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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 -
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 -
Theoretical Biology & Medical Modelling Apr 2020CpGs, the major methylation sites in vertebrate genomes, exhibit a high mutation rate from the methylated form of CpG to TpG/CpA and, therefore, influence the evolution...
BACKGROUND
CpGs, the major methylation sites in vertebrate genomes, exhibit a high mutation rate from the methylated form of CpG to TpG/CpA and, therefore, influence the evolution of genome composition. However, the quantitative effects of CpG to TpG/CpA mutations on the evolution of genome composition in terms of the dinucleotide frequencies/proportions remain poorly understood.
RESULTS
Based on the neutral theory of molecular evolution, we propose a methylation-driven model (MDM) that allows predicting the changes in frequencies/proportions of the 16 dinucleotides and in the GC content of a genome given the known number of CpG to TpG/CpA mutations. The application of MDM to the 10 published vertebrate genomes shows that, for most of the 16 dinucleotides and the GC content, a good consistency is achieved between the predicted and observed trends of changes in the frequencies and content relative to the assumed initial values, and that the model performs better on the mammalian genomes than it does on the lower-vertebrate genomes. The model's performance depends on the genome composition characteristics, the assumed initial state of the genome, and the estimated parameters, one or more of which are responsible for the different application effects on the mammalian and lower-vertebrate genomes and for the large deviations of the predicted frequencies of a few dinucleotides from their observed frequencies.
CONCLUSIONS
Despite certain limitations of the current model, the successful application to the higher-vertebrate (mammalian) genomes witnesses its potential for facilitating studies aimed at understanding the role of methylation in driving the evolution of genome dinucleotide composition.
Topics: Animals; Base Sequence; DNA Methylation; Dinucleoside Phosphates; Evolution, Molecular; Genome; Humans; Mutation
PubMed: 32264909
DOI: 10.1186/s12976-020-00122-x -
Nucleic Acids Research Feb 2021Regulatory protein access to the DNA duplex 'interior' depends on local DNA 'breathing' fluctuations, and the most fundamental of these are thermally-driven base...
Regulatory protein access to the DNA duplex 'interior' depends on local DNA 'breathing' fluctuations, and the most fundamental of these are thermally-driven base stacking-unstacking interactions. The smallest DNA unit that can undergo such transitions is the dinucleotide, whose structural and dynamic properties are dominated by stacking, while the ion condensation, cooperative stacking and inter-base hydrogen-bonding present in duplex DNA are not involved. We use dApdA to study stacking-unstacking at the dinucleotide level because the fluctuations observed are likely to resemble those of larger DNA molecules, but in the absence of constraints introduced by cooperativity are likely to be more pronounced, and thus more accessible to measurement. We study these fluctuations with a combination of Molecular Dynamics simulations on the microsecond timescale and Markov State Model analyses, and validate our results by calculations of circular dichroism (CD) spectra, with results that agree well with the experimental spectra. Our analyses show that the CD spectrum of dApdA is defined by two distinct chiral conformations that correspond, respectively, to a Watson-Crick form and a hybrid form with one base in a Hoogsteen configuration. We find also that ionic structure and water orientation around dApdA play important roles in controlling its breathing fluctuations.
Topics: Circular Dichroism; DNA; Dinucleoside Phosphates; Ions; Markov Chains; Models, Molecular; Sodium Chloride; Water
PubMed: 33503257
DOI: 10.1093/nar/gkab015 -
Chembiochem : a European Journal of... Mar 2021Cyclic dinucleotide signaling systems, which are found ubiquitously throughout nature, allow organisms to rapidly and dynamically sense and respond to alterations in...
Cyclic dinucleotide signaling systems, which are found ubiquitously throughout nature, allow organisms to rapidly and dynamically sense and respond to alterations in their environments. In recent years, the second messenger, cyclic di-(3',5')-adenosine monophosphate (c-di-AMP), has been identified as an essential signaling molecule in a diverse array of bacterial genera. We and others have shown that defects in c-di-AMP homeostasis result in severe physiological defects and virulence attenuation in many bacterial species. Despite significant advancements in the field, there is still a major gap in the understanding of the environmental and cellular factors that influence c-di-AMP dynamics due to a lack of tools to sensitively and rapidly monitor changes in c-di-AMP levels. To address this limitation, we describe here the development of a luciferase-based coupled enzyme assay that leverages the cyclic nucleotide phosphodiesterase, CnpB, for the sensitive and high-throughput quantification of 3'3'-c-di-AMP. We also demonstrate the utility of this approach for the quantification of the cyclic oligonucleotide-based anti-phage signaling system (CBASS) effector, 3'3'-cGAMP. These findings establish CDA-Luc as a more affordable and sensitive alternative to conventional c-di-AMP detection tools with broad utility for the study of bacterial cyclic dinucleotide physiology.
Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Adenosine Monophosphate; Bacteria; Bacterial Proteins; Dinucleoside Phosphates; Enzyme Assays; High-Throughput Screening Assays; Hydrolysis; Luciferases; Luminescent Measurements; Mycobacterium tuberculosis
PubMed: 33142009
DOI: 10.1002/cbic.202000667 -
Journal of Chemical Theory and... Jun 2022RNA modulation via small molecules is a novel approach in pharmacotherapies, where the determination of the structural properties of RNA motifs is considered a promising...
RNA modulation via small molecules is a novel approach in pharmacotherapies, where the determination of the structural properties of RNA motifs is considered a promising way to develop drugs capable of targeting RNA structures to control diseases. However, due to the complexity and dynamic nature of RNA molecules, the determination of RNA structures using experimental approaches is not always feasible, and computational models employing force fields can provide important insight. The quality of the force field will determine how well the predictions are compared to experimental observables. Stacking in nucleic acids is one such structural property, originating mainly from London dispersion forces, which are quantum mechanical and are included in molecular mechanics force fields through nonbonded interactions. Geometric descriptions are utilized to decide if two residues are stacked and hence to calculate the stacking free energies for RNA dinucleoside monophosphates (DNMPs) through statistical mechanics for comparison with experimental thermodynamics data. Here, we benchmark four different stacking definitions using molecular dynamics (MD) trajectories for 16 RNA DNMPs produced by two different force fields (RNA-IL and ff99OL3) and show that our stacking definition better correlates with the experimental thermodynamics data. While predictions within an accuracy of 0.2 kcal/mol at 300 K were observed in RNA CC, CU, UC, AG, GA, and GG, stacked states of purine-pyrimidine and pyrimidine-purine DNMPs, respectively, were typically underpredicted and overpredicted. Additionally, population distributions of RNA UU DNMPs were poorly predicted by both force fields, implying a requirement for further force field revisions. We further discuss the differences predicted by each RNA force field. Finally, we show that discrete path sampling (DPS) calculations can provide valuable information and complement the MD simulations. We propose the use of experimental thermodynamics data for RNA DNMPs as benchmarks for testing RNA force fields.
Topics: DNA; Dinucleoside Phosphates; Molecular Dynamics Simulation; Nucleic Acid Conformation; Purines; Pyrimidines; RNA; Thermodynamics
PubMed: 35652685
DOI: 10.1021/acs.jctc.2c00178 -
Biochemistry Aug 2023Adenylate kinases play a crucial role in cellular energy homeostasis through the interconversion of ATP, AMP, and ADP in all living organisms. Here, we explore how...
Adenylate kinases play a crucial role in cellular energy homeostasis through the interconversion of ATP, AMP, and ADP in all living organisms. Here, we explore how adenylate kinase (AdK) from interacts with diadenosine tetraphosphate (AP4A), a putative alarmone associated with transcriptional regulation, stress, and DNA damage response. From a combination of EPR and NMR spectroscopy together with X-ray crystallography, we found that AdK interacts with AP4A with two distinct modes that occur on disparate time scales. First, AdK dynamically interconverts between open and closed states with equal weights in the presence of AP4A. On a much slower time scale, AdK hydrolyses AP4A, and we suggest that the dynamically accessed substrate-bound open AdK conformation enables this hydrolytic activity. The partitioning of the enzyme into open and closed states is discussed in relation to a recently proposed linkage between active site dynamics and collective conformational dynamics.
Topics: Escherichia coli; Adenylate Kinase; Hydrolysis; Dinucleoside Phosphates; Catalysis; Catalytic Domain
PubMed: 37418448
DOI: 10.1021/acs.biochem.3c00189 -
Bacterial Second Messenger Cyclic di-AMP Modulates the Competence State in Streptococcus pneumoniae.Journal of Bacteriology Jan 2020(the pneumococcus) is a naturally competent organism that causes diseases such as pneumonia, otitis media, and bacteremia. The essential bacterial second messenger...
