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Angewandte Chemie (International Ed. in... Feb 2023Diadenosine polyphosphates (Ap As) are non-canonical nucleotides whose cellular concentrations increase during stress and are therefore termed alarmones, signaling...
Diadenosine polyphosphates (Ap As) are non-canonical nucleotides whose cellular concentrations increase during stress and are therefore termed alarmones, signaling homeostatic imbalance. Their cellular role is poorly understood. In this work, we assessed Ap As for their usage as cosubstrates for protein AMPylation, a post-translational modification in which adenosine monophosphate (AMP) is transferred to proteins. In humans, AMPylation mediated by the AMPylator FICD with ATP as a cosubstrate is a response to ER stress. Herein, we demonstrate that Ap A is proficiently consumed for AMPylation by FICD. By chemical proteomics using a new chemical probe, we identified new potential AMPylation targets. Interestingly, we found that AMPylation targets of FICD may differ depending on the nucleotide cosubstrate. These results may suggest that signaling at elevated Ap A levels during cellular stress differs from when Ap A is present at low concentrations, allowing response to extracellular cues.
Topics: Humans; Guanosine Pentaphosphate; Proteins; Adenosine Monophosphate; Dinucleoside Phosphates; Protein Processing, Post-Translational
PubMed: 36524454
DOI: 10.1002/anie.202213279 -
MBio Aug 2020The facultative intracellular pathogen , like many related , uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability,...
The facultative intracellular pathogen , like many related , uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP is essential; however, mutations in , the gene encoding c-di-AMP binding protein B, suppress essentiality. In this study, we identified that the reason for -dependent essentiality is through induction of the stringent response by RelA. RelA is a bifunctional RelA/SpoT homolog (RSH) that modulates levels of (p)ppGpp, a secondary messenger that orchestrates the stringent response through multiple allosteric interactions. We performed a forward genetic suppressor screen on bacteria lacking c-di-AMP to identify genomic mutations that rescued growth while was constitutively expressed and identified mutations in the synthetase domain of RelA. The synthetase domain of RelA was also identified as an interacting partner of CbpB in a yeast-2-hybrid screen. Biochemical analyses confirmed that free CbpB activates RelA while c-di-AMP inhibits its activation. We solved the crystal structure of CbpB bound and unbound to c-di-AMP and provide insight into the region important for c-di-AMP binding and RelA activation. The results of this study show that CbpB completes a homeostatic regulatory circuit between c-di-AMP and (p)ppGpp in Bacteria must efficiently maintain homeostasis of essential molecules to survive in the environment. We found that the levels of c-di-AMP and (p)ppGpp, two nucleotide second messengers that are highly conserved throughout the microbial world, coexist in a homeostatic loop in the facultative intracellular pathogen Here, we found that cyclic di-AMP binding protein B (CbpB) acts as a c-di-AMP sensor that promotes the synthesis of (p)ppGpp by binding to RelA when c-di-AMP levels are low. Addition of c-di-AMP prevented RelA activation by binding and sequestering CbpB. Previous studies showed that (p)ppGpp binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. This pathway is controlled via direct enzymatic regulation and indicates an additional mechanism of ribosome-independent stringent activation.
Topics: Animals; Bacterial Proteins; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Guanosine Pentaphosphate; Homeostasis; Listeria monocytogenes; Mice; Protein Binding; Second Messenger Systems; Signal Transduction
PubMed: 32843560
DOI: 10.1128/mBio.01625-20 -
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 -
Scientific Reports Feb 2022The zinc finger antiviral protein (ZAP) is known to restrict viral replication by binding to the CpG rich regions of viral RNA, and subsequently inducing viral RNA...
The zinc finger antiviral protein (ZAP) is known to restrict viral replication by binding to the CpG rich regions of viral RNA, and subsequently inducing viral RNA degradation. This enzyme has recently been shown to be capable of restricting SARS-CoV-2. These data have led to the hypothesis that the low abundance of CpG in the SARS-CoV-2 genome is due to an evolutionary pressure exerted by the host ZAP. To investigate this hypothesis, we performed a detailed analysis of many coronavirus sequences and ZAP RNA binding preference data. Our analyses showed neither evidence for an evolutionary pressure acting specifically on CpG dinucleotides, nor a link between the activity of ZAP and the low CpG abundance of the SARS-CoV-2 genome.
Topics: Animals; Base Sequence; Binding Sites; COVID-19; Dinucleoside Phosphates; Evolution, Molecular; Genome, Viral; Host-Pathogen Interactions; Humans; Nucleotide Motifs; Protein Binding; RNA, Viral; RNA-Binding Proteins; SARS-CoV-2; Virus Replication
PubMed: 35165300
DOI: 10.1038/s41598-022-06046-5 -
International Journal of Molecular... Jun 2022AppA, the periplasmic phytase of clade 2 of the histidine phosphatase (HP2) family, has been well-characterized and successfully engineered for use as an animal feed...
