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Nature Communications May 2024c-di-AMP is an essential and widespread nucleotide second messenger in bacterial signaling. For most c-di-AMP synthesizing organisms, c-di-AMP homeostasis and the...
c-di-AMP is an essential and widespread nucleotide second messenger in bacterial signaling. For most c-di-AMP synthesizing organisms, c-di-AMP homeostasis and the molecular mechanisms pertaining to its signal transduction are of great concern. Here we show that c-di-AMP binds the N-acetylglucosamine (GlcNAc)-sensing regulator DasR, indicating a direct link between c-di-AMP and GlcNAc signaling. Beyond its foundational role in cell-surface structure, GlcNAc is attractive as a major nutrient and messenger molecule regulating multiple cellular processes from bacteria to humans. We show that increased c-di-AMP levels allosterically activate DasR as a master repressor of GlcNAc utilization, causing the shutdown of the DasR-mediated GlcNAc signaling cascade and leading to a consistent enhancement in the developmental transition and antibiotic production in Saccharopolyspora erythraea. The expression of disA, encoding diadenylate cyclase, is directly repressed by the regulator DasR in response to GlcNAc signaling, thus forming a self-sustaining transcriptional feedback loop for c-di-AMP synthesis. These findings shed light on the allosteric regulation by c-di-AMP, which appears to play a prominent role in global signal integration and c-di-AMP homeostasis in bacteria and is likely widespread in streptomycetes that produce c-di-AMP.
Topics: Acetylglucosamine; Allosteric Regulation; Signal Transduction; Bacterial Proteins; Gene Expression Regulation, Bacterial; Dinucleoside Phosphates; Saccharopolyspora
PubMed: 38714645
DOI: 10.1038/s41467-024-48063-0 -
Acta Crystallographica. Section D,... May 2024CdaA is the most widespread diadenylate cyclase in many bacterial species, including several multidrug-resistant human pathogens. The enzymatic product of CdaA, cyclic...
CdaA is the most widespread diadenylate cyclase in many bacterial species, including several multidrug-resistant human pathogens. The enzymatic product of CdaA, cyclic di-AMP, is a secondary messenger that is essential for the viability of many bacteria. Its absence in humans makes CdaA a very promising and attractive target for the development of new antibiotics. Here, the structural results are presented of a crystallographic fragment screen against CdaA from Listeria monocytogenes, a saprophytic Gram-positive bacterium and an opportunistic food-borne pathogen that can cause listeriosis in humans and animals. Two of the eight fragment molecules reported here were localized in the highly conserved ATP-binding site. These fragments could serve as potential starting points for the development of antibiotics against several CdaA-dependent bacterial species.
Topics: Listeria monocytogenes; Crystallography, X-Ray; Binding Sites; Bacterial Proteins; Models, Molecular; Dinucleoside Phosphates; Anti-Bacterial Agents; Humans; Phosphorus-Oxygen Lyases; Protein Conformation
PubMed: 38682668
DOI: 10.1107/S205979832400336X -
Journal of the American Chemical Society Apr 2024Nonenzymatic template-directed RNA copying requires catalysis by divalent metal ions. The primer extension reaction involves the attack of the primer 3'-hydroxyl on the...
Nonenzymatic template-directed RNA copying requires catalysis by divalent metal ions. The primer extension reaction involves the attack of the primer 3'-hydroxyl on the adjacent phosphate of a 5'-5'-imidazolium-bridged dinucleotide substrate. However, the nature of the interaction of the catalytic metal ion with the reaction center remains unclear. To explore the coordination of the catalytic metal ion with the imidazolium-bridged dinucleotide substrate, we examined catalysis by oxophilic and thiophilic metal ions with both diastereomers of phosphorothioate-modified substrates. We show that Mg and Cd exhibit opposite preferences for the two phosphorothioate substrate diastereomers, indicating a stereospecific interaction of the divalent cation with one of the nonbridging phosphorus substituents. High-resolution X-ray crystal structures of the products of primer extension with phosphorothioate substrates reveal the absolute stereochemistry of this interaction and indicate that catalysis by Mg involves inner-sphere coordination with the nonbridging phosphate oxygen in the pro- position, while thiophilic cadmium ions interact with sulfur in the same position, as in one of the two phosphorothioate substrates. These results collectively suggest that during nonenzymatic RNA primer extension with a 5'-5'-imidazolium-bridged dinucleotide substrate the interaction of the catalytic Mg ion with the pro- oxygen of the reactive phosphate plays a crucial role in the metal-catalyzed S2(P) reaction.
