = 1.07 to 1.23, P < .001). For Levine's clock, each 5-year acceleration in biological age corresponded with a 15% increase in breast cancer risk. Although biological age may accelerate with menopausal transition, age acceleration in premenopausal women independently predicted breast cancer. Case-only analysis suggested that, among women who develop breast cancer, increased age acceleration is associated with invasive cancer (odds ratio for invasive = 1.09, 95% CI = 0.98 to 1.22, P = .10).
CONCLUSIONS
DNA methylation-based measures of biological age may be important predictors of breast cancer risk.
Topics: Adult; Aged; Aging; Breast Neoplasms; DNA Methylation; Dinucleoside Phosphates; Disease Susceptibility; Epigenesis, Genetic; Female; Gene Expression Regulation; Genetic Predisposition to Disease; Humans; Middle Aged; Odds Ratio; Prognosis; Proportional Hazards Models; Risk Assessment; Risk Factors
PubMed: 30794318
DOI: 10.1093/jnci/djz020 -
Protein Science : a Publication of the... Mar 2023Cyclic-di-nucleotide-based secondary messengers regulate various physiological functions, including stress responses in bacteria. Cyclic diadenosine monophosphate...
Cyclic-di-nucleotide-based secondary messengers regulate various physiological functions, including stress responses in bacteria. Cyclic diadenosine monophosphate (c-di-AMP) has recently emerged as a crucial second messenger with implications in processes including osmoregulation, antibiotic resistance, biofilm formation, virulence, DNA repair, ion homeostasis, and sporulation, and has potential therapeutic applications. The contrasting activities of the enzymes diadenylate cyclase (DAC) and phosphodiesterase (PDE) determine the equilibrium levels of c-di-AMP. Although c-di-AMP is suspected of playing an essential role in the pathophysiology of bacterial infections and in regulating host-pathogen interactions, the mechanisms of its regulation remain relatively unexplored in mycobacteria. In this report, we biochemically and structurally characterize the c-di-AMP synthase (MsDisA) from Mycobacterium smegmatis. The enzyme activity is regulated by pH and substrate concentration; conditions of significance in the homoeostasis of c-di-AMP levels. Substrate binding stimulates conformational changes in the protein, and pApA and ppApA are synthetic intermediates detectable when enzyme efficiency is low. Unlike the orthologous Bacillus subtilis enzyme, MsDisA does not bind to, and its activity is not influenced in the presence of DNA. Furthermore, we have determined the cryo-EM structure of MsDisA, revealing asymmetry in its structure in contrast to the symmetric crystal structure of Thermotoga maritima DisA. We also demonstrate that the N-terminal minimal region alone is sufficient and essential for oligomerization and catalytic activity. Our data shed light on the regulation of mycobacterial DisA and possible future directions to pursue.
Topics: Mycobacterium smegmatis; Bacterial Proteins; Dinucleoside Phosphates; Bacillus subtilis
PubMed: 36660887
DOI: 10.1002/pro.4568 -
Current Protocols Nov 2022This article describes a simple, reliable, efficient, and improved solution-phase method for the gram-scale chemical synthesis of RNA dinucleotides such as pA pA, pA pG,...
This article describes a simple, reliable, efficient, and improved solution-phase method for the gram-scale chemical synthesis of RNA dinucleotides such as pA pA, pA pG, and pA pU that utilizes phosphoramidite chemistry. The overall synthetic strategy involves three steps. The first step involves the coupling reaction between 5'-O-MMT protected nucleoside-3'-O-phosphoramidite and a protected nucleoside containing a free 5'-OH group in the presence of tetrazole, followed by the oxidation of phosphite triester using tert-butyl hydroperoxide to give the corresponding protected N pN. Next, the 5'-O-MMT is cleaved under 3% trichloroacetic acid in dichloromethane conditions. Finally, the 5'-hydroxyl group is phosphorylated by the use of an activated bis(2-cyanoethyl)-N,N-diisopropyl phosphoramidite using tetrazole, followed by the oxidation of trivalent to pentavalent phosphorus using tert-butyl hydroperoxide and subsequent deprotection using ammonium hydroxide to afford the corresponding RNA dinucleotide, pN pN, in good yields with high purity (>99.5%). © 2022 Wiley Periodicals LLC.