AppA, the periplasmic phytase of clade 2 of the histidine phosphatase (HP2) family, has been well-characterized and successfully engineered for use as an animal feed supplement. AppA is a 1D-6-phytase and highly stereospecific but transiently accumulates 1D--Ins(2,3,4,5)P and other lower phosphorylated intermediates. If this bottleneck in liberation of orthophosphate is to be obviated through protein engineering, an explanation of its rather rigid preference for the initial site and subsequent cleavage of phytic acid is required. To help explain this behaviour, the role of the catalytic proton donor residue in determining AppA stereospecificity was investigated. Four variants were generated by site-directed mutagenesis of the active site HDT amino acid sequence motif containing the catalytic proton donor, D304. The identity and position of the prospective proton donor residue was found to strongly influence stereospecificity. While the wild-type enzyme has a strong preference for 1D-6-phytase activity, a marked reduction in stereospecificity was observed for a D304E variant, while a proton donor-less mutant (D304A) displayed exclusive 1D-1/3-phytase activity. High-resolution X-ray crystal structures of complexes of the mutants with a non-hydrolysable substrate analogue inhibitor point to a crucial role played by D304 in stereospecificity by influencing the size and polarity of specificity pockets A and B. Taken together, these results provide the first evidence for the involvement of the proton donor residue in determining the stereospecificity of HP2 phytases and prepares the ground for structure-informed engineering studies targeting the production of animal feed enzymes capable of the efficient and complete dephosphorylation of dietary phytic acid.
Topics: 6-Phytase; Acid Phosphatase; Animals; Dinucleoside Phosphates; Escherichia coli; Escherichia coli Proteins; Phytic Acid; Prospective Studies; Protons
PubMed: 35683026
DOI: 10.3390/ijms23116346 -
Nucleic Acids Research Jul 2023The nonenzymatic copying of RNA is thought to have been necessary for the transition between prebiotic chemistry and ribozyme-catalyzed RNA replication in the RNA World....
The nonenzymatic copying of RNA is thought to have been necessary for the transition between prebiotic chemistry and ribozyme-catalyzed RNA replication in the RNA World. We have previously shown that a potentially prebiotic nucleotide activation pathway based on phospho-Passerini chemistry can lead to the efficient synthesis of 2-aminoimidazole activated mononucleotides when carried out under freeze-thaw cycling conditions. Such activated nucleotides react with each other to form 5'-5' 2-aminoimidazolium bridged dinucleotides, enabling template-directed primer extension to occur within the same reaction mixture. However, mononucleotides linked to oligonucleotides by a 5'-5' 2-aminoimidazolium bridge are superior substrates for nonenzymatic primer extension; their higher intrinsic reactivity and their higher template affinity enable faster template copying at lower substrate concentrations. Here we show that eutectic phase phospho-Passerini chemistry efficiently activates short oligonucleotides and promotes the formation of monomer-bridged-oligonucleotide species during freeze-thaw cycles. We then demonstrate that in-situ generated monomer-bridged-oligonucleotides lead to efficient nonenzymatic template copying in the same reaction mixture. Our demonstration that multiple steps in the pathway from activation chemistry to RNA copying can occur together in a single complex environment simplifies this aspect of the origin of life.
Topics: RNA; Oligonucleotides; RNA, Catalytic; Nucleotides; Dinucleoside Phosphates
PubMed: 37247941
DOI: 10.1093/nar/gkad439 -
International Journal of Molecular... Oct 2021DNA lesions that impede fork progression cause replisome stalling and threaten genome stability. RecA, at a lesion-containing gap, interacts with and facilitates DisA...
DNA lesions that impede fork progression cause replisome stalling and threaten genome stability. RecA, at a lesion-containing gap, interacts with and facilitates DisA pausing at these branched intermediates. Paused DisA suppresses its synthesis of the essential c-di-AMP messenger. The RuvAB-RecU resolvasome branch migrates and resolves formed Holliday junctions (HJ). We show that DisA prevents DNA degradation. DisA, which interacts with RuvB, binds branched structures, and reduces the RuvAB DNA-dependent ATPase activity. DisA pre-bound to HJ DNA limits RuvAB and RecU activities, but such inhibition does not occur if the RuvAB- or RecU-HJ DNA complexes are pre-formed. RuvAB or RecU pre-bound to HJ DNA strongly inhibits DisA-mediated synthesis of c-di-AMP, and indirectly blocks cell proliferation. We propose that DisA limits RuvAB-mediated fork remodeling and RecU-mediated HJ cleavage to provide time for damage removal and replication restart in order to preserve genome integrity.