(the pneumococcus) is a naturally competent organism that causes diseases such as pneumonia, otitis media, and bacteremia. The essential bacterial second messenger cyclic di-AMP (c-di-AMP) is an emerging player in the stress responses of many pathogens. In , c-di-AMP is produced by a diadenylate cyclase, CdaA, and cleaved by phosphodiesterases Pde1 and Pde2. c-di-AMP binds a transporter of K (Trk) family protein, CabP, which subsequently halts K uptake via the transporter TrkH. Recently, it was reported that Pde1 and Pde2 are essential for pneumococcal virulence in mouse models of disease. To elucidate c-di-AMP-mediated transcription that may lead to changes in pathogenesis, we compared the transcriptomes of wild-type (WT) and Δ Δ strains by transcriptome sequencing (RNA-Seq) analysis. Notably, we found that many competence-associated genes are significantly upregulated in the Δ Δ strain compared to the WT. These genes play a role in DNA uptake, recombination, and autolysis. Competence is induced by a quorum-sensing mechanism initiated by the secreted factor competence-stimulating peptide (CSP). Surprisingly, the Δ Δ strain exhibited reduced transformation efficiency compared to WT bacteria, which was c-di-AMP dependent. Transformation efficiency was also directly related to the [K] in the medium, suggesting a link between c-di-AMP function and the pneumococcal competence state. We found that a strain that possesses a V76G variation in CdaA produced less c-di-AMP and was highly susceptible to CSP. Deletion of and restored the growth of these bacteria in medium with CSP. Overall, our study demonstrates a novel role for c-di-AMP in the competence program of Genetic competence in bacteria leads to horizontal gene transfer, which can ultimately affect antibiotic resistance, adaptation to stress conditions, and virulence. While the mechanisms of pneumococcal competence signaling cascades have been well characterized, the molecular mechanism behind competence regulation is not fully understood. The bacterial second messenger c-di-AMP has previously been shown to play a role in bacterial physiology and pathogenesis. In this study, we provide compelling evidence for the interplay between c-di-AMP and the pneumococcal competence state. These findings not only attribute a new biological function to this dinucleotide as a regulator of competence, transformation, and survival under stress conditions in pneumococci but also provide new insights into how pneumococcal competence is modulated.
Topics: Bacterial Proteins; DNA-Binding Proteins; Dinucleoside Phosphates; Glycine; Hydrogen-Ion Concentration; Potassium; Second Messenger Systems; Sequence Analysis, RNA; Streptococcus pneumoniae; Transcriptome
PubMed: 31767779
DOI: 10.1128/JB.00691-19 -
Nature Communications Feb 2021Many bacteria use cyclic di-AMP as a second messenger to control potassium and osmotic homeostasis. In Bacillus subtilis, several c-di-AMP binding proteins and RNA...
Many bacteria use cyclic di-AMP as a second messenger to control potassium and osmotic homeostasis. In Bacillus subtilis, several c-di-AMP binding proteins and RNA molecules have been identified. Most of these targets play a role in controlling potassium uptake and export. In addition, c-di-AMP binds to two conserved target proteins of unknown function, DarA and DarB, that exclusively consist of the c-di-AMP binding domain. Here, we investigate the function of the c-di-AMP-binding protein DarB in B. subtilis, which consists of two cystathionine-beta synthase (CBS) domains. We use an unbiased search for DarB interaction partners and identify the (p)ppGpp synthetase/hydrolase Rel as a major interaction partner of DarB. (p)ppGpp is another second messenger that is formed upon amino acid starvation and under other stress conditions to stop translation and active metabolism. The interaction between DarB and Rel only takes place if the bacteria grow at very low potassium concentrations and intracellular levels of c-di-AMP are low. We show that c-di-AMP inhibits the binding of DarB to Rel and the DarB-Rel interaction results in the Rel-dependent accumulation of pppGpp. These results link potassium and c-di-AMP signaling to the stringent response and thus to the global control of cellular physiology.
Topics: Bacillus subtilis; Bacterial Proteins; Dinucleoside Phosphates; Guanosine Pentaphosphate; Hydrolases; Models, Biological; Protein Binding; Protein Domains; Second Messenger Systems; Signal Transduction
PubMed: 33619274
DOI: 10.1038/s41467-021-21306-0