Topics: RNA; Metals; Dinucleoside Phosphates; Phosphates; Catalysis; Oxygen; Ions; RNA, Catalytic
PubMed: 38579124
DOI: 10.1021/jacs.4c00323 -
International Journal of Molecular... Dec 2023To improve the chemical regulation on the activity of cyclic dinucleotides (CDNs), we here designed a reduction-responsive dithioethanol (DTE)-based dCDN prodrug...
To improve the chemical regulation on the activity of cyclic dinucleotides (CDNs), we here designed a reduction-responsive dithioethanol (DTE)-based dCDN prodrug (DTE-dCDN). Prodrug improved the cell permeability with the intracellular levels peaking in 2 h in THP-1 cells. Under the reductive substance such as GSH or DTT, prodrug could be quickly decomposed in 30 min to release the parent dCDN. In THP1-Lucia cells, prodrug also retained a high bioactivity with the EC of 0.96 μM, which was 51-, 43-, and 3-fold more than the 2',3'-cGAMP (EC = 48.6 μM), the parent compound 3',3'-c-di-dAMP (EC = 41.3 μM), and ADU-S100 (EC = 2.9 μM). The high bioactivity of prodrug was validated to be highly correlated with the activation of the STING signaling pathway. Furthermore, prodrug could also improve the transcriptional expression levels of , , and in THP-1 cells. These results will be helpful to the development of chemically controllable CDN prodrugs with a high cellular permeability and potency.
Topics: Deoxyribose; Alarmins; Dinucleoside Phosphates; Permeability; Prodrugs
PubMed: 38203256
DOI: 10.3390/ijms25010086 -
International Journal of Molecular... Nov 2023Dinucleoside polyphosphates (NpNs) are considered novel signalling molecules involved in the induction of plant defence mechanisms. However, NpN signal recognition and...
Dinucleoside polyphosphates (NpNs) are considered novel signalling molecules involved in the induction of plant defence mechanisms. However, NpN signal recognition and transduction are still enigmatic. Therefore, the aim of our research was the identification of the NpN receptor and signal transduction pathways evoked by these nucleotides. Earlier, we proved that purine and pyrimidine NpNs differentially affect the phenylpropanoid pathway in suspension-cultured cells. Here, we report, for the first time, that both diadenosine tetraphosphate (ApA) and dicytidine tetraphosphate (CpC)-induced stomatal closure in . Moreover, we showed that plasma membrane purinoreceptor P2K1/DORN1 (does not respond to nucleotide 1) is essential for ApA-induced stomata movements but not for CpC. Wild-type Col-0 and the knockout mutant were used. Examination of the leaf epidermis mutant provided evidence that P2K1/DORN1 is a part of the signal transduction pathway in stomatal closure evoked by extracellular ApA but not by CpC. Reactive oxygen species (ROS) are involved in signal transduction caused by ApA and CpC, leading to stomatal closure. ApA induced and CpC suppressed the transcriptional response in wild-type plants. Moreover, in leaves, the effect of ApA on gene expression was impaired. The interaction between P2K1/DORN1 and ApA leads to changes in the transcription of signalling hubs in signal transduction pathways.
Topics: Arabidopsis; Dinucleoside Phosphates; Signal Transduction; Cell Membrane; Receptors, Purinergic
PubMed: 38069010
DOI: 10.3390/ijms242316688 -
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 -
Nature Communications Jun 2023Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In...
Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In Bacillus subtilis, the K/H symporter KimA of the KUP family is inactivated by c-di-AMP. KimA sustains survival at potassium limitation at low external pH by mediating potassium ion uptake. However, at elevated intracellular K concentrations, further K accumulation would be toxic. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K binding site and therefore traps KimA in an inward-occluded conformation.
Topics: Cyclic AMP; Protons; Bacterial Proteins; Second Messenger Systems; Membrane Transport Proteins; Potassium; Dinucleoside Phosphates
PubMed: 37344476
DOI: 10.1038/s41467-023-38944-1 -
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 -
Purinergic Signalling Apr 2024During the establishment of neuronal circuits, axons and dendrites grow and branch to establish specific synaptic connections. This complex process is highly regulated...
During the establishment of neuronal circuits, axons and dendrites grow and branch to establish specific synaptic connections. This complex process is highly regulated by positive and negative extracellular cues guiding the axons and dendrites. Our group was pioneer in describing that one of these signals are the extracellular purines. We found that extracellular ATP, through its selective ionotropic P2X7 receptor (P2X7R), negatively regulates axonal growth and branching. Here, we evaluate if other purinergic compounds, such as the diadenosine pentaphosphate (ApA), may module the dynamics of dendritic or axonal growth and branching in cultured hippocampal neurons. Our results show that ApA negatively modulates the dendrite's growth and number by inducing transient intracellular calcium increases in the dendrites' growth cone. Interestingly, phenol red, commonly used as a pH indicator in culture media, also blocks the P2X1 receptors, avoided the negative modulation of ApA on dendrites. Subsequent pharmacological studies using a battery of selective P2X1R antagonists confirmed the involvement of this subunit. In agreement with pharmacological studies, P2X1R overexpression caused a similar reduction in dendritic length and number as that induced by ApA. This effect was reverted when neurons were co-transfected with the vector expressing the interference RNA for P2X1R. Despite small hairpin RNAs reverting the reduction in the number of dendrites caused by ApA, it did not avoid the dendritic length decrease induced by the polyphosphate, suggesting, therefore, the involvement of a heteromeric P2X receptor. Our results are indicating that ApA exerts a negative influence on dendritic growth.
Topics: Adenosine Triphosphate; Receptors, Purinergic P2; Neurons; Dendrites; Hippocampus; Dinucleoside Phosphates
PubMed: 37246192
DOI: 10.1007/s11302-023-09944-z -
Current Diabetes Reports Jun 2023Type 2 diabetes mellitus (T2DM) is one of the leading causes of death and disability in the world. The majority of diabetes deaths (> 80%) occur in low- and... (Review)
Review
PURPOSE OF REVIEW
Type 2 diabetes mellitus (T2DM) is one of the leading causes of death and disability in the world. The majority of diabetes deaths (> 80%) occur in low- and middle-income countries, which are predominant in Latin America. Therefore, the purpose of this article is to compare the clinical practice guideline (CPG) for the pharmacological management of T2DM in Latin America (LA) with international reference guidelines.
RECENT FINDINGS
Several LA countries have recently developed CPGs. However, the quality of these guidelines is unknown according to the AGREE II tool and taking as reference three CPGs of international impact: American Diabetes Association (ADA), European Diabetes Association (EASD), and Latin American Diabetes Association (ALAD). Ten CPGs were selected for analysis. The ADA scored > 80% on the AGREE II domains and was selected as the main comparator. Eighty percent of LA CPGs were developed before 2018. Only one was not recommended (all domains < 60%). The CPGs in LA have good quality but are outdated. They have significant gaps compared to the reference. There is a need for improvement, as proposing updates every three years to maintain the best available clinical evidence in all guidelines.
Topics: Humans; Diabetes Mellitus, Type 2; Latin America; Dinucleoside Phosphates
PubMed: 37126189
DOI: 10.1007/s11892-023-01504-4