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ELife Sep 2021Cyclic-di-guanosine monophosphate (c-di-GMP) is an important effector associated with acute-chronic infection transition in . Previously, we reported a signaling network...
Cyclic-di-guanosine monophosphate (c-di-GMP) is an important effector associated with acute-chronic infection transition in . Previously, we reported a signaling network SiaABCD, which regulates biofilm formation by modulating c-di-GMP level. However, the mechanism for SiaD activation by SiaC remains elusive. Here we determine the crystal structure of SiaC-SiaD-GpCpp complex and revealed a unique mirror symmetric conformation: two SiaD form a dimer with long stalk domains, while four SiaC bind to the conserved motifs on the stalks of SiaD and stabilize the conformation for further enzymatic catalysis. Furthermore, SiaD alone exhibits an inactive pentamer conformation in solution, demonstrating that SiaC activates SiaD through a dynamic mechanism of promoting the formation of active SiaD dimers. Mutagenesis assay confirmed that the stalks of SiaD are necessary for its activation. Together, we reveal a novel mechanism for DGC activation, which clarifies the regulatory networks of c-di-GMP signaling.
Topics: Bacterial Proteins; Binding Sites; Biofilms; Catalysis; Dinucleoside Phosphates; Enzyme Activation; Escherichia coli Proteins; Phosphorus-Oxygen Lyases; Protein Binding; Protein Conformation; Pseudomonas aeruginosa; Signal Transduction; Structure-Activity Relationship
PubMed: 34498587
DOI: 10.7554/eLife.67289 -
PLoS Biology Sep 2021Antiviral defenses can sense viral RNAs and mediate their destruction. This presents a challenge for host cells since they must destroy viral RNAs while sparing the host...
Antiviral defenses can sense viral RNAs and mediate their destruction. This presents a challenge for host cells since they must destroy viral RNAs while sparing the host mRNAs that encode antiviral effectors. Here, we show that highly upregulated interferon-stimulated genes (ISGs), which encode antiviral proteins, have distinctive nucleotide compositions. We propose that self-targeting by antiviral effectors has selected for ISG transcripts that occupy a less self-targeted sequence space. Following interferon (IFN) stimulation, the CpG-targeting antiviral effector zinc-finger antiviral protein (ZAP) reduces the mRNA abundance of multiple host transcripts, providing a mechanistic explanation for the repression of many (but not all) interferon-repressed genes (IRGs). Notably, IRGs tend to be relatively CpG rich. In contrast, highly upregulated ISGs tend to be strongly CpG suppressed. Thus, ZAP is an example of an effector that has not only selected compositional biases in viral genomes but also appears to have notably shaped the composition of host transcripts in the vertebrate interferome.
Topics: A549 Cells; Cell Line; Dinucleoside Phosphates; Humans; Interferon Regulatory Factors; Interferon-beta; RNA, Messenger; RNA, Viral; RNA-Binding Proteins; Virus Physiological Phenomena; Viruses
PubMed: 34491982
DOI: 10.1371/journal.pbio.3001352 -
ACS Chemical Biology Sep 2021Many pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) and lipoteichoic acid, are potent immunostimulatory molecules and promote the...
Many pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) and lipoteichoic acid, are potent immunostimulatory molecules and promote the expression of cyclooxygenase 2 (COX-2). While the production of COX-2, and ultimately prostaglandin E, could be protective, persistent induction of COX-2 leads to inflamed environments that can result in septic shock and death. Bacterial derived cyclic dinucleotides (CDNs), c-di-GMP and c-di-AMP, are also PAMPs and have been shown to produce inflamed environments via the production of pro-inflammatory cytokines such as type I interferons. The well-characterized CDN immunostimulatory mechanism involves binding to stimulator of interferon genes (STING), which ultimately results in the phosphorylation of IRF3 or release of NF-κB to promote expression of type I IFN or pro-inflammatory cytokines. In this study, we sought to investigate if CDNs promote COX-2 expression. Using RAW macrophages as a model system, we reveal that c-di-GMP, but not c-di-AMP or the host-derived 2',3'-cGAMP, promotes COX-2 expression. Using analogues of CDNs, we show that the presence of two guanines and two 3',5'-phosphodiester linkages are requirements for the promotion of COX-2 expression by cyclic dinucleotides. Both c-di-GMP and LPS inductions of COX-2 expression in RAW macrophages are STING-independent and are regulated by Tpl2-MEK-ERK-CREB signaling; inhibitors of Tpl2, MEK, and ERK could attenuate COX-2 expression promoted by c-di-GMP. This work adds to the growing body of evidence that cyclic dinucleotides regulate pathways other than the STING-TBK1-IRF3 axis. Additionally, the differential COX-2 induction by c-di-GMP but not c-di-AMP or cGAMP suggests that the type and level of inflammation could be dictated by the nucleotide signature of the invading pathogen.