Topics: Dinucleoside Phosphates; Nucleosides; RNA; tert-Butylhydroperoxide; Tetrazoles
PubMed: 36342272
DOI: 10.1002/cpz1.583 -
ACS Sensors Sep 2022Fluorescence-guided cancer surgery can dramatically improve recurrence rates and postoperative quality of life of patients by accurately distinguishing the boundary...
Fluorescence-guided cancer surgery can dramatically improve recurrence rates and postoperative quality of life of patients by accurately distinguishing the boundary between normal and cancer tissues during surgery, thereby minimizing excision of normal tissue. One promising target in early stage cancer is fragile histidine triad (FHIT), a cancer suppressor protein with dinucleoside triphosphate hydrolase activity. In this study, we have developed fluorescence probes containing a nucleoside diphosphate moiety, which dramatically improves the reactivity and specificity for FHIT, and a moderately lipophilic ester moiety to increase the membrane permeability. The ester moiety is cleaved by ubiquitous intracellular esterases, and then, FHIT in the cells specifically cleaves nucleoside monophosphate. The remaining phosphate moiety is rapidly cleaved by ubiquitous intracellular phosphatases to release the fluorescent dye. We confirmed that this probe can detect FHIT activity in living cells. A comprehensive evaluation of the effects of various ester moieties revealed that probes with CLogP = 5-7 showed good membrane permeability and were good substrates of the target enzyme; these findings may be helpful in the rational design of other multiple phosphate-containing probes targeting intracellular enzymes.
Topics: Acid Anhydride Hydrolases; Dinucleoside Phosphates; Diphosphates; Esterases; Esters; Fluorescence; Fluorescent Dyes; Histidine; Humans; Hydrophobic and Hydrophilic Interactions; Neoplasm Proteins; Nucleosides; Phosphoric Monoester Hydrolases; Quality of Life
PubMed: 35981239
DOI: 10.1021/acssensors.2c01273 -
The Journal of Biological Chemistry Jul 2019Cyclic di-AMP (c-di-AMP) is the only second messenger known to be essential for bacterial growth. It has been found mainly in Gram-positive bacteria, including...
Cyclic di-AMP (c-di-AMP) is the only second messenger known to be essential for bacterial growth. It has been found mainly in Gram-positive bacteria, including pathogenic bacteria like CdaA is the sole diadenylate cyclase in , making this enzyme an attractive target for the development of novel antibiotic compounds. Here we report crystal structures of CdaA from in the apo state, in the post-catalytic state with bound c-di-AMP and catalytic Co ions, as well as in a complex with AMP. These structures reveal the flexibility of a tyrosine side chain involved in locking the adenine ring after ATP binding. The essential role of this tyrosine was confirmed by mutation to Ala, leading to drastic loss of enzymatic activity.
Topics: Bacterial Proteins; Binding Sites; Catalytic Domain; Cobalt; Crystallography, X-Ray; Dinucleoside Phosphates; Ligands; Listeria monocytogenes; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Phosphorus-Oxygen Lyases; Recombinant Proteins
PubMed: 31118276
DOI: 10.1074/jbc.RA119.009246 -
Patient Education and Counseling Aug 2021Double antiplatelet therapy (DAPT) is indicated for the treatment of coronary artery diseases (CAD). The optimal duration of therapy with DAPT continues to be a subject...
INTRODUCTION
Double antiplatelet therapy (DAPT) is indicated for the treatment of coronary artery diseases (CAD). The optimal duration of therapy with DAPT continues to be a subject of debate in the scientific community. To improve adherence to DAPT, the FDC (fixed dose combination) of Acetylsalicylic acid (ASA) and clopidogrel was developed into a single pill instead of two separate pills thus facilitating the dosage and administration of the therapy and increasing compliance. The aim of this study was to assess adherence and persistence over a period of two years in patients treated with DAPT composed of: ASA/clopidogrel, ASA/prasugrel, ASA/ticagrelor and FDC with ASA and clopidogrel in real life and to assess whether the use of ASA and clopidogrel FDC is associated with improved adherence.
MATERIALS AND METHODS
In the following retrospective pharmacological-observational non-interventional study, all patients treated with DAPT in the Hospital of Pescara from January 2010 to October 2019 were considered. Persistence to treatment is defined as the duration of time from initiation to discontinuation of treatment. Adherence was calculated as the ratio between Received Daily Dose (RDD) and Prescribed Daily Dose (PDD).