Topics: Adenosine Triphosphatases; Bacillus subtilis; Bacterial Proteins; Chromosome Breakage; DNA Helicases; DNA Replication; DNA, Bacterial; DNA, Cruciform; DNA-Binding Proteins; Dinucleoside Phosphates; Escherichia coli; Holliday Junction Resolvases; Magnesium; Phosphorus-Oxygen Lyases
PubMed: 34768753
DOI: 10.3390/ijms222111323 -
MBio Feb 2021In Bacillus subtilis and other Gram-positive bacteria, cyclic di-AMP is an essential second messenger that signals potassium availability by binding to a variety of...
In Bacillus subtilis and other Gram-positive bacteria, cyclic di-AMP is an essential second messenger that signals potassium availability by binding to a variety of proteins. In some bacteria, c-di-AMP also binds to the pyruvate carboxylase to inhibit its activity. We have discovered that in B. subtilis the c-di-AMP target protein DarB, rather than c-di-AMP itself, specifically binds to pyruvate carboxylase both and . This interaction stimulates the activity of the enzyme, as demonstrated by enzyme assays and metabolite determinations. Both the interaction and the activation of enzyme activity require apo-DarB and are inhibited by c-di-AMP. Under conditions of potassium starvation and corresponding low c-di-AMP levels, the demand for citric acid cycle intermediates is increased. Apo-DarB helps to replenish the cycle by activating both pyruvate carboxylase gene expression and enzymatic activity via triggering the stringent response as a result of its interaction with the (p)ppGpp synthetase Rel and by direct interaction with the enzyme, respectively. If bacteria experience a starvation for potassium, by far the most abundant metal ion in every living cell, they have to activate high-affinity potassium transporters, switch off growth activities such as translation and transcription of many genes or replication, and redirect the metabolism in a way that the most essential functions of potassium can be taken over by metabolites. Importantly, potassium starvation triggers a need for glutamate-derived amino acids. In many bacteria, the responses to changing potassium availability are orchestrated by a nucleotide second messenger, cyclic di-AMP. c-di-AMP binds to factors involved directly in potassium homeostasis and to dedicated signal transduction proteins. Here, we demonstrate that in the Gram-positive model organism Bacillus subtilis, the c-di-AMP receptor protein DarB can bind to and, thus, activate pyruvate carboxylase, the enzyme responsible for replenishing the citric acid cycle. This interaction takes place under conditions of potassium starvation if DarB is present in the apo form and the cells are in need of glutamate. Thus, DarB links potassium availability to the control of central metabolism.
Topics: Cyclic AMP; Bacillus subtilis; Pyruvate Carboxylase; Bacterial Proteins; Second Messenger Systems; Dinucleoside Phosphates; Glutamic Acid; Potassium
PubMed: 35130724
DOI: 10.1128/mbio.03602-21 -
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 -
International Journal of Biological... 2022Chronic Hepatitis B virus (CHB) infection is a global public health problem. Oligodeoxynucleotides (ODNs) containing class C unmethylated cytosine-guanine dinucleotide...
Chronic Hepatitis B virus (CHB) infection is a global public health problem. Oligodeoxynucleotides (ODNs) containing class C unmethylated cytosine-guanine dinucleotide (CpG-C) motifs may provide potential adjuvants for the immunotherapeutic strategy against CHB, since CpG-C ODNs stimulate both B cell and dendritic cell (DC) activation. However, the efficacy of CpG-C ODN as an anti-HBV vaccine adjuvant remains unclear. In this study, we demonstrated that CpG M362 (CpG-C ODN) as an adjuvant in anti-HBV vaccine (cHBV-vaccine) successfully and safely eliminated the virus in HBV-carrier mice. The cHBV-vaccine enhanced DC maturation both and , overcame immune tolerance, and recovered exhausted T cells in HBV-carrier mice. Furthermore, the cHBV-vaccine elicited robust hepatic HBV-specific CD8 and CD4 T cell responses, with increased cellular proliferation and IFN-γ secretion. Additionally, the cHBV-vaccine invoked a long-lasting follicular CXCR5 CD8 T cell response following HBV re-challenge. Taken together, CpG M362 in combination with rHBVvac cleared persistent HBV and achieved long-term virological control, making it a promising candidate for treating CHB.
Topics: Adjuvants, Immunologic; Animals; Dinucleoside Phosphates; Disease Models, Animal; Hepatitis B Vaccines; Hepatitis B, Chronic; Male; Mice; Mice, Inbred C57BL; Oligodeoxyribonucleotides
PubMed: 34975324
DOI: 10.7150/ijbs.62424