Topics: Animals; Beclin-1; Cell Line; Cyclic GMP; Cyclooxygenase 2; Dinucleoside Phosphates; Gene Expression Regulation; Guanine; Immunity, Innate; Interferon Regulatory Factor-3; Interferon Type I; Macrophages; Mice; NF-kappa B; Oligonucleotides; Phosphorylation; Prostaglandins; Signal Transduction
PubMed: 34478263
DOI: 10.1021/acschembio.1c00342 -
Analytical and Bioanalytical Chemistry Nov 2021Cyclic dinucleotides (CDNs) are key secondary messenger molecules produced by cyclic dinucleotide synthases that trigger various cellular signaling cascades from...
Cyclic dinucleotides (CDNs) are key secondary messenger molecules produced by cyclic dinucleotide synthases that trigger various cellular signaling cascades from bacteria to vertebrates. In mammals, cyclic GMP-AMP synthase (cGAS) has been shown to bind to intracellular DNA and catalyze the production of the dinucleotide 2'3' cGAMP, which signals downstream effectors to regulate immune function, interferon signaling, and the antiviral response. Despite the importance of CDNs, sensitive and accurate methods to measure their levels in vivo are lacking. Here, we report a novel LC-MS/MS method to quantify CDNs in vivo. We characterized the mass spectrometric behavior of four different biologically relevant CDNs (c-di-AMP, c-di-GMP, 3'3' cGAMP, 2'3' cGAMP) and provided a means of visually representing fragmentation resulting from collision-induced dissociation at different energies using collision energy breakdown graphs. We then validated the method and quantified CDNs in two in vivo systems, the bacteria Escherichia coli OP50 and the killifish Nothobranchius furzeri. We found that optimization of LC-MS/MS parameters is crucial to sensitivity and accuracy. These technical advances should help illuminate physiological and pathological roles of these CDNs in in vivo settings. Graphical abstract.
Topics: Animals; Chromatography, Liquid; Cyclic GMP; Dinucleoside Phosphates; Escherichia coli; Fundulidae; Nucleotides, Cyclic; Tandem Mass Spectrometry
PubMed: 34476522
DOI: 10.1007/s00216-021-03628-6 -
Nucleic Acids Research Sep 2021The cyclic dinucleotide second messenger c-di-AMP is a major player in regulation of potassium homeostasis and osmolyte transport in a variety of bacteria. Along with...
The cyclic dinucleotide second messenger c-di-AMP is a major player in regulation of potassium homeostasis and osmolyte transport in a variety of bacteria. Along with various direct interactions with proteins such as potassium channels, the second messenger also specifically binds to transcription factors, thereby altering the processes in the cell on the transcriptional level. We here describe the structural and biochemical characterization of BusR from the human pathogen Streptococcus agalactiae. BusR is a member of a yet structurally uncharacterized subfamily of the GntR family of transcription factors that downregulates transcription of the genes for the BusA (OpuA) glycine-betaine transporter upon c-di-AMP binding. We report crystal structures of full-length BusR, its apo and c-di-AMP bound effector domain, as well as cryo-EM structures of BusR bound to its operator DNA. Our structural data, supported by biochemical and biophysical data, reveal that BusR utilizes a unique domain assembly with a tetrameric coiled-coil in between the binding platforms, serving as a molecular ruler to specifically recognize a 22 bp separated bipartite binding motif. Binding of c-di-AMP to BusR induces a shift in equilibrium from an inactivated towards an activated state that allows BusR to bind the target DNA, leading to transcriptional repression.