RESULTS
277 patients treated with ASA/clopidogrel, 77 patients treated with ASA/prasugrel, 57 patients treated with ASA/ticagrelor and 108 patients treated with FDC of ASA/clopidogrel were analysed. Persistence curves at two years showed a statistically significant difference (p < 0.001). Adherence to therapy was optimal with an absolute value at two years of 0.96. Adherence was better in patients treated with ASA/prasugrel with a value of 0.98 and with 97 % of patients with an adherence value greater than or equal to 0.8, while, it was worse in patients treated with FDC ASA/clopidogrel with an absolute value of 0.94 and with 88 % of patients with an optimal adherence value. No statistically significant difference was found between the ASA/clopidogrel FDC in comparison to each component taken as a separate pill (p = 0.0752).
CONCLUSION
DAPT along with ASA/clopidogrel showed a statistically significant better persistence than ASA/ticagrelor and ASA/prasugrel. Whereas, to our knowledge and as per the current literature no statistically significant differences were found, in terms of adherence in real life, between the use of ASA/Clopidogrel FDC and the use of two different pills.
Topics: Acute Coronary Syndrome; Clopidogrel; Dinucleoside Phosphates; Drug Therapy, Combination; Humans; Platelet Aggregation Inhibitors; Prasugrel Hydrochloride; Retrospective Studies; Treatment Outcome
PubMed: 33461875
DOI: 10.1016/j.pec.2021.01.005 -
Cardiovascular Revascularization... Aug 2022Guidelines recommend individualization of dual antiplatelet therapy (DAPT) duration. Whether to guide decisions based on bleeding risk, ischemic risk or a combination is...
BACKGROUND
Guidelines recommend individualization of dual antiplatelet therapy (DAPT) duration. Whether to guide decisions based on bleeding risk, ischemic risk or a combination is not known.
AIMS
To compare a bleeding prediction model, an ischemic prediction model, and the DAPT score in guiding DAPT duration.
METHODS
11,648 patients in the DAPT Study were categorized into higher and lower risk using a bleeding model, an ischemic model, and the DAPT score. Effect of 30 vs. 12 months of DAPT on bleeding events, ischemic events, and the combination (net-adverse clinical events [NACE]) was assessed.
RESULTS
Among patients stratified with the bleeding model, 30 vs. 12 months of DAPT resulted in similar ischemic and bleeding event rates. With the ischemic model, however, higher risk patients had a greater reduction in ischemic events with extended duration of DAPT (difference in risk differences [DRD]: -2.6%, 95% CI: -3.9 to -1.3%; p < 0.01), and a smaller increase in bleeding (DRD: -1.0%, 95% CI: -2.1-0.0%; p = 0.04). Similarly, high DAPT score patients had a greater reduction in ischemic events with extended DAPT duration (DRD: -2.4%, 95%: CI: -3.6 to -1.1%; p < 0.01) and a smaller increase in bleeding (DRD: -1.2%, 95%: CI: -2.2-0.0%; p = 0.02). Although NACE was similar for bleeding risk groups, NACE was significantly reduced with extended DAPT in the higher ischemic risk and high DAPT score groups.
CONCLUSIONS
In this low-bleeding risk population, stratifying patients based on predicted ischemic risk and the DAPT score best discerned benefit versus harm of extended DAPT duration on ischemic events, bleeding events, and NACE.
CONDENSED ABSTRACT
Duration of dual antiplatelet therapy (DAPT) should be guided by an individualized risk assessment. Bleeding risk tools have emerged to identify patients at high bleeding risk for whom truncated DAPT therapy may be safest. In a lower bleeding risk population, however, whether DAPT duration should be guided by bleeding risk, ischemic risk, or a combination is unknown. In this analysis, implementation of a score based on ischemic risk prediction and the DAPT score (a combination of ischemic and bleeding risk) best predicted ischemic events, bleeding events, and net-adverse clinical events (NACE).
Topics: Dinucleoside Phosphates; Drug Therapy, Combination; Dual Anti-Platelet Therapy; Hemorrhage; Humans; Ischemia; Percutaneous Coronary Intervention; Platelet Aggregation Inhibitors; Treatment Outcome
PubMed: 35045941
DOI: 10.1016/j.carrev.2022.01.006