Topics: ATP-Binding Cassette Transporters; Biological Transport; Crystallography, X-Ray; DNA, Bacterial; DNA-Binding Proteins; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Potassium; Protein Domains; Second Messenger Systems; Streptococcus agalactiae; Transcription Factors
PubMed: 34432045
DOI: 10.1093/nar/gkab736 -
Infection and Immunity Oct 2021Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial...
Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis () and degradation ( and ). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (Δ strain) or c-di-AMP accumulation (Δ, Δ, and Δ Δ strains) drastically impaired general cell fitness and virulence of E. faecalis. In particular, the Δ strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine , and was virtually avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of the Δ strain also could be attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both and conditions. Collectively, these results demonstrate that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.
Topics: Animals; Biofilms; Dinucleoside Phosphates; Enterococcus faecalis; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Gram-Positive Bacterial Infections; Humans; Virulence
PubMed: 34424750
DOI: 10.1128/IAI.00365-21 -
Biochimica Et Biophysica Acta. General... Nov 2021Human HINT2 is an important mitochondrial enzyme involved in many processes such as apoptosis and bioenergetics, but its endogenous substrates and the three-dimensional...
Biochemical, crystallographic and biophysical characterization of histidine triad nucleotide-binding protein 2 with different ligands including a non-hydrolyzable analog of Ap4A.
BACKGROUND
Human HINT2 is an important mitochondrial enzyme involved in many processes such as apoptosis and bioenergetics, but its endogenous substrates and the three-dimensional structure of the full-length protein have not been identified yet.
METHODS
An HPLC assay was used to test the hydrolytic activity of HINT2 against various adenosine, guanosine, and 2'-deoxyguanosine derivatives containing phosphate bonds of different types and different leaving groups. Data on binding affinity were obtained by microscale thermophoresis (MST). Crystal structures of HINT2, in its apo form and with a dGMP ligand, were resolved to atomic resolution.
RESULTS
HINT2 substrate specificity was similar to that of HINT1, but with the major exception of remarkable discrimination against substrates lacking the 2'-hydroxyl group. The biochemical results were consistent with binding affinity measurements. They showed a similar binding strength of AMP and GMP to HINT2, and much weaker binding of dGMP, in contrast to HINT1. A non-hydrolyzable analog of Ap4A (JB419) interacted with both proteins with similar K and Ap4A is the signaling molecule that can interact with hHINT1 and regulate the activity of some transcription factors.
CONCLUSIONS
Several forms of homo- and heterodimers of different lengths of N-terminally truncated polypeptides resulting from degradation of the full-length protein were described. Ser144 in HINT2 appeared to be functionally equivalent to Ser107 in HINT1 by supporting the protonation of the leaving group in the hydrolytic mechanism of HINT2.
SIGNIFICANCE
Our results should be considered in future studies on the natural function of HINT2 and its role in nucleotide prodrug processing.
Topics: Dinucleoside Phosphates; Humans; Ligands; Mitochondrial Proteins
PubMed: 34329705
DOI: 10.1016/j.bbagen.2021.129968 -
Journal of Bacteriology Sep 20213'3'-Cyclic di-AMP (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. c-di-AMP is essential in many bacteria and...
3'3'-Cyclic di-AMP (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. c-di-AMP is essential in many bacteria and regulates a diverse array of effector proteins controlling pathogenesis, cell wall homeostasis, osmoregulation, and central metabolism. Despite the ubiquity and importance of c-di-AMP, methods to detect this signaling molecule are limited, particularly at single-cell resolution. In this work, crystallization of the Listeria monocytogenes c-di-AMP effector protein Lmo0553 enabled structure-guided design of a Förster resonance energy transfer (FRET)-based biosensor, which we have named CDA5. CDA5 is a fully genetically encodable, specific, and reversible biosensor which allows the detection of c-di-AMP dynamics both and within live cells in a nondestructive manner. Our initial studies identified a distribution of c-di-AMP in Bacillus subtilis populations first grown in Luria broth and then resuspended in diluted Luria broth compatible with fluorescence analysis. Furthermore, we found that B. subtilis mutants lacking either a c-di-AMP phosphodiesterase and cyclase have higher and lower FRET responses, respectively. These findings provide novel insight into the c-di-AMP distribution within bacterial populations and establish CDA5 as a powerful platform for characterizing new aspects of c-di-AMP regulation. c-di-AMP is an important nucleotide second messenger for which detection methods are severely limited. In this work we engineered and implemented a c-di-AMP-specific FRET biosensor to remedy this dearth. We present this biosensor, CDA5, as a versatile tool to investigate previously intractable facets of c-di-AMP biology.
Topics: Bacillus subtilis; Bacterial Proteins; Biosensing Techniques; Dinucleoside Phosphates; Fluorescence Resonance Energy Transfer; Gene Expression Regulation, Bacterial; Listeria monocytogenes; Models, Molecular; Mutation; Nucleotides; Protein Conformation
PubMed: 34309402
DOI: 10.1128/JB.00080-21 -
Nucleic Acids Research Sep 2021The most common form of DNA methylation involves the addition of a methyl group to a cytosine base in the context of a cytosine-phosphate-guanine (CpG) dinucleotide....
The most common form of DNA methylation involves the addition of a methyl group to a cytosine base in the context of a cytosine-phosphate-guanine (CpG) dinucleotide. Genomes from more primitive organisms are more abundant in CpG sites that, through the process of methylation, deamination and subsequent mutation to thymine-phosphate-guanine (TpG) sites, can produce new transcription factor binding sites. Here, we examined the evolutionary history of the over 36 000 glucocorticoid receptor (GR) consensus binding motifs in the human genome and identified a subset of them in regulatory regions that arose via a deamination and subsequent mutation event. GR can bind to both unmodified and methylated pre-GR binding sequences (GBSs) that contain a CpG site. Our structural analyses show that CpG methylation in a pre-GBS generates a favorable interaction with Arg447 mimicking that made with a TpG in a GBS. This methyl-specific recognition arose 420 million years ago and was conserved during the evolution of GR and likely helps fix the methylation on the relevant cytosines. Our study provides the first genetic, biochemical and structural evidence of high-affinity binding for the likely evolutionary precursor of extant TpG-containing GBS.
Topics: Binding Sites; DNA Methylation; DNA-Binding Proteins; Dinucleoside Phosphates; Evolution, Molecular; Genome, Human; Humans; Nucleic Acid Conformation; Receptors, Glucocorticoid; Regulatory Sequences, Nucleic Acid; Thymine
PubMed: 34289059
DOI: 10.1093/nar/gkab605 -
Toxicological Sciences : An Official... Sep 2021Molecular signatures are being increasingly integrated into predictive biology applications. However, there are limited studies comparing the overall predictivity of...
Molecular signatures are being increasingly integrated into predictive biology applications. However, there are limited studies comparing the overall predictivity of transcriptomic versus epigenomic signatures in relation to perinatal outcomes. This study set out to evaluate mRNA and microRNA (miRNA) expression and cytosine-guanine dinucleotide (CpG) methylation signatures in human placental tissues and relate these to perinatal outcomes known to influence maternal/fetal health; namely, birth weight, placenta weight, placental damage, and placental inflammation. The following hypotheses were tested: (1) different molecular signatures will demonstrate varying levels of predictivity towards perinatal outcomes, and (2) these signatures will show disruptions from an example exposure (ie, cadmium) known to elicit perinatal toxicity. Multi-omic placental profiles from 390 infants in the Extremely Low Gestational Age Newborns cohort were used to develop molecular signatures that predict each perinatal outcome. Epigenomic signatures (ie, miRNA and CpG methylation) consistently demonstrated the highest levels of predictivity, with model performance metrics including R2 (predicted vs observed) values of 0.36-0.57 for continuous outcomes and balanced accuracy values of 0.49-0.77 for categorical outcomes. Top-ranking predictors included miRNAs involved in injury and inflammation. To demonstrate the utility of these predictive signatures in screening of potentially harmful exogenous insults, top-ranking miRNA predictors were analyzed in a separate pregnancy cohort and related to cadmium. Key predictive miRNAs demonstrated altered expression in association with cadmium exposure, including miR-210, known to impact placental cell growth, blood vessel development, and fetal weight. These findings inform future predictive biology applications, where additional benefit will be gained by including epigenetic markers.
Topics: DNA Methylation; Dinucleoside Phosphates; Female; Humans; Infant, Newborn; Methylation; MicroRNAs; Placenta; Pregnancy
PubMed: 34255065
DOI: 10.1093/toxsci/kfab089